Introduction and guidance

ALT_1 Understanding antibiotic resistance About this free course This free course provides a sample of level 1 study in Science www.open.ac.uk/courses/find/science This version of the content may include video, images and interactive content that may not be optimised for your device. You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University: There you’ll also be able to track your progress via your activity record, which you can use to demonstrate your learning. Copyright © 2018 The Open University

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What is a badged course? While studying Understanding antibiotic resistance you have the option to work towards gaining a digital badge. Badged courses are a key part of The Open University’s mission to promote the educational wellbeing of the community. The courses also provide another way of helping you to progress from informal to formal learning. Completing a course will require about 24 hours of study time. However, you can study the course at any time and at a pace to suit you. Badged courses are available on The Open University’s OpenLearn website and do not cost anything to study. They differ from Open University courses because you do not receive support from a tutor, but you do get useful feedback from the interactive quizzes. What is a badge? Digital badges are a new way of demonstrating online that you have gained a skill. Colleges and universities are working with employers and other organisations to develop open badges that help learners gain recognition for their skills, and support employers to identify the right candidate for a job. Badges demonstrate your work and achievement on the course. You can share your achievement with friends, family and employers, and on social media. Badges are a great motivation, helping you to reach the end of the course. Gaining a badge often boosts confidence in the skills and abilities that underpin successful study. So, completing this course could encourage you to think about taking other courses.

How to get a badge Getting a badge is straightforward! Here’s what you have to do: read each week of the course score 50% or more in the two badge quizzes in Week 4 and Week 8. For all the quizzes, you can have three attempts at most of the questions (for true or false type questions you usually only get one attempt). If you get the answer right first time you will get more marks than for a correct answer the second or third time. Therefore, please be aware that for the two badge quizzes it is possible to get all the questions right but not score 50% and be eligible for the badge on that attempt. If one of your answers is incorrect you will often receive helpful feedback and suggestions about how to work out the correct answer. For the badge quizzes, if you’re not successful in getting 50% the first time, after 24 hours you can attempt the whole quiz, and come back as many times as you like. We hope that as many people as possible will gain an Open University badge – so you should see getting a badge as an opportunity to reflect on what you have learned rather than as a test. If you need more guidance on getting a badge and what you can do with it, take a look at the OpenLearn FAQs. When you gain your badge you will receive an email to notify you and you will be able to view and manage all your badges in My OpenLearn within 24 hours of completing the criteria to gain a badge. Get started with Week 1.

Week 1: A future without antibiotics? Introduction Welcome to Week 1 of this free course, Understanding antibiotic resistance. In this week you will read about common bacterial pathogens and how antibiotics can be used to treat bacterial infections. You will then step back in time, through the medium of video, to consider how and with what success infections were once treated. After a brief review of key scientific advances that heralded the discovery of antibiotics, you will consider how these life-saving drugs revolutionised medical care. Back in the present, you will analyse data from different countries highlighting the growing problem of antibiotic resistance worldwide. You will be introduced to factors, covered in more detail later in the course, that have contributed to this problem. Finally, you will listen to scientists discussing the potential threat to modern medicine of antibiotic resistance and will begin to form your own opinion about it. Start by watching the video below which reveals how the natural processes of bacteria are exploited to fight infections – and how bacteria fight back! Video 1 Antibiotics. NARRATOR: Microbes, mostly tiny bacteria, make up around 90% of the cells in a typical human body and 10% of our body weight. Most of them are in our gut and on our skin. Many microbes are beneficial, for example, helping us to digest our food. Only a tiny fraction cause disease, and are usually kept in check by our immune system. But when they aren't, microbes also help us to fight back. Bacteria cause disease when they are able to reproduce in the body. They produce harmful substances called toxins, which damage tissues and organs. But in nature, microbes can also produce agents called antibiotics to protect themselves against competitors. PROFESSOR CHARLES COCKELL: It's a tough world out there. And you might think that you just see competition in the savannas of Africa. But in fact, the microbes fight each other as well. And in fact, like martial arts, they have ways of fighting other microbes with particular moves. And one move they have is to produce antibiotics. These are compounds that allow them to kill other microbes and take all the food for themselves, all the resources that they need. And so the competition between microbes results in these very sophisticated antibiotic molecules. NARRATOR: The discovery of antibiotics and their power to fight bacterial disease began with Alexander Fleming. He observed the mould penicillium notatum accidentally growing on a sample of staphylococci, and saw it had killed the surrounding colonies of disease causing bacteria. DR PAULA SALDAGO: All antibiotics work by disrupting a critical function in the bacterial cell. For example, penicillin, discovered in 1928, prevents the cell from renewing it's cell wall during growth. Eventually, the cell wall weakens and bursts. NARRATOR: By the 1950s, the use of antibiotics had revolutionised the treatment of previously untreatable infectious diseases. In 1967, the Surgeon General of the United States of America, William Stewart, declared, ‘The time has come to close the book on infectious diseases. We have basically wiped out infection in the United States.’ But Stewart's optimism proved premature. PROFESSOR CHARLES COCKELL: The bad news is that microbes can become resistant to antibiotics, and they can change their biochemistry in order to adapt to these antibiotics and prevent the antibiotics from damaging the cell. NARRATOR: It's standard evolutionary behaviour. When bacteria reproduce, chance mutations occur. Most will be useless. But sometimes there will be one that will protect the bacterium against a particular antibiotic. While most of the bacteria succumb to the antibiotic, the one that survives goes on to reproduce and replicate the resistance. And bacteria reproduce very fast. DR PAULA SALDAGO: The good news is that scientists are developing new synthetic antibiotics that target resistant bacteria. NARRATOR: But who knows whether one day a mutated bacterium might become resistant to all synthetic antibiotics, a super, super bug. [MUSIC PLAYING]

By the end of this week, you should be able to: recall why pathogenic bacteria pose a threat to human health define the term antibiotic and give examples describe the importance of antibiotics in modern health care analyse antibiotic data and make simple deductions about antibiotic use and resistance patterns discuss the consequences of a future without antibiotics. Although this is an introductory course to antibiotic resistance, it assumes that you have a basic understanding of DNA and proteins. If you are unfamiliar with these concepts, you may want to try our free OpenLearn course What do genes do? or listen to our set of audios on DNA, RNA and protein formation before you start this course. 1 Bacteria and infectious disease Bacteria are the smallest and most numerous organisms living on Earth and are found in every conceivable habitat. The secret of their success is their relatively simple, single-cell structure which allows them to reproduce quickly and efficiently. In the next section you will look at the process that allows bacteria to reproduce so quickly.

1.1 Bacterial growth Bacteria reproduce by a straightforward process in which each cell splits into two identical, new cells. This process is called binary fission and you will learn more about it in Week 4. Individual bacterial cells can divide, and the bacterial population can double, very quickly – in as little as 20 minutes in some species. Watch the following video to see a speeded-up film of binary fission. Video 2 Speeded-up film of bacterial growth. NARRATOR: These rod-shaped bacteria are about to replicate. There are eight in this culture, but each bacterial cell is about to divide into two. Now there are 16 bacteria, and soon there will be 32. Under ideal conditions, some bacteria can divide in as little as 20 minutes. A bacterial infection can take hold so quickly because bacteria can double their numbers in such a short time unless something keeps them in check. Handwashing with soap, vaccination to boost the immune system, and anti-bacterial drugs may halt this replication cycle. But left unchecked, in less than four days, this bacterial culture would weigh as much as the Earth.

However, bacteria do not continue growing at such a rapid rate indefinitely. This is because factors such as the availability of nutrients and rising toxin levels start to have an effect. You can explore this in the first activity. Don’t worry if you don’t understand all the terms, as they will be explained later. Activity 1 Bacterial growth phases Allow about 10 minutes In nature, bacterial growth follows a typical pattern shown in Figure 1. The growth curve is comprised of four phases. Lag phase: during the lag phase, the bacteria are adapting to their environment; nutrients are plentiful and the cells grow in size without dividing. Cell number remains constant. Exponential phase: the exponential phase marks a big increase in cell number. Maximum growth rate is achieved with a constant doubling of the bacterial population. Growth then slows as nutrients become depleted and bacterial waste products build up to toxic levels. Stationary phase: the bacteria enter the stationary phase when the number of new cells equals the number of cells dying. The total number of cells in the population remains constant. Death phase: unless nutrients are replenished and waste products removed, the bacteria progress to the death phase. More cells die than are produced and the number of cells in the population declines. (a) Using drag-and-drop, match the descriptions above to the correct phase of the growth curve. Don’t worry if you are not sure of the correct answer at this stage. You can check your answers by clicking on the buttons below Figure 1. Figure 1 Graph of bacterial growth showing how the number of cells changes with time in a culture in which the bacteria are reproducing by binary fission. This figure is a simple graph in which the horizontal axis is labelled time and the vertical axis is labelled number of cells. The four phases of the growth curve are distinguished using background shading of different colours. In the lag phase (yellow), cell number increases very slowly with time; cell number increases progressively rapidly during the exponential phase (red), levels off to a constant value in the stationary phase (blue), then falls during the death phase (grey). During the lag phase, the bacteria are adapting to their environment; nutrients are plentiful and the cells grow in size. The exponential phase marks a big increase in cell number. Maximum growth rate is achieved with a constant doubling of the bacterial population. Growth then slows as nutrients become depleted and bacterial waste products build up to toxic levels. The bacteria enter the stationary phase when the number of new cells equals the number of cells dying. Unless nutrients are replenished and waste products removed, the bacteria progress to the death phase. More cells die than are produced and the population declines. Because they can divide so rapidly, bacteria adapt quickly to changes in their surroundings. Advantageous characteristics which allow the bacteria to flourish in the new conditions are passed on to successive generations and the species evolves rapidly. In Week 4 you will learn about the genetic mechanisms underlying this process.

1.2 Common bacterial pathogens of humans Most bacteria found in or on the human body are harmless commensals living on the body without having any detrimental effect. However, a tiny proportion – about 500 species – are pathogenic, that is they are capable of causing disease. These bacteria may evade the body’s normal defences to colonise or invade body cells and tissues, or they may produce harmful toxins. Many bacteria are opportunistic pathogens. These take advantage of an unusually vulnerable host and adapt quickly to the changed conditions. Activity 2 Common bacterial pathogens and infectious diseases Allow about 10 minutes You might recognise the names of some important pathogenic bacteria shown in Figure 2. Note how different species of bacteria have characteristic shapes: for example, the spherical (coccus)-shaped Streptococci and the rod-shaped Klebsiella. Can you name the infectious diseases that they cause? Click on reveal to see the answer. Figure 2 High magnification images of common bacterial pathogens in humans taken using a scanning electron microscope. The figure shows scanning electron micrographs of common bacterial pathogens in humans. Part (a) is a scanning electron micrograph of Streptococcus pneumoniae which appear as chains of red oval shaped cells on a grey background. Part (b) is a scanning electron micrograph of Escherichia coli bacteria which appear as yellow rod shaped cell clustered on a grey background. Part (c) is a scanning electron micrograph of methicillin-resistant Staphylococcus aureus which appear as a chain of yellow spheres on a dark background. Part (d) is a scanning electron micrograph of Mycobacterium tuberculosis which appear as a large clump of red rod shaped bacteria on a dark background. Part (e) is a scanning electron micrograph of two Klebsiella pneumoniae bacteria which appear as two red, rod shaped cells wrapped in a long blue filament. Part (f) is a scanning electron micrograph of Neisseria gonorrhoeae which appear as individual purple kidney shaped cells on a dark background. S. pneumoniae is a common cause of pneumonia and ear infections. Pathogenic strains of E. coli are a common cause of diarrhoeal disease, for example as a result of food poisoning, and of urinary infections. MRSA is a particular threat in healthcare settings where it can cause sepsis and death if not treated quickly. M. tuberculosis causes tuberculosis (TB). K. pneumoniae is a common cause of many healthcare-associated infections including pneumonia, and bloodstream and wound infections. N. gonorrhoeae causes the sexually transmitted infection gonorrhoea. Until the mid-twentieth century, bacterial infections were notoriously difficult to treat and were a leading cause of human morbidity and mortality worldwide. Then, in the 1930s, antibiotics were introduced and the outcomes for bacterial infection improved dramatically. The next section introduces these new wonder drugs of the twentieth century – antibiotics.

2 Antibiotics Antibiotics are chemicals which kill bacteria, that is they are bactericidal, or inhibit bacterial growth, that is they are bacteriostatic. They are produced naturally by soil-living bacteria and fungi in order to stop rival bacteria competing for nutrients and other resources. Antibiotics specifically target bacteria – a characteristic that humans have exploited for their own advantage to manage infectious diseases. Narrow spectrum antibiotics affect only a few bacterial types. Broad spectrum antibiotics affect a wider range of bacteria. In Section 1 you learned about the different phases of bacterial growth. Most antibiotics target the exponential phase of growth. Can you suggest a reason for this? The exponential phase is when bacterial cells are at their most active, continually growing, dividing and forming new cells. The various metabolic processes which underpin this period of growth – such as the synthesis of DNA/RNA, proteins and the cell wall – are good opportunities for antibiotics to disrupt and/or kill cells. You will learn how antibiotics work in Week 2.

2.1 Classification There are numerous different antibiotics, some of which are naturally occurring while others are semi- or fully synthetic. Don’t worry if you don’t understand these terms, as they will be explained later. One of the most useful ways of classifying antibiotics is by chemical structure because structurally similar antibiotics tend to have similar antibacterial activity. Examples of common antibiotic classes are shown in Table 1. Table 1 Common classes of antibiotic* You will learn more about these cellular processes in Week 2.** Common effect but partly depends on the concentration at which the antibiotic is used. (Source: OpenStax College Microbiology, n.d.)

Antibiotic class	Example	Cellular process targeted*	Effect on bacteria**
ß-Lactams (penicillins)	ampicillin	bacterial cell wall synthesis	bactericidal
ß-Lactams (cephalosporins)	cephazolin	bacterial cell wall synthesis	bactericidal
ß-Lactams (carbapenems)	imipenem	bacterial cell wall synthesis	bactericidal
Glycopeptides	vancomycin	bacterial cell wall synthesis	bactericidal

Aminoglycosides	streptomycin	protein synthesis	bactericidal
Macrolides	azithromycin	protein synthesis	bacteriostatic
Tetracyclines	tetracycline	protein synthesis	bacteriostatic
Oxazolidinones	linezolid	protein synthesis	bacteriostatic

Fluoroquinolones	ciprofloxacin	DNA synthesis	bactericidal
Rifamycins	rifampicin	RNA synthesis	bactericidal

Not applicable	trimethoprim	metabolic reactions	bactericidal

2.2 How much do you know about antibiotics? Try this short quiz to find out how much you know about antibiotics. Don’t worry if you don’t know the answers to all the questions. By the end of this course you should be able to answer them all. Activity 3 The antibiotics quiz Allow about 5 minutes 1 Antibiotics can be used to treat infections caused by: bacteria and viruses bacteria viruses all microorganisms Antibiotics specifically target bacteria. They are not effective against infections such as the common cold and flu which are caused by viruses. 2 Antibiotics: have non-therapeutic uses are only active against pathogens do not cause side effects stimulate the body’s immune system Antibiotics are used for many non-therapeutic purposes, for example as growth promoters in farm animals. Antibiotics are not selective and will inhibit or kill ‘good’ bacteria along with ‘bad’ bacteria in the gut. This can lead to common side effects such as upset stomach and loose stools. Antibiotics neither enhance nor inhibit the body’s immune response. 3 What should you do with left-over antibiotics that have been prescribed by your doctor? Return them to your doctor to dispose of Throw them away Nothing – you should always complete the course Save them for when you get another infection It is important to take antibiotics at the dose prescribed and to complete the full course. Otherwise, the effectiveness of the drug may be reduced which could lead to antibiotic resistance. 4 Antibiotic resistance occurs when: bacteria are no longer susceptible to the antibiotic a person develops an allergic reaction to a prescribed antibiotic the drug stops working in the individual the antibiotic changes in some way It is the bacterial pathogen that develops antibiotic resistance and is no longer susceptible to its effects. 5 Antibiotic-resistant infections: may require treatment with more expensive and more powerful drugs may require hospital treatment may take longer to cure all of these Antibiotic-resistant infections may not respond to common antibiotics and/or may require treatment with combinations of drugs. Some antibiotic-resistant infections may be fatal. In the next section you will learn how the medical profession managed before antibiotics were available.

3 Pre-antibiotic era Before antibiotics were discovered, the treatment options for bacterial infections were limited, as you will see next. Activity 4 Life without antibiotics Allow about 15 minutes Watch the following video about infection in the pre-antibiotic era. As you watch, consider: which bacterial infections were common and what the usual outcome was how bacterial infections were treated. Video 3 Living in the eighteenth century. MICHAEL MOSLEY: On Saturday the 14th of December, 1799, George Washington, one of the founding fathers of the United States of America, lay dying. A couple of days earlier, he'd been out riding in cold and wet weather. He developed a sore throat. Early on the Saturday morning he said to his wife, I'm feeling very ill. Within hours, his personal physicians had arrived, the finest in the country, the best money could buy. They had all sorts of suggestions as to what was making him ill-- his humours were unbalanced, or perhaps he breathed in miasmas, foul air. His doctors gave him the standard medical treatment for someone who was as severely ill as he obviously was. They took a knife, found a vein, and bled him-- repeatedly. They drained him of more than four pints of blood. By nightfall, Washington was fading fast, so his physicians applied even more scientific treatments. This Spanish fly, although it's actually a green beetle. They would have ground it up and then applied the paste to his throat. But all that did was blister the skin. He said to his doctors, I die hard, but I'm not afraid to go. By late that evening, the first president of the United States was dead. Washington probably died from a simple infection. At the end of the 18th century, it made no difference if you were a pauper or the president. What you got was little more than quackery. The Centres for Disease Control and Prevention in Atlanta is not for those of a nervous disposition. It holds some of the world's most deadly life forms. This facility contains some of the greatest evils ever collected in one place. This bacteria causes the plague. Your flesh dies and rots while you're still alive. The Black Death of the 14th century killed a quarter of Europe's population. And then there's tuberculosis, a slow and deadly killer, the creator of oozing lung abscesses. The poet Keats, all three Bronte sisters, and Chopin are a few of its more artistic victims. And this is gangrene caused by any number of bacterial infections that lurk unseen in every dirty bullet, scalpel and delivery wood. Some bacteria don't need a wound to get inside you. They're already there, waiting patiently. Patiently for our defences to drop, and then they pounce. That's probably what did poor George Washington. DR DRUIN BURCH: In 1790s America, sudden death was utterly common. And you clustered round people's bedside when they got cold and when they got chills because they could die. MICHAEL MOSLEY: Washington's doctors would have laughed in your face if you'd told them that microscopic life was killing him. They still clung to theories passed down from the ancient Greeks. DR WALTER SNEADER: They believed that if you had a disease, the humours were out of balance. If you had a fever, you were considered to have an excess of a blood humour-- well, you were flushed after all.

It is striking that common infections such as sore throats, which are considered ‘non-serious’ today, were often killers in the pre-antibiotic age. Routine procedures such as childbirth were also dangerous. People were not only vulnerable to potentially deadly infections like TB (tuberculosis) and meningitis, but also the infection of simple cuts or more serious wounds by opportunistic pathogens could lead to sepsis and death. In the absence of powerful, antibacterial drugs, treatment was largely ineffectual. Examples referred to in the video are ‘bloodletting’, which was an established treatment until the 1940s, and the draining of pus from wounds and sores. Other treatments from this period, but not mentioned in the video, include herbal remedies, noxious chemicals such as mercury – used as a treatment for syphilis – and fresh air for TB. While these approaches could have helped relieve a patient’s symptoms, they did little to treat the underlying cause of the infection and outcomes were generally poor. Unlike George Washington’s physicians, we now know that infectious diseases are caused by microorganisms. This discovery, known as germ theory, was a pivotal moment in medicine. Activity 5 Proving germ theory Allow about 10 minutes Read the short article below about the work of Louis Pasteur and Robert Koch who provided the scientific proof of germ theory. Why was their work so important to understanding how infectious diseases could be successfully managed? Article 1 The history of germ theory. Pasteur discovered the link between microorganisms and disease, while Koch established that a particular type of bacteria was responsible for a specific disease. Being able to identify the pathogen responsible prompted research into potential tailor-made treatments for specific infections. By the early twentieth century, efforts to tackle infectious diseases were focused on finding drugs that killed the bacterial pathogen without harming the patient – so-called ‘magic bullets’. Alexander Fleming’s chance discovery in 1928 of the first antibiotic – penicillin – paved the way for research into other ‘magic bullets’ to cure bacterial infections. This was the start of the antibiotic era, which you will look at in Section 4. 4 Modern times Since their introduction in the 1930s, antibiotics have saved millions of lives (Figure 3). Once-deadly diseases such as pneumonia and TB are now treatable and everyday infections and minor injuries are no longer potentially life-threatening.

Antibiotics are used extensively in medicine, for example to improve the survival rates of transplant and cancer patients, or for antibiotic prophylaxis, that is they are taken before routine surgical procedures to prevent infection. They are also used in dentistry and veterinary medicine, for agriculture and for many other non-therapeutic purposes. You will learn more about how antibiotics are used in Week 5. Unfortunately, antibiotics are no longer the ‘magic bullets’ they once were. Our over-reliance on these drugs to prevent and/or treat a range of infections in both humans and animals, and for multiple other purposes, has left many antibiotics powerless as bacteria become resistant to them. Antibiotic resistance can develop naturally in bacteria. However, the widespread use of antibiotics increases the selective pressure on bacteria to adapt and survive – that is, to develop resistance. You will learn more about this Weeks 4 and 5. It is no coincidence that as antibiotic use has risen, so too has antibiotic resistance. For example, between 2000 and 2010, total global antibiotic consumption increased by over 30%, although there were country and regional variations (CDDEP, 2015).

4.1 The rise of antibiotic resistance By analysing country-specific data, we can build up a picture of antibiotic use and resistance worldwide. Activity 6 Antibiotic consumption Allow about 10 minutes Review Figure 4 which shows country-specific antibiotic consumption data for the period 2000 to 2010. What trend(s) can you identify?

High-income countries, for example in Western Europe and the USA, maintained or even reduced antibiotic consumption between 2000 and 2010. In contrast, antibiotic consumption increased in low and middle income countries (LMICs) such as South Africa and India. You will explore some of the reasons for the changing patterns of antibiotic use later in the course. Global levels of antibiotic resistance have similarly increased this century. However, the resistance shown by individual bacterial species to a particular antibiotic can vary considerably between, and even within, countries (Figure 5).

4.2 Superbugs An increasing and serious concern is that the more antibiotics are used, the greater the likelihood that bacteria develop resistance to multiple antibiotics and so become even more difficult to treat. Watch this short BBC video about the rise of superbugs and then read an excerpt from the accompanying BBC article. Video 4 What is a superbug? [TEXT ON SCREEN: What is a ‘superbug’?] There is a constant arms race going on between doctors and the bacteria that they're trying to kill. A superbug is one that's got extra defences, extra abilities, to ignore the impact of antibiotics. [TEXT ON SCREEN: What is the warning?] The warning here is simple. The very last line of defence, the last drug doctors use when all other antibiotics has failed, no longer works, because bacteria become resistant to it. [TEXT ON SCREEN: How big could the impact be?] At the moment, some antibiotics can already be used to treat this infection. The concern is that it goes global, combines with other types of antibiotic resistance, to create truly untreatable infections. In that circumstance, you'll have infections that can no longer be treated, but also cancer therapies and things like surgery will no longer be effective.

Accompanying article: Article 2 Bacteria that resist ‘last antibiotic’ found in UK. In 2017, the World Health Organization (WHO) published a list of antibiotic-resistant bacterial pathogens for which alternative treatments are urgently required. Five of the bacteria featured in Activity 2 are on the list (see Table 2). The sixth – M. tuberculosis – continues to be a serious health threat but was not included on the WHO list for operational reasons. Table 2 WHO ‘priority pathogens’ in 2017

Bacterium	Antibiotic resistance	Priority rating
K. pneumoniae	multi-drug	criticalcritical
E. coli	multi-drug	criticalcritical
S. aureus	methicillin, vancomycin	highhigh
N. gonorrhoeae	cephalosporins, fluoroquinolones	highhigh
S. pneumoniae	penicillin	medium

In the next section you will find out about a particular class of antibiotics.

5 Case study: the link between antibiotic use and antibiotic resistance In this case study you will further explore the link between antibiotic use and antibiotic resistance, by looking at a specific class of antibiotics, the cephalosporins. Cephalosporins are a group of ß-lactam antibiotics which target cell wall synthesis. Discovered in the late 1940s, cephalosporins have a wide range of activity, have few side effects, and are one of the most commonly used antibiotics in the world. Activity 7 Finding out about cephalosporins Allow about 25 minutes The images provided for this case study are taken from Resistance Map, a web-based programme that allows antibiotic use and antibiotic resistance data from different countries to be compared. Study Figures 6a and 6b which compare cephalosporin use from 2000 to 2015 in the UK and South Africa. Then answer the following questions. How did cephalosporin use change over time in each country? Which country had the lowest consumption of cephalosporins in 2015? What factor is most likely to have accounted for the change in cephalosporin consumption in the UK since 2007?

Over the period 2000 to 2015, antibiotic consumption decreased in the UK and remained relatively stable in South Africa. In 2015, the UK and South Africa had similar levels of cephalosporin consumption – about 700 standard units per 1000 population. The fall in cephalosporin consumption use in the UK since 2007 was probably due to a switch in prescribing away from cephalosporins towards other antibiotic alternatives. Now study Figures 7a and 7b which compare cephalosporin resistance among K. pneumoniae and E. coli clinical isolates in the UK and South Africa. Then answer the following questions. What was the resistance pattern for each organism in each country? Which country had the highest levels of cephalosporin resistance for these bacterial species in 2015?

In the UK, cephalosporin resistance remained relatively stable in K. pneumoniae over the period 2000–2015, but increased from about 2% to 12% in E. coli.In South Africa, there was a slight increase in cephalosporin resistance in both K. pneumoniae and E. coli from 2011 to 2015. Levels of cephalosporin resistance were higher in South Africa than in the UK – considerably so for K. pneumoniae. What conclusions, if any, can you draw from the data in Figures 6 and 7? The link between antibiotic use and antibiotic resistance is complex and factors other than the amount of antibiotics used can affect the levels of resistance found. For example, the underlying mechanism by which the bacterial population becomes resistant to the antibiotic and the frequency at which resistance spreads is also important. You will learn more about this in Weeks 3 and 4. Note also that the resistance data in Figures 6 and 7 are for clinical isolates from healthcare settings, whereas antibiotics are increasingly used for non-therapeutic purposes such as agriculture. In the next section you will explore what the rising global levels of antibiotic resistance mean for us now and in the future. 6 What does the future hold? How serious a threat to public health is antibiotic resistance? In this final section, some scientists give their views. Activity 8 The view of the UK’s Chief Medical Officer Allow about 10 minutes First, watch the short video of Professor Sally Davis, the UK’s Chief Medical Officer, talking in 2013 about antibiotic resistance. Then read the more recent interview with Professor Davis published in 2017. Finally, answer the following questions. What does Professor Davis warn against? What explanation does Professor Davis give for public complacency about the perceived threat(s) of antibiotic resistance? Video 5 ‘A very serious issue’: Sally Davis talking in 2013. INTERVIEWER: So what scale are we talking about? How serious is this really? DAME SALLY DAVIES: I've described an apocalyptic scenario where you could go in for the little operation I had earlier this week to release a nerve in my hand, get an infection, but that antibiotics don't work, and I die of it, or a hip replacement. Let alone cancer patients who won't be treatable, and will die early in their treatments, and organ transplants-- kidneys, for instance-- where we may not be able to do them. It is a very serious issue for mankind.

Article 3 Sally Davis’ 2017 interview in The Guardian. Professor Davis warns of a post-antibiotic apocalypse if antibiotic resistance is not tackled on a global scale. Medical interventions we currently take for granted could become a thing of the past and simple infections could once again become killers. Antibiotic resistance is a ‘hidden’ problem. People are not aware that deaths from infectious diseases are the result of treatment failure. What does all of this mean for you? Activity 9 Personal reflections Allow about 10 minutes First, listen to the following short audio clip of four scientists’ opinions about what the future holds for the treatment of bacterial infections. Audio 1 Scientists’ perspective on the antibiotic resistance threat. MAN 1 I'm bloody petrified, to be honest. I don't think there's the economic or political impetus to properly get behind it at the moment. There's been a lot of noise. And there's a little bit more funding coming through. But I don't think it's nearly enough. And it's also not joined up. WOMAN I think it's terrifying. I think that you can definitely imagine a situation where people are coming in with bacteria that we don't currently have tools for. MAN 2 By 2050, 10 million people will die from drug resistant infections around the world, which is more people than die of cancer at the moment. MAN 3 I'm not too worried for people in the rich West. I think bringing new, broad-spectrum antibiotics to market for poor parts of the world, where they're needed, is going to be much more difficult. Second, use the following questions to help form your own opinion. On a scale of 1 (low) to 10 (high), how serious a problem is antibiotic resistance? What, if anything, can be done about antibiotic resistance? Whose responsibility is it to address this problem? You might like to think about:whether individuals should take some responsibility or whether it is up to the medical profession, governments, etc.the extent to which different countries and regions should work together to address this problem. How urgent a problem is it? How soon should action(s) be taken? At the end of the course you will be asked these questions again to see if what you have learned throughout the course has changed your opinion. 7 This week’s quiz Well done – you have reached the end of Week 1 and can now do the weekly quiz to test your learning. Week 1 practice quiz Open the quiz in a new tab or window by holding down Ctrl (or Cmd on a Mac) when you click on the link. Return here when you have finished. 8 Summary This week introduced the key themes discussed in this course. You now know what antibiotics are and which pathogens they are active against. You have learned that our over-reliance on antibiotics has encouraged the development of resistance and led to many drugs becoming powerless against common bacterial infections. You have also heard from eminent scientists that antibiotic resistance poses one of the biggest threats to public health today. Having had a glimpse of how limited medical options were in the pre-antibiotic age, you can now begin to speculate on what the future might be like without these ‘wonder’ drugs. You should now be able to: recall why pathogenic bacteria pose a threat to human health define the term antibiotic and give examples describe the importance of antibiotics in modern health care analyse antibiotic data and make simple deductions about antibiotic use and resistance patterns discuss the consequences of a future without antibiotics. Next week you will find out how different types of antibiotic work and how they can target bacteria in the body yet leave body cells unharmed. You will also explore why some antibiotics are active against a wide range of bacteria but others are not. You can now go to Week 2. Week 2: How do antibiotics work? Introduction In Week 1 you learned that antibiotics are used to treat bacterial infections. They either kill the bacteria outright or prevent them from growing and replicating. You were also introduced to the concept of ‘magic bullets’ – drugs such as antibiotics that are highly effective at treating infections without unduly harming the patient. This second week of the course looks in more detail at how antibiotics work. You will start by exploring how antibiotics can exert powerful antibacterial effects and yet be generally well tolerated by people and animals. You will then study the different modes of antibiotic action, looking in more detail in this week’s case study at the precise mechanism used by ß-lactam antibiotics. Finally, you will consider factors that determine antibiotic type, such as spectrum of activity and bactericidal or bacteriostatic nature. Begin this week by watching the video below about the pioneering work of Paul Ehrlich (1854–1915). He discovered the first ‘magic bullet’ in 1909 – Salvarsan, a derivative of arsenic – which could cure syphilis. Ehrlich hoped that other ‘magic bullets’ which could be safely used to treat bacterial infections would swiftly follow. However, the world had to wait another ten years for penicillin to be accidentally discovered! Video 1 In pursuit of ‘magic bullets’: the seminal work of Paul Ehrlich. MICHAEL MOSLEY: At the start of the 20th century, diseases you might have associated with mediaeval times were still rampant. Syphilis, for example. Now for centuries, doctors had used mercury to treat it. But being extremely toxic, it tended to kill the patients. MAN 1: Paul Ehrlich studied medicine in the early 1870s. But he spent an awful lot of time in the laboratory rather than the clinic, where he should have been. MICHAEL MOSLEY: One of the things Ehrlich was doing was playing around with artificial dyes. The first had been discovered in 1856. And soon, people went dye crazy. His favourite colour was methylene blue. And with this, he made a remarkable discovery, one which would set him on the path to medical greatness. MICHAEL MOSLEY: When Ehrlich added a drop of methylene blue to tissue infected with bacteria, he noticed something astonishing-- only the bacteria was stained by the dye, not the tissue around them. Now the fact that an artificial dye will selectively stain bacteria was remarkable. But it's what Ehrlich thought next that was truly revolutionary. MAN 2: What he did was he noted that some compounds were toxic. And he said, what if you create selective toxicity so that you can give somebody a compound that will kill off what's making them unwell and leave them unharmed. And he famously coined the phrase from a German folk story, you could create these magic bullets, which is what we've been trying to do ever since. MICHAEL MOSLEY: Initially, he tried finding a cure for sleeping sickness. But with the help of his Japanese assistant, Sahachiro Hata, he switched his attention to a pathogen that was rather more common in Germany. Common, but horribly disfiguring-- syphilis. There were no cures. And the only treatment, mercury, made your hair and teeth fall out before eventually destroying your entire nervous system. [MUSIC PLAYING] Ehrlich hoped to find a magic bullet that would be more selective, poisoning the bacteria but not the rest of the body. Ehrlich thought that arsenic might be effective against syphilis. Arsenic is notoriously poisonous, but by this point, German chemists had made hundreds of different compounds of arsenic. So Ehrlich asked his assistant, Hata to work his way systematically through them, hoping that amongst would be one that was safe and effective. Hata had found a way to infect rabbits with syphilis. He now set about the un-enviable task of testing arsenic compounds on them one after the other. Some compounds killed both bacteria and rabbit. Some killed neither. Hata went through hundreds and hundreds of compounds, until, finally, he found one that was rather special. Compound 606, it killed the bacteria, but, best of all, it left the dear old rabbit intact. This was the magic bullet they had been hoping for. MAN 2: Salvarsan 606 showed that Ehrlich was right. That these things were out there. All you have to do is methodically screen for them, and you would find them. It showed the power of methodically screening lots of compounds. 606-- 606 compounds to see what worked. MAN 1: The newspapers at the time carried it on the front pages. The medical profession were stunned. MAN 2: To be able to treat syphilis was miraculous, absolutely miraculous. MICHAEL MOSLEY: By the 1920s, Salvarsan was the world's most popular drug, particularly with men. But Ehrlich's hope that it would be just the first of many magic bullets proved ill founded. Infections caused by the most minor of injuries were still uncontrollable.

By the end of this week, you should be able to: recognise different types of commonly used antibiotics recall the characteristic features of bacterial and human or animal cells explain why antibiotics have selective toxicity demonstrate how commonly used antibiotics affect bacterial growth summarise the main mechanisms by which antibiotics stop infections from spreading and kill bacteria. 1 Selective toxicity As you saw in Video 1, Ehrlich’s seminal work on syphilis proved that ‘magic bullets’ existed. It also led to wide acceptance of the principle that drugs should demonstrate ‘selective toxicity’. That is, they target the disease-causing organism while causing no or minimal harm to the patient. This principle is still firmly entrenched in medical research and practice today (Valent et al., 2016). Underpinning selective toxicity are the differences between pathogens and human or animal cells. It is these differences that antibiotics (and other drugs) exploit to exert their specific effects.

1.1 Cell structure There are two distinct types of cell. Bacteria are prokaryotes while human and animal cells are eukaryotes. Activity 1 Exploring cell basics Allow about 15 minutes (a) Study the general organisation of prokaryotic and eukaryotic cells in Figure 1. Then read more about each component below.

Cell membrane A complex structure enclosing the cytosol of living cells which controls the passage of substances into and out of the cell. Cell wall A protective outer layer which provides mechanical support to the cell. It also prevents harmful surges of water moving into the cell by osmosis which could cause it to burst (lysis). Cytosol A watery fluid in which many chemical reactions take place. Ribosome A structure where proteins are made in the cell. Each ribosome consists of a large and a small subunit which have distinct roles in protein synthesis. Mitochondrion The place in a cell where chemical energy derived from nutrients is converted to a form that can be used by the cell (plural, mitochondria). Endoplasmic reticulum The place in a cell that sorts proteins and ensures they are transported to the correct part of the cell. The rough endoplasmic reticulum is studded with ribosomes. Nucleus A membrane-bound structure that encloses the genetic material (DNA). DNA Deoxyribonucleic acid is a large molecule containing the cell’s genetic information. This is the complete set of instructions needed for an organism to grow, survive and reproduce. (b) What structural differences can you see between the bacterial and the animal cell? The bacterial cell has a cell wall; the animal cell lacks one. The bacterial cell lacks membrane-bound organelles such as mitochondria and endoplasmic reticulum. DNA is free in the cytosol of the bacterial cell; DNA in the animal cell is in the nucleus. In the animal cell, ribosomes are both free in the cytosol and attached to the membrane of the rough endoplasmic reticulum. Although there are similarities between the two cell types, eukaryotic cells are structurally much more complex. Prokaryotes and eukaryotes carry out the same essential processes necessary for survival, such as making new proteins, metabolism and reproduction, but these processes are not identical. For example, different proteins might be produced or different enzymes used to drive key chemical reactions. Antibiotics are selectively toxic because they target structural features or cellular processes in the bacterial pathogen that are different or lacking in the host’s cells.

1.2 Potential bacterial targets for antibiotics In Activity 2 you will discover which essential cell processes in the bacterial pathogen are potential targets for antibiotics. Activity 2 When are bacteria vulnerable to antibiotics? Allow about 20 minutes Watch the video about key bacterial cell processes and answer the related questions. You can pause the video to work through this activity at your own pace. Video 2 How do antibiotics work? NARRATOR: How do antibiotics work? Pathogenic bacteria in the body cause infections, which can be treated by antibiotics. TEXT ON SCREEN: Antibiotics can be bacteriostatic or bactericidal. Statis = to stop, Cidal = to kill Antibiotics can be bacteriostatic or bactericidal. Bacteriostatic antibiotics slow the growth of bacteria by interfering with the processes the bacteria need to multiply. These processes include: DNA replication. TEXT ON SCREEN: DNA replication. Typically a bacterial DNA takes the form of a single, circular DNA molecule called a chromosome. Along the length of the chromosome are many short sections of DNA called genes which carry the instructions to make one of the thousands of proteins that cells need to grow and function. Before the bacterial cell divides, the DNA must make a copy of itself (replicate) so that each daughter cell can receive a copy of this chromosome. Suggest a likely consequence for the cell if DNA replication is blocked. Answer: Blocking DNA replication would impair cell division and kill the bacterial cell. Metabolism, e.g., enzyme activity. TEXT ON SCREEN: Metabolism, e.g. enzyme activity. Metabolism refers to the chemical reactions that occur within a cell to ensure an organism obtains the energy and nutrients it needs. Enzymes are proteins that are required for metabolism; they bind in a specific manner to another molecule (the substrate) in order to help it undergo a chemical reaction. Protein production. TEXT ON SCREEN: Protein production. Proteins, which are composed of amino acid building blocks, are synthesised in two stages. First, the instructions carried by the gene are transferred to a messenger ribose nucleic acid molecule (mRNA) and taken to a ribosome for processing. Second, the instructions are used to create a long chain of amino acids - the order of the amino acid building blocks is unique to the protein being made. Once complete, the amino acid chain folds up into a complex, three-dimensional protein. Which stage or stages of protein synthesis could be targeted by antibiotics? Answer: Interference with either stages of protein synthesis could result in faulty enzymes and/or structural proteins. DNA replication, metabolic reactions and protein synthesis also occur in eukaryotic cells. Suggest why antibiotics that target these bacterial processes demonstrate selecting toxicity. Answer: Although cellular processes of prokaryotic and eukaryotic cells have many similarities, antibiotics are selected for clinical use that target those process that are wholly or partly unique to the bacterial pathogen. This minimises the risk of side-effects in the patient. Bactericidal antibiotics kill the bacteria. For example, by preventing the bacteria from making a cell wall. TEXT ON SCREEN: What might happen to a cell that can no longer make a cell wall? Answer: Bacterial cells that lack a cell wall are in danger of osmotic damange and lysis. Explain why antibiotics that target cell wall synthesis leave eukaryotic cells unharmed. Answer: Eukaryotic cells lack a cell wall. Penicillin's a bactericidal. Penicillin's include Penicillin V for sore throats, amoxicillin for chest infections, and fluctoxacillin for skin infections. Antibiotics can be so-called broad spectrum, affecting many different bacteria in your body, including useful bacteria in your gut. Some antibiotics are more narrow spectrum, only affecting one or two types of bacteria. It is better to use narrow-spectrum antibiotics where possible. Most antibiotics have no effect on your immune system. Antibiotics do not work on viruses, because viruses have a different structure to bacteria. Viruses incorporate themselves into a host cell in your body in order to multiply. Bacteriostatic antibiotics that affect bacterial DNA, metabolism, or protein production do not attack body cells. And therefore, do not slow the growth of viruses. Viruses do not have a cell wall. And therefore, bactericidal antibiotics that act on cell walls cannot kill viruses.

(a) Suggest a likely consequence for the cell if DNA replication is blocked. Blocking DNA replication would impair cell division and kill the bacterial cell. (b) Which stage or stages of protein synthesis could be targeted by antibiotics? Interference with either stage of protein synthesis could result in faulty enzymes and/or structural proteins. (c) DNA replication, metabolic reactions and protein synthesis also occur in eukaryotic cells. Suggest why antibiotics that target these bacterial processes demonstrate selective toxicity. Although cellular processes of prokaryotic and eukaryotic cells have many similarities, antibiotics are selected for clinical use that target those processes that are wholly or partly unique to the bacterial pathogen. This minimises the risk of side effects in the patient. (d) What might happen to a cell that can no longer make a cell wall? Bacterial cells that lack a cell wall are in danger of bursting if too much water moves into the cell by osmosis (e) Why do antibiotics that target cell wall synthesis leave eukaryotic cells unharmed? Eukaryotic cells lack a cell wall. (f) A relatively small number of antibiotics target the bacterial cell membrane. Such antibiotics are often highly toxic to the host. Can you suggest a reason for this? The membrane of animal and human cells has a very similar structure to that of bacteria. The potential for such antibiotics to adversely affect eukaryotic cells is therefore greater and these antibiotics generally demonstrate poor selective toxicity. This increases the risk of harmful side effects for the patient. In Week 1 you learned that structurally similar antibiotics tend to have similar antibacterial activity and are grouped together in the same class. You should by now appreciate that each class of antibiotic has a specific mode of action, affecting susceptible bacterial cells in a way that depends on the drug’s affinity for a specific target or process in the bacterial cell. You will explore different modes of antibiotic action in Section 2.

2 Antibiotic modes of action This section focuses on the four main modes of antibiotic action that lead to inhibition of one of the following: cell wall synthesis protein synthesis nucleic acid synthesis metabolic reactions. Don’t worry if you don’t understand all of these terms, as they will be explained in the next sections.

Members of the same class of antibiotics share a characteristic structural feature that determines the drug’s affinity and specificity for target molecules in susceptible bacteria. You will now look in more detail at antibiotics that exemplify each of these four main modes of action.

2.1 Inhibitors of cell wall synthesis As you saw in Activity 2, the cell wall is essential for normal functioning of the bacterial cell. Antibiotic inhibitors of cell wall synthesis block the production of peptidoglycan, the main component of the cell wall. Cross-linking between peptidoglycan chains forms a strong, mesh-like structure that gives the cell wall structure and rigidity, and protects the underlying cell membrane from osmotic damage when water moving into the cell by osmosis could cause it to burst, or lyse. Disruption of the peptidoglycan layer of the cell wall can therefore result in cell lysis (Figure 3).

Examples of cell wall synthesis inhibitors are the ß-lactam antibiotics. These include penicillin and its derivatives, and the cephalosporins. All ß-lactam antibiotics contain a core chemical structure called a ß-lactam ring (Figure 4) which determines the mode of action of this class of antibiotics.

The ß-lactam antibiotics interfere with the formation of the peptidoglycan cross-links, thereby weakening the cell wall. You will learn more about the precise mechanism in this week’s case study (Section 3). For now, you can see the effect of the disrupted cell wall on bacterial growth in Video 3. Video 3 A ß-lactam antibiotic in action. INSTRUCTOR: This is cephalosporin C, the first compound in one of the most important classes of beta-lactams-- the cephalosporins. It occurs in nature and was originally isolated from a fungus found growing in a sewage outlet in Sardinia in 1947. It's the lead compound from which a whole family of antibiotics has been made, but it isn't itself a particularly powerful antibiotic. Why? It all hinges in the way that antibiotics work. What they do is disrupt the process of cell wall biosynthesis. In a growing colony, this process is taking place all the time, both to maintain the existing cell wall and to make new wall as the bacteria divide. When an antibiotic is added to a colony, this inhibitory action has two effects. Firstly, the bacteria are prevented from successfully dividing, so they start to form long, spaghetti-like strings. The other effect arises from the fact that the existing cell walls routinely break down and are in constant need of repair. As the process of repair is prevented, small flaws appear, large enough to allow water to start moving into the cell due to the high internal osmotic pressure. As a result, the cells start to swell and eventually burst, a process called lysis.

2.2 Inhibitors of protein synthesis You learned in Activity 1 that cells synthesise new proteins in ribosomes which are made up of one large and one small subunit. These subunits differ structurally and chemically between prokaryotic and eukaryotic ribosomes (Figure 5). This provides antibiotic targets in the bacterial pathogen which are not present in the host cells.

Table 1 Examples of protein synthesis inhibitor antibiotic classes(OpenStax College Microbiology, n.d.)

Ribosomal target	Outcome	Antibiotic class	Structure	Example drug
Small (30S) subunit	Errors give rise to faulty proteins that disrupt the cell membrane	Aminoglycosides	All contain amino sugar substructures (red)	Streptomycin
Large (50S) subunit	First steps of protein synthesis (initiation) are impaired and bacteria cannot grow and divide	Oxazolidines	All contain 2-oxazolidone (red) somewhere in their structure	Linezolid

2.3 Inhibitors of nucleic acid synthesis Differences between enzymes that carry out the synthesis of nucleic acids in eukaryotic and prokaryotic cells allow antibiotics to target these processes in bacterial pathogens. For example, fluoroquinolones (Figure 6) specifically inhibit bacterial enzymes that unwind the DNA double helix, separating the two strands so that the DNA can make a copy of itself. If this process does not happen, replication is blocked.

Another class of antibiotics – rifamycins – inhibits RNA synthesis by binding to and inhibiting an enzyme called RNA polymerase. This enzyme transfers the instructions carried by genes to the intermediary molecule, mRNA. Interference in this process ultimately stops new proteins being made.

2.4 Inhibitors of metabolic reactions Antibiotics that disrupt essential bacterial metabolic pathways are acting as antimetabolites. These chemicals are structurally similar to natural metabolites but just different enough to interfere with normal cell function. For example, trimethoprim inhibits the synthesis of folic acid, a vitamin which bacteria, unlike humans, must make themselves. Trimethoprim is a structural analogue of dihydrofolic acid, an intermediate compound in the folic acid pathway. Trimethoprim out-competes dihydrofolic acid to react with a specific bacterial enzyme in the pathway, thereby interrupting folic acid synthesis and inhibiting bacterial growth (Figure 7).

The action of trimethoprim illustrated in Figure 7b exemplifies the specific interaction between antibiotic and bacterial target at a molecular level which disrupts a particular cellular process. You will return to this topic in Week 3 in relation to the development of antibiotic resistance. In the next section, you will look in detail at the mechanism of ß-lactam antibiotics.

3 Case study: mechanism of ß-lactams The β-lactam antibiotics target the bacterial cell wall (Figure 8) by inhibiting the enzymes responsible for cross-linking adjacent molecules in the peptidoglycan layer. The ß-lactam antibiotics bind to these enzymes, collectively known as penicillin-binding proteins (PBPs), and prevent them from forming cross-links. As the bacterial cell grows, the effect of the antibiotic is to progressively weaken the cell wall until the cell lyses as a result of osmotic damage.

In Section 2 you learned that the activity of penicillins and cephalosporins resides in the ß-lactam ring. Activity 3 looks more closely at this. Activity 3 Mechanism of ß-lactam antibiotics Allow about 15 minutes First, watch the short video below which describes the inherent instability of the ß-lactam ring structure which makes it highly reactive. The video refers to penicillin, but the same is true of the ß-lactam ring in cephalosporins and all other antibiotics of this class. Video 4 The inherent instability of the ß-lactam ring structure. INSTRUCTOR: This is a model of the four-membered ring, referred to as a beta-lactam ring in the text, that's part of a penicillin molecule. And if you take a look at it, you can see that it's composed of three carbon atoms and one nitrogen atom. But even more importantly, note that as a four-membered ring, the bond angle now between the carbons-- instead of being like the tetrahedral carbon bond angle of 109 degrees, it's constrained to be just 90 degrees. That introduces what we call ring strain into the molecule. And it's a way in which molecules are encouraged, therefore, to react. And indeed, the reaction of penicillin with bacteria and an enzyme in bacteria is to open up this four-membered ring and release that ring strain. I think you can see before I demonstrate that that the bonds are already in the four-membered ring quite bent. But watch what happens when I break one of these bonds and simulate the reaction. You can see how it will spring apart to release that ring strain. So another important feature in the understanding of why chemical reactions take place is to release this ring strain that we're seeing in the four-membered ring and is demonstrated when the enzymes in bacteria react with penicillin.

Figure 9 shows what happens when a ß-lactam antibiotic, in this case penicillin, binds to an active PBP.

The reaction shown in Figure 9 results in a new PBP side chain which is much larger than the original -NH₂ group and effectively deactivates the PBP. Can you suggest why? The large side chain means that there is no longer sufficient space for the enzyme (PBP) to bind to its normal substrate during the peptidoglycan cross-linking process. You will learn more about how disrupting the interaction between ß-lactam antibiotics and PBP contributes to antibiotic resistance mechanisms in Week 3. Next, however, you will see how antibiotics of the same class, and with the same mode of action, can have a different spectrum of activity and exert different effects. 4 Types of antibiotic So far in this course you have learned that antibiotics may be active against a wide range of bacteria (broad-spectrum) or just a few types (narrow-spectrum). You also know that antibiotics either kill bacterial cells (bactericidal) or stop them growing and dividing (bacteriostatic). Factors that determine the spectrum of antibiotic activity include: ability to penetrate the bacterial cell – since most bacterial targets are located in the cell’s interior how widespread the target is among different bacterial species bacterial resistance to the antibiotic (discussed in Weeks 3 and 4).

4.1 Gram-positive and Gram-negative bacteria Bacteria are divided into two groups based on how the cell wall appears when they are stained using Gram straining. This procedure allows the composition of the wall to be visualised. In Gram-positive bacteria, the cell wall has a thick peptidoglycan layer which is relatively porous, allowing substances to pass through it quite easily. In Gram-negative bacteria, this peptidoglycan layer is greatly reduced and is further protected by a second, outer membrane (Figure 10).

This second, outer membrane of Gram-negative bacteria is an effective barrier, regulating the passage of large molecules such as antibiotics into the cell. In contrast, the thick, porous peptidoglycan layer in the cell wall of Gram-positive bacteria gives greater access to antibiotics, allowing them to more easily penetrate the cell and/or interact with the peptidoglycan itself. You will learn more about the strategies antibiotics use to cross the cell wall in Week 3.

4.2 Activity against Gram-positive and Gram-negative bacteria Narrow-spectrum antibiotics are effective against either Gram-positive or Gram-negative bacteria, whereas broad-spectrum antibiotics are effective against both types. Not all Gram-positive and/or Gram-negative bacteria are affected by a single antibiotic. Why is this? This is because of the specificity of the antibiotic/bacterial target interaction, whether the bacterial species has the target in question and whether the bacteria are resistant to the antibiotic.

4.3 Bactericidal versus bacteriostatic antibiotics While some antibiotic classes have consistent antibacterial effects, such as ß-lactams which are nearly always bactericidal, the activity of other classes may depend on the dose of antibiotic prescribed or how long the treatment lasts. For example, fluoroquinolones and aminoglycosides, while usually bactericidal, may be bacteriostatic when used at low concentration. You should by now have a good idea of how antibiotics interact with bacterial cells. Activity 4 looks at what happens to the bacterial population as a whole when antibiotics are administered. Activity 4 Effect of antibiotics on bacterial growth Allow about 10 minutes In Week 1 you learned that bacteria are at their most susceptible to antibiotic attack when they are actively growing. In this activity you consider what happens to a bacterial culture when antibiotics are introduced during this exponential phase of growth. Bacteriostatic antibiotics (a) Figure 11a shows the normal growth curve of a bacterium which is sensitive to the bacteriostatic antibiotic ‘A’. Explain what you would expect to happen to the rate of bacterial growth when A is added to the culture in high concentration. You should assume that growth conditions are otherwise optimal.

(b) Predict what will happen to bacterial growth if antibiotic A is removed from the culture at the point indicated on the graph.

Bactericidal antibiotics Figure 12a shows the normal growth curve of a bacterium which is sensitive to the bactericidal antibiotic ‘B’. Explain what you would expect to happen to the rate of bacterial growth when B is added to the culture in high concentration. You should assume that growth conditions are otherwise optimal.

Bactericidal antibiotics kill susceptible bacteria during the exponential phase of growth and cure the infection. Bacteriostatic antibiotics stop bacterial growth even though the cells remain viable. This allows time for the host’s immune system to be activated and target the bacterial pathogen – again effecting a cure.

5 This week’s quiz Well done – you have reached the end of Week 2 and can now do the quiz to test your learning. Week 2 practice quiz Open the quiz in a new tab or window by holding down Ctrl (or Cmd on a Mac) when you click on the link. Return here when you have finished it. 6 Summary This week introduced some of the basic biology and chemistry that underpins antibiotic activity. You looked at the main modes of antibiotic action and learned why these drugs demonstrate selective toxicity. You should now be able to: recognise different types of commonly used antibiotics recall the characteristic features of bacterial and human or animal cells explain why antibiotics have selective toxicity demonstrate how commonly used antibiotics affect bacterial growth summarise the main mechanisms by which antibiotics stop infections from spreading and kill bacteria. Having explored different types of antibiotic in some detail, you should now be well prepared to move on to Week 3 which discusses antibiotic resistance mechanisms. You can now go to Week 3. Week 3: How do bacteria become resistant to antibiotics? Introduction In Week 2 you looked at how antibiotics target bacteria, either killing them or preventing their growth. But bacteria are constantly fighting back against this threat to their survival. Antibiotic resistance is the ability of pathogenic bacteria to resist the action of antibiotics so that they survive exposure to antibiotics that would normally kill them or stop their growth (CDC, 2017; PHE, 2017). You could be forgiven for thinking that antibiotic resistance has been caused by our use, and misuse, of antibiotics. However, as you will see in the following video, bacteria that have not interacted with humans have acquired resistance to many of the antibiotic medicines we use to treat infections. Video 1 Antibiotic resistance is a natural bacterial defence mechanism. You will return to look at how resistance has evolved and spread in Week 4. In this week, you will focus on how bacteria develop resistance in order to protect themselves from antibiotics. You will start by considering several mechanisms of antibiotic resistance before moving on to look at the differences between intrinsic and acquired antibiotic resistance. You will end the week by returning to the case study to explore the mechanisms responsible for resistance to third generation cephalosporins. By the end of this week, you should be able to: state what is meant by the term ‘antibiotic resistance’ recognise that antibiotic resistance evolved to protect bacteria describe the three main mechanisms of resistance that bacteria have developed to counteract the action of antibiotics give examples of these resistance mechanisms distinguish between intrinsic and acquired antibiotic resistance. 1 Antibiotic resistance mechanisms Bacteria have evolved several sophisticated antibiotic resistance mechanisms. Figure 1 gives an overview of the major mechanisms by which bacteria become resistant to the action of antibiotics. Don’t worry if you don’t understand all of these terms, as they will be explained in the following sections.

In this section you will look at the three main mechanisms of antibiotic resistance: modifying the antibiotic target destroying or modifying the antibiotic preventing the antibiotic from reaching its target. Although you will look at each of these mechanisms in turn, it is worth remembering that bacteria may use multiple resistance strategies simultaneously to survive exposure to antibiotics.

1.1 Modifying the antibiotic target As you saw in Week 2, antibiotics are selectively toxic because they target structural features or cellular processes in the bacterial pathogen that are different or lacking in the host’s cells. Recall how penicillin and other related β-lactam antibiotics work by binding to penicillin-binding proteins (PBPs), preventing them from binding to their normal target, peptidoglycan. Or how trimethoprim prevents dihydrofolate reductase reacting with dihydrofolic acid. Changes to the structure of the target that prevent efficient antibiotic binding but still enable the target to carry out its normal function will confer antibiotic resistance (Figure 2).

This resistance strategy is widespread among bacteria. For example, the oxazolidinone class antibiotic linezolid disrupts bacterial growth by preventing the initiation of protein synthesis. The target of linezolid is the bacterial large (50S) ribosomal subunit. Changes to the 50S ribosomal subunit structure have been identified in clinical isolates of S. aureus and S. pneumoniae that are resistant to linezolid (Woodford and Ellington, 2007). As you will see in Week 4, changes to the structure of antibiotic targets are often caused by genetic mutations. However, the structure of antibiotic targets can also be modified to prevent antibiotic binding by adding chemical groups. For example, resistance to linezolid can be caused by either genetic mutations (see Week 4) or the addition of chemical groups to the bacterial 50S ribosomal subunit, both of which prevent or reduce linezolid binding (Long et al., 2006). You will return to look at how changes to the structure of penicillin-binding protein (PBP) contributes to resistance to cephalosporins in the case study at the end of this week.

1.2 Destroying or modifying the antibiotic molecule The second mechanism of antibiotic resistance you will look at is the destruction or modification of the antibiotic by bacterial enzymes. Probably the most well studied example of enzymes that destroy antibiotics are the β-lactamases. As you may recall from Week 2, β-lactamases deactivate the β-lactam ring of β-lactam antibiotics, preventing them from binding to their target (Figure 3).

The β-lactamases can deactivate almost all of the β-lactam antibiotics currently in therapeutic use. As you will see in the case study at the end of this week, this includes cephalosporins. Consequently, their presence significantly reduces the available treatment options for infections caused by bacteria expressing β-lactamase. One successful strategy for treating these infections is to combine antibiotic treatment with a β-lactamase inhibitor. How might a β-lactamase inhibitor help the treatment of infections caused by β-lactamase-expressing bacteria? The β-lactamase inhibitor will block the ability of the β-lactamase to deactivate the β-lactam antibiotic so that it can bind to its target molecule. Other antibiotic-modifying enzymes do not destroy or target the core chemical structure that confers antibacterial activity. Instead they modify the antibiotic’s structure by adding chemical groups to prevent it from binding to its target. One group of antibiotics that are particularly susceptible to modification are the aminoglycoside antibiotics which include streptomycin (Figure 4).

Aminoglycoside-modifying enzymes add bulky chemical groups to the exposed hydroxyl (-OH) and amino (-NH₂) groups of the antibiotic, which prevent it from binding to its target.

1.3 Preventing entry, increasing exit Antibiotics are only effective if they can reach their target. Preventing antibiotics from reaching their target is the final mechanism of antibiotic resistance that you will look at this week. As you should recall from Week 2, the cell wall protects bacteria from osmotic and mechanical damage. To reach their targets inside the cell, antibiotics must cross this cell wall. In Activity 1 you will look at the mechanisms that antibiotics use to cross this bacterial cell wall. Activity 1 Transporting antibiotics across the bacterial cell wall Allow 15 minutes First, watch the following animation which describes how antibiotics are transported across the bacterial cell wall. Video 1 Animation of the mechanisms of transport of antibiotics across the membrane. INSTRUCTOR: In this activity, you'll look at how altering the transport of antibiotics across the membrane can result in antibiotic resistance. The cell walls of gram-positive bacteria are permeable to most antibiotics, represented here as blues spheres and triangles. gram-positive bacteria are susceptible to these antibiotics because the antibiotic can cross the membrane and reach their targets, here shown in dark green inside the bacterial cell. However, the outer membrane of gram-negative bacteria, like e. coli, forms a permeability barrier that prevents antibiotics from entering the bacterial cell and reaching their target. To reach their target inside gram-negative bacteria, antibiotics must overcome this permeability barrier. Embedded in the outer membrane of gram-negative bacteria are proteins that form channels known as porins, shown here in light green. Antibiotics cross the outer membrane of gram-negative bacteria by diffusing through these porin channels. Porin channels are fairly nonspecific and can transport many antibiotics across the membrane. The presence of porin channels in the outer membrane makes bacteria susceptible to antibiotics. Some antibiotics can be efficiently removed from bacteria by efflux. Efflux is the movement of molecules out of the cell. Antibiotics are transported out of the bacterial cell by efflux pumps in the membrane, shown here in purple. Removing the antibiotic from the cell prevents it from binding to its target, so bacteria expressing efflux pumps are resistant to antibiotics. Some efflux pumps are specific and only transport one class of antibiotics, but many transport a wide range of molecules. These efflux pumps are known as multi-drug-resistant efflux pumps. Porins and efflux pumps have opposite effects on the concentration of antibiotic inside the cell. In the following animations, the concentration of antibiotic inside the cell is shown by the brown colour. As the concentration of antibiotics increases, the colour becomes darker. The number of porins and efflux pumps on the outer membrane of bacteria can be altered, and these changes can affect the concentration of antibiotic inside the cell, and therefore, the susceptibility of bacteria to antibiotics. Watch what happens to the concentration of antibiotics as they enter the cell via porins and are removed by efflux pumps, and then answer the following questions.

Now answer the following questions. 1 Decreasing the number of porin channels on the outer membrane: (a) decreases the amount of antibiotic entering Gram-negative bacteria Your answer is correct. Most antibiotics cannot cross the outer membrane of Gram-negative bacteria and therefore enter the cell via porin channels. Decreasing the number of porin channels will decrease the amount of antibiotic entering the bacteria. (b) increases the amount of antibiotic entering Gram-negative bacteria Your answer is incorrect. Most antibiotics cannot cross the outer membrane of Gram-negative bacteria and therefore enter the cell via porin channels. Decreasing the number of porin channels will decrease the amount of antibiotic entering the bacteria. (c) has no effect on the amount of antibiotic entering Gram-negative bacteria. Your answer is incorrect. Most antibiotics cannot cross the outer membrane of Gram-negative bacteria and therefore enter the cell via porin channels. Decreasing the number of porin channels will decrease the amount of antibiotic entering the bacteria. 2 Bacteria that are resistant to penicillin are likely to have: (a) very few porin channels on their outer membrane or have replaced their porin channels with channels that exclude penicillin Your answer is correct. If an antibiotic cannot reach its target, bacteria will be resistant to its action. Decreasing the expression of porins, or replacing them with channels that cannot transport the antibiotic, will prevent the antibiotic from crossing the outer membrane and reaching its target, therefore these bacteria will be resistant. (b) numerous porin channels on their outer membrane Your answer is incorrect. If an antibiotic cannot reach its target, bacteria will be resistant to its action. Decreasing the expression of porins, or replacing them with channels that cannot transport the antibiotic, will prevent the antibiotic from crossing the outer membrane and reaching its target, therefore these bacteria will be resistant. (c) replaced their porin channels with channels that selectively transport penicillin. Your answer is incorrect. If an antibiotic cannot reach its target, bacteria will be resistant to its action. Decreasing the expression of porins, or replacing them with channels that cannot transport the antibiotic, will prevent the antibiotic from crossing the outer membrane and reaching its target, therefore these bacteria will be resistant. 3 Increasing the rate of active transport of penicillin through the efflux pump would: (a) increase the amount of penicillin in the bacterial cell Your answer is incorrect. Efflux pumps actively transport antibiotics out of the bacterial cell. Therefore, increasing transport through these channels will decrease the amount of antibiotic inside the cell. (b) decrease the amount of penicillin in the bacterial cell Your answer is correct. Efflux pumps actively transport antibiotics out of the bacterial cell. Therefore, increasing transport through these channels will decrease the amount of antibiotic inside the cell. (c) have no effect on the amount of penicillin in the bacterial cell. Your answer is incorrect. Efflux pumps actively transport antibiotics out of the bacterial cell. Therefore, increasing transport through these channels will decrease the amount of antibiotic inside the cell. 4 Bacteria that are resistant to penicillin are likely to have: (a) efflux pumps that are unable to transport penicillin Your answer is incorrect. If an antibiotic cannot reach its target, bacteria will be resistant to its action. Actively transporting antibiotics out of the cell decreases their concentration inside the cell, so that they cannot build up to a high enough concentration to exert the effect on their target. Increasing active transport by expressing more efflux pumps that can actively transport the antibiotic out of the cell decreases the amount of antibiotic inside the cell and prevents it from acting on its target. (b) efflux pumps that transport penicillin Your answer is correct. If an antibiotic cannot reach its target, bacteria will be resistant to its action. Actively transporting antibiotics out of the cell decreases their concentration inside the cell, so that they cannot build up to a high enough concentration to exert the effect on their target. Increasing active transport by expressing more efflux pumps that can actively transport the antibiotic out of the cell decreases the amount of antibiotic inside the cell and prevents it from acting on its target. (c) no efflux pumps. Your answer is incorrect. If an antibiotic cannot reach its target, bacteria will be resistant to its action. Actively transporting antibiotics out of the cell decreases their concentration inside the cell, so that they cannot build up to a high enough concentration to exert the effect on their target. Increasing active transport by expressing more efflux pumps that can actively transport the antibiotic out of the cell decreases the amount of antibiotic inside the cell and prevents it from acting on its target. As you should now appreciate, bacteria can prevent antibiotics from reaching their target by decreasing the permeability of their outer membrane or by actively transporting antibiotics out of the cell (Activity 1). Both decreased porin expression and increased efflux pump expression have been reported in antibiotic-resistant clinical isolates. For example, S. aureus that overexpresses multidrug-resistant efflux pumps, which transport a wide range of antibiotics, have been isolated from patients (Kosmidis et al., 2012). You will look at an example of how altering porin expression contributes to antibiotic resistance in the case study at the end of this week.

2 Intrinsic and acquired resistance There are two types of antibiotic resistance: intrinsic (or inherent) resistance acquired resistance. In this section, you will look at each type in turn.

2.1 Intrinsic resistance Intrinsic resistance is the innate ability of a type of bacteria species to resist the action of an antibiotic as a consequence of the bacteria’s structural or functional characteristics. In contrast to acquired resistance, which you will look at next, intrinsic resistance is ‘normal’ for bacteria of a given type. Intrinsic resistance may occur because bacteria lack the target for a particular antibiotic or because the drug can’t get to its target. It reduces the pool of antibiotics available to treat infections. In addition, as you will see in Week 4, resistance elements that are intrinsic to one bacterial type can be transferred to another one. In this way, intrinsic antibiotic resistance in non-pathogenic bacteria (like the ones you saw in Video 1) can be transferred to a pathogenic bacterium where it can restrict the treatment options for infections caused by these bacteria.

2.2 Introducing acquired resistance As its name suggests, acquired resistance is not innate to a bacterial type. It occurs when a bacterium acquires the ability to resist the actions of a particular antibiotic. Unlike intrinsic resistance, acquired resistance is only found in some populations of a bacterial type. This makes acquired resistance harder to track since each new outbreak or isolate may have acquired resistance to a different spectrum of antibiotics. Acquired resistance is a very significant healthcare concern. Infections caused by bacteria that have acquired resistance to an antibiotic can no longer be treated with that antibiotic. Consequently, identifying the type of pathogenic bacteria causing an infection may not always be sufficient to determine which antibiotics will be effective treatments. Resistant isolates must be tested to determine which antibiotics are effective before treatment can be prescribed. Activity 2 Acquiring multidrug resistance Allow 15 minutes The treatment options for infections caused by bacteria with acquired resistance can be further limited because bacteria can accumulate resistance to a variety of antibiotics over time. This is known as multidrug resistance (MDR). Perhaps the most often cited example of intrinsic resistance is the multidrug resistance of Gram-negative bacteria. Can you suggest why Gram-negative bacteria might be intrinsically resistant to many antibiotics? Unlike Gram-positive bacteria, Gram-negative bacteria have an outer membrane which is impermeable to many antibiotics. Now read the following BBC news article which highlights that, although multidrug resistance is rare, it can have a devastating impact. Article 1 Bug resistant to all antibiotics kills woman While you read the article, note down the answers to the following questions. 1 Which bacterium caused the patient’s infection? The patient’s infection was caused by the Gram-negative bacterium Klebsiella pneumoniae. 2 How many antibiotics was the infection resistant to? It was resistant to 26 different antibiotics, including the ‘drug of last resort’ – colistin. 3 Is resistance to all antibiotics a common occurrence? No, infections that are resistant to all antibiotics are uncommon. Acquired resistance can occur as a result of genetic mutations or the transfer of resistance elements from other bacteria through a process called horizontal gene transfer. Don’t worry if you don’t understand these terms yet. You will return to these processes in Week 4. Activity 3 Comparing intrinsic and acquired resistance Allow 15 minutes Look at the following statements in the table. Decide whether they are about intrinsic or acquired resistance or both and type your answer into the right-hand column.

Statement	Intrinsic resistance, acquired resistance, or both?
Mechanism only present in a subpopulation of bacteria of a given type
Difficult to track
Can be identified if the bacterial type is known
Normal for bacteria of that type
Limits treatment options
Mechanism present in all bacteria of a given type
Occurs as a result of genetic mutation or horizontal gene transfer

Statement	Intrinsic resistance, acquired resistance, or both?
Mechanism only present in a subpopulation of bacteria of a given species	Acquired resistance
Difficult to track	Acquired resistance
Can be identified if the bacterial species is known	Intrinsic resistance
Normal for bacteria of that species	Intrinsic resistance
Limits treatment options	Both
Mechanism present in all bacteria of a given species	Intrinsic resistance
Occurs as a result of genetic mutation or horizontal gene transfer	Acquired resistance

3 Case study: resistance to third-generation cephalosporins In Weeks 1 and 2 you learned about the cephalosporin antibiotics and their mechanism of action. You may recall from Week 1 that the proportion of E. coli isolates that are resistant to cephalosporins has been increasing in the UK. In this case study, you will look at some the molecular mechanisms underlying this resistance.

3.1 Intrinsic resistance to cephalosporins Several bacteria are intrinsically resistant to cephalosporins. As a result, infections caused by these bacteria cannot be treated with cephalosporins. Some of these intrinsically resistant bacteria are summarised in Table 1. Table 1 Bacteria intrinsically resistant to cephalosporins* you will learn more about different generations of cephalosporins in Week 6

Type	Infectious disease	Resistance mechanism (Cox and Wright, 2013)	Resistance
Pseudomonas aeruginosa	Nosocomial infections including pneumonia, urinary tract infections and bacteraemia	Expresses cephalosporinase which deactivates cephalosporins	Resistant to 1st and 2nd generation cephalosporins*
Enterococci spp.	Urinary tract infections, bacteraemia, bacterial endocarditis, diverticulitis and meningitis	Expresses a modified antibiotic target (PBP) that binds to β-lactams poorly	Resistant to 1st and 2nd generation cephalosporins. Some resistance to 3rd generation cephalosporins*
Listeria monocytogenes	Listeriosis	Expresses a modified antibiotic target (PBP) that binds to cephalosporins poorly	Resistant to 1st, 2nd and 3rd generation cephalosporins*

While intrinsic resistance limits the treatment options for infections caused by these pathogens, a greater concern is cephalosporin resistance being acquired by other intrinsically susceptible bacterial types. Some of these bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), are of huge clinical importance. The massive use of cephalosporin antibiotics to treat infections has led to the emergence of these bacteria, as you will see in the case studies in Weeks 4 and 5. But next you will look at the mechanisms of resistance to cephalosporins.

3.2 Mechanisms of cephalosporin resistance Bacteria use multiple different mechanisms to resist the effects of cephalosporin antibiotics. In the final section of this week, you will look at three examples that illustrate the resistance mechanisms described in Section 2: modifying the target destroying the antibiotic preventing the antibiotic from reaching its target. 3.2.1 PBP2a – a PBP that doesn’t bind cephalosporins Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most β-lactam antibiotics, including cephalosporins. This is one of the reasons why infections caused by MRSA are extremely challenging to treat. This resistance results from the expression of penicillin-binding protein 2a (PBP2a). PBP2a binds β-lactams more poorly than other PBPs because differences in its structure prevent β-lactam antibiotics from reaching the binding site (Figure 5).

You may recall from Week 2 that cephalosporins exert their bactericidal action by binding to penicillin-binding proteins and preventing them from cross-linking the bacterial cell wall. Since cephalosporins do not bind to PBP2a, its presence in MRSA allows cell wall biosynthesis to occur in the presence of most cephalosporins. Fortunately, more recently developed cephalosporins, including ceftaroline (Duplessis and Crum-Cianflone, 2011) and ceftobiprole (Kisgen and Whitney, 2008) can bind to and inhibit the activity of PBP2a. These cephalosporins have been licensed for the treatment of community- and hospital-acquired pneumonia and complicated skin and soft tissue infections (NICE, 2017). You will learn more about the development of cephalosporin antibiotics in Week 6. 3.2.2 Extended spectrum β-lactamases In Section 1.2 you saw how β-lactamases can hydrolyse β-lactam antibiotics in order to destroy them. The first β-lactamase to be identified was penicillinase. As its name suggests, penicillinase can hydrolyse penicillin but not cephalosporins. In the 1980s, a new group of β-lactamase enzymes were detected in Europe that hydrolyse cephalosporins. Because of their ability to hydrolyse a wider range of β-lactams, the name for these enzymes is extended spectrum β-lactamase (ESBL). In the next activity, you will look at how the presence of ESBLs in E. coli is associated with cephalosporin resistance. Activity 4 Cephalosporin resistance and ESBLs Allow 20 minutes The data in this activity are from Pfizer’s antimicrobial testing leadership and surveillance (ATLAS) (Pfizer, 2017). E. coli bacteria were isolated from infections and tested to determine whether they produced ESBLs and whether they were resistant to cephalosporins. Figure 6 shows the percentage of ESBL- and non-ESBL-producing E. coli isolates that were resistant to the cephalosporin cefepime between 2004 and 2016. Note that this figure compares ESBL-producing and non-ESBL-producing isolates for resistance to cefepime. However, it does not give any information on the number of E. coli isolates that produce ESBLs. As you will see in the case study in Week 4, the percentage of ESBL-producing E. coli isolates in the UK during this period remained below 15% (BSAC UK, 2014).

Now answer the following questions based on the data in Figure 6. 1 How has the proportion of (a) ESBLs and (b) non-ESBLs resistant to cefepime changed over time? (a) ESBL-producing E. coli have higher levels of resistance to cefepime than non-ESBL-producing E. coli isolates over the entire period. In 2004, approximately 50% of ESBL isolates were resistant. This increased to a peak of approximately 100% in 2007 and 2008. Resistance decreased between 2008 and 2009 and then rose again until 2011. Resistance reached its lowest level in 2013 before increasing again. Resistance never fell below 50% with the lowest levels in 2004 and 2013. (b) non-ESBL producing bacteria display hardly any resistance to cefepime with low levels of resistance (less than 10%) only being observed in 2007, 2009, 2012 and 2015. 2 Explain the difference in resistance between ESBL- and non-ESBL-producing E. coli? The presence of an ESBL in ESBL-producing E. coli results in the hydrolysis, and therefore destruction, of cefepime. Since cefepime can no longer inhibit PBP, these bacteria are resistant. Non-ESBL-producing E. coli lack the ESBL and cannot hydrolyse cefepime, therefore it can exert its bactericidal effects by binding to and inhibiting its target PBP. 3 Do you think the expression of ESBLs is a major determinant of resistance to cephalosporins in E. coli? Resistance to cefepime remains between 50 and 100% in ESBL-producing E. coli whereas almost all non-ESBL-producing bacteria are susceptible to cefepime. This suggests that the presence of an ESBL is a major determinant of cephalosporin resistance. Since they were first described in the early 1980s, the frequency of infections caused by ESBL-producing bacteria has been increasing (Figure 7). Resistance to cephalosporins limits the treatment options for these infections. Consequently, ESBLs represent an ever-growing healthcare challenge.

Several ESBL classes have been identified. Of them, the CTX-M class of ESBLs, has become the most common worldwide. You will learn more about the origin and spread of CTX-M ESBLs in the case study in Week 4. 3.2.3 Porin expression and cephalosporin resistance in K. pneumoniae K. pneumoniae bacteria express two major porins called OmpK35 and OmpK36. Expression of either of them is sufficient for the transport of β-lactam antibiotics across the cell membrane. You may remember from Section 1.3 that reducing the number of porins in the cell wall can confer antibiotic resistance by preventing the antibiotic from crossing the membrane to reach its target. Therefore, you might expect that loss of either OmpK35 or OmpK36 from K. pneumoniae would result in resistance to cephalosporins. However, multiple studies have suggested that the loss of porins from the cell wall of K. pneumoniae does not result in clinically relevant resistance (Hernández-Allés et al., 2000). Therefore, infections caused by K. pneumoniae lacking porins can still be treated using β-lactam antibiotics. Porin loss can contribute to cephalosporin resistance in bacteria with additional mechanisms of resistance. For example, the loss of OmpK35 and OmpK36 from the cell wall of ESBL-producing K. pneumoniae results in resistance to cefoxitin, a cephalosporin which is a poor ESBL substrate. How might this affect the treatment of ESBL-producing K. pneumoniae? Cefoxitin could be used to treat ESBL-producing K. pneumoniae because it is a poor substrate for ESBLs. However, if these K. pneumoniae strains do not express OmpK35 or OmpK36, cefoxitin will not be able to cross the outer membrane to reach its target. Although you have largely considered each resistance mechanism separately this week, this example illustrates how bacteria may rely on multiple resistance mechanisms to protect themselves.

4 This week’s quiz Well done – you have reached the end of Week 3 and can now do the quiz to test your learning. Week 3 practice quiz Open the quiz in a new tab or window by holding down Ctrl (or Cmd on a Mac) when you click on the link. Return here when you have finished it. 5 Summary This week introduced the mechanisms of antibiotic resistance. You should now be able to explain how antibiotic resistance protects bacteria from both natural and synthetic antibiotics and give examples of the main mechanisms of antibiotic resistance. You should now be able to: state what is meant by the term ‘antibiotic resistance’ recognise that antibiotic resistance evolved to protect bacteria describe the three main mechanisms of resistance that bacteria have developed to counteract the action of antibiotics give examples of these resistance mechanisms distinguish between intrinsic and acquired antibiotic resistance. Having seen how antibiotic resistance can be either intrinsic or acquired, next week you will look in more detail at the processes of mutation and gene transfer that lead to acquired resistance. You can now go to Week 4. Week 4: Why are so many bacteria resistant to antibiotics? Introduction Last week you learned that acquired resistance can result from mutation or horizontal gene transfer. This week you will learn more about both of these processes before considering why antibiotic resistance arises and spreads so rapidly. Begin this week by watching the first 2.40 minutes of the following Ted Talks video which shows how bacteria can acquire and spread antibiotic resistance. Video 1 An introduction to the acquisition and spread of antibiotic resistance. [MUSIC PLAYING] NARRATOR: What if I told you there were trillions of tiny bacteria all around you? It's true. Microorganisms called bacteria were some of the first life forms to appear on Earth. Though they consist of only a single cell, their total biomass is greater than that of all plants and animals combined. And they live virtually everywhere, on the ground, in the water, on your kitchen table, on your skin, even inside you. Don't reach for the panic button just yet. Although you have 10 times more bacterial cells inside you than your body has human cells, many of these bacteria are harmless or even beneficial, helping digestion and immunity. But there are a few bad apples that can cause harmful infections, from minor inconveniences to deadly epidemics. Fortunately, there are amazing medicines designed to fight bacterial infections. Synthesised from chemicals or occurring naturally in things like mould, these antibiotics kill or neutralise bacteria by interrupting cell wall synthesis, or interfering with vital processes, like protein synthesis, all while leaving human cells unharmed. The deployment of antibiotics over the course of the 20th century has rendered many previously dangerous diseases easily treatable. But today, more and more of our antibiotics are becoming less effective. Did something go wrong to make them stop working? The problem is not with the antibiotics, but the bacteria they were made to fight. And the reason lies in Darwin's theory of natural selection. Just like any other organisms, individual bacteria can undergo random mutations. Many of these mutations are harmful or useless. But every now and then, one comes along that gives its organism an edge in survival. And for a bacterium, a mutation making it resistant to a certain antibiotic gives quite the edge. As the nonresistant bacteria are killed off, which happens especially quickly in antibiotic-rich environments, like hospitals, there is more room and resources for the resistant ones to thrive, passing along only the mutated genes that help them do so. Reproduction isn't the only way to do this. Some can release their DNA upon death to be picked up by other bacteria while others use a method called conjugation, connecting through pili to share their genes. Over time, the resistant genes proliferate, creating entire strains of resistant super bacteria. So how much time do we have before these superbugs take over? Well, in some bacteria, it's already happened. For instance, some strands of staphylococcus aureus, which causes everything from skin infections to pneumonia and sepsis, have developed into MRSA, becoming resistant to beta lactam antibiotics, like penicillin, methicillin, and oxacillin. Thanks to a gene that replaces the protein beta lactams normally target and bind to, MRSA can keep making its cell walls unimpeded. Other super bacteria, like salmonella, even sometimes produce enzymes, like beta lactamase, that break down antibiotic attackers before they can do any damage. And E.coli, a diverse group of bacteria that contain strains that cause diarrhoea and kidney failure, can prevent the function of antibiotics, like quinolones, by actively booting any invaders that managed to enter the cell. But there is good news. Scientists are working to stay one step ahead of the bacteria. And although development of new antibiotics has slowed in recent years, the World Health Organization has made it a priority to develop novel treatments. Other scientists are investigating alternate solutions, such as phage therapy or using vaccines to prevent infections. Most importantly, curbing the excessive and unnecessary use of antibiotics, such as for minor infections that can resolve on their own, as well as changing medical practice to prevent hospital infections, can have a major impact, by keeping more nonresistant bacteria live as competition for resistant strains. In the war against super bacteria, de-escalation may sometimes work better than an evolutionary arms race.

By the end of this week, you should be able to: explain how genetic mutations can give rise to antibiotic resistance that can be inherited describe the horizontal gene transfer mechanisms that allow antibiotic resistance to be transferred between bacteria discuss how evolution and natural selection maintain antibiotic resistance in bacteria. 1 How do mutations lead to resistance? A bacterium can acquire antibiotic resistance through genetic mutations which are permanent changes in the deoxyribonucleic acid (DNA) sequence that makes up a gene. Perhaps the best example of acquisition of resistance by mutation is Mycobacterium tuberculosis where resistance to all therapeutic agents is caused by mutation. So how does altering the sequence of a bacteria’s DNA result in antibiotic resistance? The answer lies in how genetic information, encoded by DNA, is converted into proteins which are required for the structure and function of bacteria. Optional activity: What do genes do? If you are unfamiliar with the terms DNA, RNA, base pair, gene, amino acid or protein, you may want to try our free OpenLearn course What do genes do? before you begin the following sections.

1.1 From genetic information to protein function Almost every process in a cell requires proteins. As you saw in Week 2, antibiotics often exert their bactericidal and bacteriostatic effects by binding to proteins that are crucial to the structure or function of the bacterial cell. The function of a protein is largely determined by its structure. Proteins are comprised of building blocks called amino acids. The sequence of these amino acids determines the structure of a protein. The amino acid sequence of a protein is specified by the DNA sequence of a gene (Figure 1). So, there is a direct relationship between DNA and the structure and function of a protein.

In 1958, Francis Crick, who helped discover the structure of DNA, proposed the central dogma to explain how genetic information, encoded in DNA, can be converted into a functional product, a protein. The following short video gives an overview of this central dogma. Note that the conversion of information, encoded in DNA, into a protein occurs via an intermediate molecule called RNA (ribonucleic acid). Video 2 An overview of the flow of information from DNA to protein. NARRATOR: To make a particular protein in the cell, the relevant gene is first switched on in the DNA. A working copy of the gene, called messenger RNA, is made. This copying process is called transcription. Next, the information in the messenger RNA is acted upon to produce a protein.

1.2 Genetic mutations and protein structure As you saw in Week 3, changes in the structure of bacterial proteins can result in antibiotic resistance. Can you think of a specific example of how changing protein structure could lead to antibiotic resistance? Structural changes to an antibiotic target protein could prevent the antibiotic from binding. This would make the target insensitive to the antibiotic and bacteria containing this protein would be resistant to the effects of the antibiotic. For example, linezolid exerts its antibiotic effects by binding to ribosomes and preventing the initiation of protein synthesis. Structural changes to the ribosome can prevent the binding of linezolid. Consequently, protein synthesis initiation is no longer blocked in the presence of linezolid and resistant bacteria can grow. Recall from Section 1.1 that the amino acid sequence, and therefore the structure of a protein, is encoded in the DNA sequence of a gene. Small changes, or mutations, in the DNA sequence within a gene can alter the amino acid sequence of the protein it encodes (Figure 2).

It only requires a very small change in the bacteria’s DNA sequence to alter the amino acid sequence and, therefore, the structure of proteins that are targeted by antibiotics. As you have seen, these changes in the structure of proteins targeted by antibiotics can have important consequences for their function. Recall from Week 3 how changing the structure of the ribosome to prevent linezolid binding results in resistance to this antibiotic. These structural changes are caused by several genetic mutations that alter the amino acid sequence, and therefore the structure, of the ribosome. In the case study later this week, you will look at how genetic mutations can cause resistance to cephalosporin antibiotics.

1.3 Transmission of mutations by vertical gene transfer Vertical gene transfer is the transfer of genetic information, including any genetic mutations, from a parent to its offspring. As you briefly saw in Week 1, bacteria reproduce by binary fission, where the cell divides into two identical daughter cells. As in humans, the genetic information in bacteria is encoded in DNA, which is packed into chromosomes. During binary fission, the chromosomal DNA is copied, so that each new daughter cell inherits an exact copy of the parent cell’s chromosomes (Figure 3).

Activity 1 Exploring vertical transmission Allow about 10 minutes Begin by watching the following animation which illustrates the process of binary fission in E. coli. Then complete the activity below. Video 3 Binary fission in E. coli. TEXT ON SCREEN Schematic representation of an E. coli bacterium. The region of the cytoplasm containing the single circular genomic DNA molecule is called the nucleoid. Between divisions, the bacterium grows by elongation from the middle of the cell, increasing cell mass. When the cell reaches a certain size, the circular DNA molecule is replicated (copied). The two DNA molecules each attach to a different part of the cell membrane, so that as the cell elongates, they segregate to opposite ends of the cell. The cell membrane constricts and new cell walls are formed at a point between the two DNA molecules. And the two identical cells finally separate.

Apply what you have learned and the information about binary fission in the animation to complete the following sentences. Select the appropriate word from the list. (a) The DNA in both of the daughter cells is [identical/similar/different] to the DNA in the parent cell. identical similar different During binary fission, the genetic material (DNA) is copied so that each new daughter cell inherits an exact copy of the parent cell's DNA. (b) If an E. coli bacterium contains a genetic mutation in a chromosomal pbp gene, both of its daughters will [always/sometimes/never] contain a mutation in the pbp gene. sometimes never always During binary fission, the genetic material (DNA) is copied, so that each new daughter cell inherits an exact copy of the parent cell’s DNA. When the parent DNA is copied during binary fission, any genetic mutations will also be copied, and consequently inherited, by both of the daughter cells. (c) If the parent bacterial cell contains a genetic mutation that results in resistance to β-lactam antibiotics, both of the daughter cells will [always/sometimes/never] be resistant to β-lactam antibiotics. sometimes always never During binary fission, the genetic material (DNA) is copied, so that each new daughter cell inherits an exact copy of the parent cell's DNA. When the parent DNA is copied during binary fission, any genetic mutations will also be copied, and consequently inherited, by both of the daughter cells. If these genetic mutations give rise to antibiotic resistance in the parent bacteria, they will also result in antibiotic resistance in both of the daughters. Vertical gene transfer is only one of the ways in which bacteria can spread antibiotic resistance genes. In the next section you will look at another – horizontal transfer.

2 Horizontal transfer Horizontal gene transfer, or horizontal transfer, is the primary mechanism of spread of antibiotic resistance that allows bacteria to spread antibiotic resistance genes rapidly between different bacterial types. As you should be starting to appreciate, the acquisition of antibiotic resistance by new bacterial types is particularly concerning because it can result in multidrug-resistant bacterial strains such as MRSA.

2.1 Plasmids In Section 1.3, you saw how chromosomal DNA can be copied and transmitted to the next generation via vertical gene transfer. Unlike humans, bacteria contain additional, non-chromosomal DNA which can be replicated independently of the genomic chromosomal DNA. These non-chromosomal genetic elements are called plasmids. Plasmids are small, circular pieces of DNA which often carry genes associated with a specific function: for example, antibiotic resistance (Figure 5).

Unlike vertical gene transmission, where chromosomal DNA is replicated and then transferred from parent cells to daughter cells through binary fission, plasmids are usually transferred by horizontal gene transfer. This is the process of swapping genetic information between two unrelated cells. In contrast to vertical gene transmission, it does not require binary fission and can occur between bacteria of the same generation, not just between parents and daughters, and even between bacteria of different types (Figure 6).

Can you suggest why horizontal gene transfer is the primary mechanism of spreading antibiotic resistance? Horizontal gene transfer allows plasmids carrying antibiotic resistance genes to spread rapidly between different types of bacteria. Thus species of bacteria that are intrinsically sensitive to a given antibiotic rapidly acquire resistance genes, making them insensitive to treatment with that antibiotic. There are three mechanisms of horizontal gene transfer: conjugation transformation transduction. You will now look at each mechanism in more detail.

2.2 Conjugation In the process of conjugation, plasmids are transferred between two contacting bacteria via a hollow tube or pilus (Figure 7).

Since antibiotic resistance genes are often located on plasmids, conjugation can result in the transfer of antibiotic resistance from one bacterium to another.

2.3 Transformation In contrast to conjugation, the process of transformation allows bacteria to take up DNA from their environment (for example, from a lysed bacterium) across the cell wall. This DNA can then be incorporated into the genome of the bacterium (Figure 8).

Transformation occurs naturally between some bacteria, such as Streptococcus pneumoniae and Haemophilus influenza. When antibiotic resistance genes in the environment are transformed into a new bacterial type, they can be incorporated into that bacterium’s genome. They are then transmitted to the next generation by binary fission, establishing a newly resistant population of bacteria.

2.4 Transduction The final mechanism of horizontal gene transfer you will look at is transduction. In this process, transfer of DNA from one bacterial cell to another is mediated by a virus. Viruses that infect bacteria are called bacteriophages. When bacteriophages infect a bacterial cell, they insert their DNA into the bacterial cell genome. When it is time for the virus to replicate, it excises its DNA from the bacterial genome. However, this excision is imperfect and some bacterial DNA is accidentally excised and incorporated into the newly made virus. When these newly made viruses infect a different bacterial species, they carry this bacterial DNA, which may contain antibiotic resistance genes, and insert it into the genome of the new host bacterium (Figure 9).

Activity 2 Comparing horizontal transfer mechanisms Allow about 10 minutes Below are some incomplete sentences. Type in the missing words using the following list of words: horizontal, binary fission, vertical, conjugation, transformation, transduction, plasmid, chromosome, vertical gene transfer. ___ gene transfer can occur between bacteria of the same generation but vertical gene transfer requires ___. Horizontal gene transfer can occur between bacteria of the same generation but vertical gene transfer requires binary fission. ___ is the only mechanism of horizontal gene transfer that requires direct contact between the donor and recipient bacteria. Conjugation is the only mechanism of horizontal gene transfer that requires direct contact between the donor and recipient bacteria. ___, ___ and ___ can occur between bacteria of different types. Conjugation, transduction and transformation can occur between bacteria of different types. ___ is the only mechanism of horizontal gene transfer which requires a virus known as a bacteriophage. Transduction is the only mechanism of horizontal gene transfer which requires a virus known as a bacteriophage. The bacterial genetic element transmitted by horizontal gene transfer is called a ___. The bacterial genetic element transmitted by horizontal gene transfer is called a plasmid. You will return to look at some specific examples of how genetic mutation and horizontal gene transfer can result in acquired resistance in the case study of cephalosporin antibiotics at the end of this week. Next you will look at how antibiotic resistance has developed through evolution and natural selection.

3 Why are so many bacteria resistant to antibiotics? So far this week, you have looked at how bacteria acquire antibiotic resistance through genetic mutation or horizontal gene transfer. But why are so many bacteria resistant to antibiotics? In this section, you will look at how antibiotic resistance spreads so quickly.

3.1 Evolution and natural selection In 1858, the British naturalists Charles Darwin and Alfred Russel Wallace both independently proposed the theory of evolution through natural selection to explain how organisms change over time. Evolution is a change over time in the inherited characteristics or traits in a population. This change is largely brought about by natural selection. This is the process by which a particular trait that confers a survival advantage for an individual becomes more frequent in the population. Although Darwin and Wallace were unaware of the existence of DNA, we now know that natural selection is the process by which genetic mutations that increase the ability of an organism to survive are selectively passed on to subsequent generations. Now listen to Audio 1 in which Professor Steve Jones from University College London explains Darwin’s theories of evolution and natural selection. Audio 1 Darwin’s theories of evolution and natural selection. INTERVIEWER What was the crux of Darwin's ideas? STEVE JONES Darwin described his own theory in a pretty tight nutshell. Evolution, he said, is descent with modification. Descent – the passage of information, we would say, today, from one generation to the next and modification – the fact that that passage is imperfect. Over time, those changes will build up and you will get change. It's inevitable. It's bound to happen. But we can rephrase that in slightly more telling terms today. We can say, evolution is genetics plus time. If you've got genetics, DNA, all that stuff – if it copies itself with mistakes, that's mutations. And if you've got time – and we got three and a half thousand million years since the origin of life– evolution is absolutely inevitable. So that's the core of Darwin's theory. It's extraordinarily simple. But Darwin had a second idea, and that's really where he was so smart, because he realised that what's being copied in biology is itself a copying machine, so that if one version inherits a change, a mutation, which makes it more likely that it will survive and reproduce itself, then that change will become more common and will spread. And over time, those differences will build up and new forms of life will emerge by what he called natural selection – inherited differences in the chances of reproducing. So that's Darwinism in one minute. In the next section, you will see how our use of antibiotics contributes to the evolution of resistance.

3.2 Evolving resistance to antibiotics How do bacteria evolve resistance to antibiotics? Activity 3 will help you to think about how evolution and natural selection contribute to the spread of antibiotic resistance. Activity 3 Evolution, natural selection and antibiotic resistance Allow about 10 minutes Darwin’s theory was that evolution by natural selection would occur if the following conditions were met: There is a struggle for existence. Survival is limited by environmental constraints, so that there is competition, and not all individuals will survive to produce offspring. There is variation between individuals. Individuals with advantageous traits will have a greater probability of survival under these conditions and are therefore more likely to reproduce. The characteristics, or traits, of an individual are inherited. Advantageous traits that promote survival will be inherited by the next generation so that these traits become increasingly common in the population. Now answer the following questions about the evolution of antibiotic resistance. 1 Which of the environmental conditions below might lead to the evolution of antibiotic resistance? (a) low nutrient supply Your answer is partially correct. Any of these could conditions could create a struggle for existence that could lead to the evolution of antibiotic resistance. (b) presence of antibiotics Your answer is partially correct. Any of these could conditions could create a struggle for existence that could lead to the evolution of antibiotic resistance. (c) low oxygen availability Your answer is partially correct. Any of these could conditions could create a struggle for existence that could lead to the evolution of antibiotic resistance. (d) all of the above Your answer is correct. Any of these could conditions could create a struggle for existence that could lead to the evolution of antibiotic resistance. 2 Would antibiotic resistance be an advantageous, or a disadvantageous, trait for bacteria growing in the presence of antibiotics? (a) Antibiotic resistance would be advantageous. Your answer is correct. Antibiotic resistance would be an advantageous trait in the presence of antibiotics because resistant bacteria in the population will have a survival advantage over sensitive bacteria. However, in the absence of antibiotics, resistance can sometimes be disadvantageous because it can result in slower growth. (b) Antibiotic resistance would be disadvantageous. Your answer is incorrect. Antibiotic resistance would be an advantageous trait in the presence of antibiotics because resistant bacteria in the population will have a survival advantage over sensitive bacteria. However, in the absence of antibiotics, resistance can sometimes be disadvantageous because it can result in slower growth. (c) Antibiotic resistance would have no effect on the survival of bacteria growing in the presence of antibiotics. Your answer is incorrect. Antibiotic resistance would be an advantageous trait in the presence of antibiotics because resistant bacteria in the population will have a survival advantage over sensitive bacteria. However, in the absence of antibiotics, resistance can sometimes be disadvantageous because it can result in slower growth. 3 How would the trait of antibiotic resistance be inherited by the next generation? (a) horizontal gene transfer Your answer is incorrect. Antibiotic resistance genes, acquired via genetic mutation or horizontal gene transfer, can be inherited by subsequent generations through binary fission. (b) transformation Your answer is incorrect. Antibiotic resistance genes, acquired via genetic mutation or horizontal gene transfer, can be inherited by subsequent generations through binary fission. (c) binary fission Your answer is correct. Antibiotic resistance genes, acquired via genetic mutation or horizontal gene transfer, can be inherited by subsequent generations through binary fission. (d) genetic mutation Your answer is incorrect. Antibiotic resistance genes, acquired via genetic mutation or horizontal gene transfer, can be inherited by subsequent generations through binary fission. Within a bacterial population, some bacteria will be sensitive to antibiotic treatment while others will have acquired resistance to antibiotics, via either genetic mutation or horizontal gene transfer. In the presence of antibiotics, the resistant bacteria have a survival advantage over the sensitive bacteria and are more likely to survive and reproduce. Because bacteria reproduce so quickly, resistant bacteria can quickly dominate the population (Figure 10).

Of course, changes to the bacteria’s environment made by us can affect the evolution of antibiotic resistance. You will return to this theme when you look at how our use of antibiotics contributes to the rise of antibiotic resistance bacteria in Week 5.

3.3 Experimentally evolving antibiotic resistance Most animals evolve over millions of years. However, because bacteria grow and evolve so rapidly, scientists can study the evolution of antibiotic resistance in the laboratory. You will now look at an experiment which shows how bacteria adapt to survive increasingly higher doses of antibiotic (Baym et al., 2016). Activity 4 Evolution in action Allow about 30 minutes Watch the following video taken from an episode of the BBC’s Horizon programme. Here, researchers from Harvard University and Technion-Israel Institute of Technology describe an experiment to evolve antibiotic-resistant bacteria in the laboratory. Video 4 Experimental evolution of antibiotic resistance. NARRATOR Scientists are now trying to understand exactly how superbugs have gained resistance, and ultimately how we can defeat them. Here at Harvard University scientists are investigating why some of our antibiotics are failing. It's an experiment that happens in Professor Roy Kishony's lab. Here they are deliberately trying to create superbugs. ROY KISHONY So this is a new device that we have developed. We call it the "morbidostat." NARRATOR Using the morbidostat they are going to produce a highly-resistant version of a harmless strain of a bacteria we all have in our gut, E. coli. ROY KISHONY In the beginning you have bacteria just going happily in the tube. They have enough food, so they're going fast. NARRATOR They start by trying to kill the E. coli by dripping in a low concentration of antibiotic. But as the millions of bacteria have been multiplying in the tubes, some, by chance, will have developed mutations that allow them to be resistant to the antibiotic. ROY KISHONY This mutant would start replicating faster than everyone else. Ultimately it would take over on the whole population. NARRATOR So now they try to kill this new mutant strain. They up the strength of the antibiotic. Again, most of them die. But a new mutation appears that can survive the even stronger antibiotic. ROY KISHONY And then see another step. Now we can go in even higher drug concentrations. So we keep iterating this process over and over and over. NARRATOR This experiment shows that bacteria become resistant by being exposed to low levels of the very thing we use to protect us – antibiotics. Now the team have created a new experiment to find out exactly what is happening in these mutant bacteria to allow them to be resistant. It starts with what is, in effect, a giant Petri dish. ROY KISHONY We're setting an experiment, really, for the first time in which we're going to let bacteria swim against an ever-increasing concentration of an antibiotic and see what happens. NARRATOR The jelly contains food for the bacteria to grow. But each slab is infused with an increasing concentration of antibiotic, which should act as a barrier, killing the bacteria. ROY KISHONY The first slab is no drug. Then about the amount that's needed to kill the bacteria. Then 10 times more, 100 times more, and 1000 times more. NARRATOR The experiment begins with a tiny drop of E. coli. ROY KISHONY They're certainly going to spread when there is no drug. But we want to see, can they actually go to the place where there is an antibiotic?

In the next video you will watch what happens when bacteria grown on a plate containing increasing concentrations of antibiotics like the one described in Video 4. This plate is known as a Microbial-Evolution and Growth Area (MEGA) plate. Before you watch the next video, note down what you think will happen to the bacteria as they grow on the MEGA plate. As bacteria grow to fill the area of the MEGA plate, with no antibiotic they begin to compete for resources, meeting one of Darwin’s conditions for evolution – a struggle for existence. At this point, mutations occur which allow some bacteria to survive in the area of the plate containing antibiotic. These bacteria have a survival advantage over the antibiotic-sensitive bacteria and grow and reproduce to cover the area of the plate containing a low antibiotic dose. As they fill this area of the plate, they also begin to compete for resources and the cycle of mutation, selection and growth repeats. Now click on the following link to watch a video showing a time-lapse recording of bacteria growing on the MEGA plate. Then answer the questions below. 1 How many different mutants have reached the 1000× antibiotic concentration at the end of the experiment? (Hint: you will need to watch until the end of the video.) Using the coloured tree diagram at the end of the video, we counted 16 different mutants that reached the 1000× antibiotic concentration at the end of the experiment. 2 Would you expect the first mutant bacteria that appear (those that occur at the no antibiotic:1× antibiotic boundary) to grow on the 1000× antibiotic region on the plate? It is unlikely that the resistance mutations that allowed bacteria to survive on the 1× dose of antibiotic would be sufficient for bacteria to survive on the 1000× dose. It is more likely that bacteria would require multiple antibiotic resistance mutations to survive on the 1000× dose. 3 Did your predictions from the first part of this activity match the experimental results? You may not have exactly predicted what would happen in the experiment but you may have been able to make some suggestions about how bacteria acquire mutations in order to cross the no antibiotic:1× antibiotic boundary. Now that you have watched the experiment, you may want to reread the discussion from the first part of this activity.

4 Case study: resistance to cephalosporins In Week 3’s case study, you looked at the molecular mechanisms of resistance to cephalosporins and were introduced to ESBLs. The most common class of ESBLs in Europe is the CTX-M-type ESBL. In this case study, you will explore how bacteria acquire resistance to cephalosporin antibiotics through horizontal gene transfer and the mutation of CTX-M-type ESBLs. You will begin by looking at how the presence of CTX-M-type ESBLs has changed in the UK over recent years. Activity 5 The rise of CTX-M-type ESBLs Allow about 20 minutes The data in this activity are from The British Society for Antimicrobial Chemotherapy’s Resistance Surveillance Project (www.bsacsurv.org) which collects annual data on antibiotic resistance rates. First, look at Figure 11 which shows the percentage of E. coli isolate producing CTX-M-type, or other, ESBLs between 2002 and 2016. Then answer the questions below.

(a) Using the data in Figure 11, how has the proportion of isolates producing CTX-M-type ESBLs changed over time? The proportion of isolates producing CTX-M-type ESBLs was low in 2002 and increased to a peak in 2006. The percentage of resistant isolates decreased between 2007 and 2010 and then began to increase again, peaking in 2013. (b) CTX-M-type ESBLs emerged in the late 1990s and were first reported in the UK in 2002. Suggest one possible reason for the change in frequency of these ESBLs in the UK over time. The increasing use of cephalosporin antibiotics could create conditions where resistance to cephalosporin antibiotics through the acquisition of CTX-M-type ESBLs is advantageous for the bacteria. Hence these conditions would satisfy Darwin’s conditions for evolution through natural selection. (c) In Activity 7 in Week 1, you looked at how resistance to cephalosporins had changed in the UK over time (Figure 12). By comparing the data in Figures 11 and 12 do you think that the occurrence of CTX-M-type ESBLs is a good indicator of the rate of resistance to cephalosporins?

Yes. Patterns of resistance to cephalosporins and the occurrence of CTX-M-type ESBLs were broadly similar between 2002 and 2016. This suggests that CTX-M occurrence is a good indicator of cephalosporin resistance and that the presence of CTX-M-type ESBLs in E. coli is the major cause of cephalosporin resistance. (d) What challenges might these changes in the prevalence of CTX-M-type ESBLs present to health care? The increasing frequency of cephalosporin-resistant bacteria and CTX-M-type ESBLs restricts the treatment options for infections caused by these bacteria.

4.1 The origin of CTX-M-type ESBLs Unlike most acquired β-lactamases, for which the source remains unknown, the source of CTX-M-type ESBLs has been identified as members of the bacterial genus Kluyvera. Kluyvera spp. are soil bacteria which are associated with plant roots and are non-pathogenic to humans. Precursors of the CTX-M genes found in E. coli have been identified as chromosomal genes in Kluyvera spp. where they can confer resistance to third-generation cephalosporins (Humeniuk et al., 2002). The resistance of Kluyvera spp. to third generation cephalosporins is an example of what type of resistance? It is an example of intrinsic resistance. These chromosomal CTX-M precursor genes have been captured and incorporated into plasmids through mechanisms that you do not need to know about in this course. In the next section, you will look at how these plasmid-encoded CTX-M genes have been rapidly spread by horizontal gene transfer to other bacterial types, including E. coli and Klebsiella pneumoniae.

4.2 The rapid spread of CTX-M genes Plasmids carrying CTX-M genes have been reported in several enterobacteria types (Figure 13). They are most commonly found in E. coli and K. pneumoniae but have also been isolated from other pathogenic bacteria. Plasmids that are found in many genetically diverse bacterial strains are termed ‘epidemic plasmids’ and can help to explain the rapid global spread of CTX-Ms. This is sometimes referred to as the ‘CTX-M pandemic’ (Cantón and Coque, 2006). However, the reasons for this CTX-M pandemic are complex. The horizontal gene transfer of plasmids containing CTX-M genes occurs in the human gut and in the environment and is fundamental to their global spread.

Plasmids carrying CTX-M genes often carry bacteriophage-related sequences (Falgenhauer et al., 2014) or genes that are required for the formation of pili (Carattoli, 2013). What horizontal gene transfer mechanisms do the presence of bacteriophage-related sequences and pili formation genes suggest? The presence of bacteriophage-related sequences in some CTX-M-containing plasmids suggests horizontal gene transfer by transduction (see Section 2.4). Alternatively, gene transfer via conjugation requires a pilus linking the donor and recipient bacteria, therefore the presence of genes required for the formation of pili suggests horizontal gene transfer via this mechanism (see Section 2.1). Perhaps the most concerning feature of these CTX-M-containing plasmids is their ability to acquire additional antibiotic resistance genes (Potron et al., 2013). If ‘epidemic plasmids’ acquire resistance to antibiotics such as carbapenems, which are frequently used to treat cephalosporin-resistant infections, the rapid spread of multidrug resistance could seriously challenge the treatment of infections.

4.3 Mutations in CTX-M-type ESBLs CTX-M-type ESBLs preferentially act on certain cephalosporin antibiotics. Cefotaxime is easily recognised and inactivated by CTX-M-type ESBLs while the bulkier cephalosporin ceftazidime is poorly recognised by CTX-M-type ESBLs (Bonnet, 2004). As a consequence, infections caused by bacteria that produce CTX-M-type ESBLs can be treated with ceftazidime. This specificity is based on the structure of the CTX-M β-lactam binding site which only allows the efficient recognition of penicillins, first-generation cephalosporins and cefotaxime (Figure 14) (Chen et al., 2005).

The specificity of CTX-M-type ESBLs can be modified by point mutations which improve the specificity of CTX-M-type ESBLs for ceftazidime. One of these mutations is indicated on Figure 14 (Cartelle et al., 2004). Altering this amino acid allows the bulkier ceftazidime to be more easily accommodated in the β-lactam binding site (Chen et al., 2005). Infections caused by bacteria producing this mutated version of the CTX-M-type ESBL are not treatable with ceftazidime. This CTX-M variant has been isolated from clinical strains of E. coli (Cartelle et al., 2004) and has probably been selected for the increasing use of ceftazidime in clinical practice.

5 This week’s quiz It’s time to complete the Week 4 badged quiz. It is similar to the previous quizzes but this time, instead of answering 5 questions, there will be 15, covering Weeks 1 to 4. Week 4 compulsory badge quiz Remember that the quiz counts towards your badge. If you’re not successful the first time, you can attempt the quiz again in 24 hours. Open the quiz in a new tab or window by holding down Ctrl (or Cmd on a Mac) when you click on the link. 6 Summary In this week you learned how bacteria acquire antibiotic resistance and how this resistance can rapidly spread, or evolve, in a population. You should now be able to explain how genetic mutations cause acquired antibiotic resistance and how these mutations can be inherited through binary fission. You have also seen how horizontal gene transfer has an important role in transmitting antibiotic resistance to different bacterial types. Having seen how antibiotic resistance evolves to protect bacteria, you can now begin to speculate on how our use of antibiotics contributes to the rise of antibiotic resistance. You should now be able to: explain how genetic mutations can give rise to antibiotic resistance that can be inherited describe the horizontal gene transfer mechanisms that allow antibiotic resistance to be transferred between bacteria discuss how evolution and natural selection maintain antibiotic resistance in bacteria. Next week you will discover how the mismanagement of antibiotics has increased the rate of resistance. You can now go to Week 5. Week 5: How antibiotic resistance has become such a big problem Introduction In Weeks 3 and 4, you learned how some bacteria have an innate ability to resist the action of a particular antibiotic. Other bacteria acquire the ability to resist one or more antibiotics through genetic mutation or horizontal gene transfer. You also learned how evolution and natural selection contribute to the rapid spread of acquired antibiotic resistance both within and between bacterial types. This week, you will discover how the mismanagement of antibiotics, coupled with behaviours that promote the spread of infections, has increased the rate of antibiotic resistance. Drug-resistant bacteria lead to infections which are difficult to treat and are a significant and growing healthcare concern. By the end of this week, it should be clear that the current global health crisis, with record levels of antibiotic resistance, has been fuelled by human activity. By the end of this week, you should be able to: describe the scale and nature of antibiotic resistance worldwide summarise how antibiotic resistance spreads explain how the overuse and misuse of antibiotics contribute to bacterial resistance list the factors which have prevented new antibiotics coming onto the market recognise how a lack of laboratory capacity and inadequate surveillance contribute to the development and spread of antibiotic resistance. 1 The antibiotic resistance crisis Start this week by completing the activity below. This will give you a sense of how serious the problem is and an introduction to the factors that drive resistance. Activity 1 The antibiotic resistance crisis Allow about 10 minutes First, watch the following video. Video 1 The antibiotic resistance crisis. TEXT ON SCREEN Why are antibiotics so important to us? Antibiotics are used extensively in medicine to treat infections and to prevent infection during surgery or cancer treatment. Antibiotics are used extensively in veterinary medicine, dentistry. Antibiotics are used extensively in global food production. ‘Antibiotic resistance is one of the biggest threats to global health, food security, and development today.’ Antibiotics are the largest and best known class of a wider group of drugs called antimicrobials which also includes antiviral drugs and antimalarial drugs. Resistance can develop to all types of antimicrobial making these drugs less effective, but antibiotic resistance poses the greatest threat to global health. Levels of antimicrobial resistance (AMR) and antibiotic resistance (AR) are monitored worldwide. Every year: 700,000 people die from AMR infection; nearly one third of these deaths are caused by multi-drug resistant TB (review on antimicrobial resistance, 2016). Every year: about two million people in the United States become infected with AR bacteria and at least 23,000 die as a direct result of these infections (Centres for Disease Control and Prevention, 2017). Every year: 25,000 people in Europe die from a drug-resistant infection caused by one of these bacteria: escherochia coli, klebsiella pneumoniae, enteroccus faecium, pseudomonas aeruginosa, methicilin-resistant Staphylococcus aereus (MRSA) (Public Health England, 2017). Resistant infections lead to higher death rates and are more expensive to treat. In Europe healthcare costs and lost productivity as a result of drug-resistant infections already cost an estimated €1.5 billion per year (Review on antimicrobial resistance, 2016). If not tackled, rising AMR could have a devastating impact. If not tackled, rising AMR could result in: 10 million deaths per year by 2050 at a global economic cost of $100 trillion … or $10,000 per person. There will be further indirect costs to society through loss of life and quality of life (Review on antimicrobial resistance, 2016). Why are antibiotics so important to us? Every time an antibiotic is used there is the potential for resistance to develop. Poor hygiene and infection control spreads infections and indirectly leads to resistance. Resistance develops more quickly when antibiotics are overused or misused. New drugs are needed to replace antibiotics that are no longer effective. The risk of resistance developing is reduced when clinicians know which antibiotic will be the most effective treatment (Review on antimicrobial resistance, 2016). The more antibiotics are used the more antibiotic resistance increased ( (Review on antimicrobial resistance, 2016). Antibiotics only work against bacterial infections. Colds and flu are caused by viruses. Nearly one third of antibiotics prescribed in the United States are to treat non-bacterial infections, meaning around 47 million prescriptoins are unnecessary each year. In the UK, antibiotics are prescribed for 60% of sore throat diagnoses, but 90% of cases are caused by viruses (The King’s Fund, 2016). During food production, antibiotics are used in healthy animals to prevent infection or speed up growth (O’Neill Report, 2015). Animals in the USA consume more than twice as many medically important antibiotics as humans. Few new antibiotics are being developed. The development of new antibiotics is costly and regulatory approval is difficult. Antimicrobial R&D is not attractive to venture capitalists. In some countries systems to track infections and AMR are inadequate or non-existant. What does the future hold? In December 2017, the UK Office of National Statistics (ONS) revised life expectancy down citing ‘fears of the re-emergence of existing diseases and increases in antimicrobial resistance’. ‘… it is entirely possible that we could see a return to a situation where 40 per cent of the population die prematurely from infections we cannot treat’, Professor Dame Sally Davis, UK Chief Medical Officer (The King’s Fund, 2016).

Consider the following statements and decide if they are true or false. Write your answer in the right-hand column.

Statement	True or false?
1 All antibiotics are antimicrobials but not all antimicrobials are antibiotics.
2 Drug resistance is only a problem in Europe, the USA and other high-income countries (HICs).
3 Ten million people each year die from antimicrobial resistance (AMR) infection.
4 AMR causes significant economic damage.
5 Using fewer antibiotics will not help reduce antibiotic resistance.
6 It is acceptable to give antibiotics to healthy animals to promote growth.
7 Few new antibiotics are being developed to replace those to which bacteria have become resistant.
8 Antibiotic resistance surveillance data is necessary to inform clinical decision making.

TRUE Antibiotics are just one type of antimicrobial drug. Antivirals, antifungals and antiprotozoans are also antimicrobials. FALSE AMR is a global problem. FALSE An estimated 700,000 people die every year from AMR infections. This number is expected to rise to 10 million deaths per year by 2050 if resistance is not tackled. TRUE For example, the estimated cost to the European Union of AMR infections is €1.5 billion per year. FALSE The more antibiotics that are used, the greater the antibiotic resistance. FALSE Using antibiotics for reasons other than to treat bacterial infection has been shown to increase antibiotic resistance. You will learn more later about why this happens. TRUE Pharmaceutical companies find the cost and regulatory challenge of developing new antibiotics prohibitive. TRUE The impact of inadequate AMR surveillance systems on the spread of antibiotic resistance is discussed in Section 6. 2 How antibiotic resistance spreads In Week 4, you learned that, in the presence of antibiotics, resistant bacteria have a survival advantage over sensitive bacteria and can quickly dominate a bacterial population. Resistance is an inevitable consequence of using antibiotics. The more that antibiotics are used, the more widespread resistance becomes. You have also learned that antibiotic resistance spreads in a bacterial population by resistance genes passing from one bacterium to another. In Activity 2, you will discover that antibiotic resistance also spreads when resistant bacteria move from one human or animal host to another. Activity 2 How does antibiotic resistance spread? Allow about 10 minutes First, watch the video below which shows how antibiotic resistance spreads in different communities. Video 2 How does antibiotic resistance spread? INSTRUCTOR: How does antibiotic resistance spread? Antibiotic resistance is the ability of a bacteria to combat the action of one or more antibiotics. Humans and animals do not become resistant to antibiotic treatments, but bacteria carried by humans and animals can. In animal farming, animals may be treated with antibiotics, and they can therefore carry antibiotic-resistant bacteria. Vegetables may also be contaminated with antibiotic-resistant bacteria from animal manure used as fertiliser. Finally, antibiotic-resistant bacteria can spread to humans through food and direct contact with animals. In a community, humans sometimes receive antibiotics prescribed to treat infections. However, bacteria develop resistance to antibiotics as a natural adaptive reaction, and then antibiotic-resistant bacteria can spread from the treated patient to other persons. In health care facilities, humans may receive antibiotics and then carry antibiotic-resistant bacteria. These can spread to other patients via unclean hands or contaminated objects. Patients who may be carrying antibiotic-resistant bacteria will ultimately be sent home and can spread these resistant bacteria to other persons. Travellers requiring hospital care while visiting a country with a high prevalence of antibiotic resistance may return home with antibiotic-resistant bacteria. Even if not in contact with health care, travellers may carry and import resistant bacteria acquired from food or the environment during travel. Bacteria have become resistant because antibiotics have been used for the wrong reasons or incorrectly in different settings. Infections with resistant bacteria are difficult to treat.

Now, answer the following questions: In which ways can people be infected by antibiotic-resistant bacteria? What roles do people moving from one geographical region to another play in spreading antibiotic resistance? Antibiotic-resistant bacteria can spread to humans through food and through direct contact with animals or other people (for example, via unclean hands or contaminated objects). Resistance may also develop naturally if a person takes antibiotics to treat a bacterial infection. You will learn more about how infections are spread between people in Week 7. The numbers of people travelling abroad for work or holidays is rising. This increases the likelihood that resistant bacteria, acquired from food or the environment, will be brought home and spread to other people. Antibiotics eventually end up in the environment. Contaminated soil and waterways become reservoirs of antibiotics and antibiotic-resistant bacteria. This creates selective pressure that encourage the development and spread of resistance and the transfer of antibiotic-resistant bacteria to people and animals (Figure 1).

Two interrelated factors contribute to the spread of antibiotic resistance. An increased rate of resistance, which results in higher numbers of antibiotic-resistant bacteria, and a greater number of cases of infectious diseases. Both factors increase the use of antibiotics which, in turn, drives the antibiotic resistance rate. In the following sections, you will look at the main drivers of antibiotic resistance: poor hygiene and infection control the overuse of antibiotics the misuse of antibiotics. You will also consider how the lack of new antibiotics and gaps in global infection and resistance data exacerbate the problem. 3 Poor hygiene and infection control Healthcare settings are hot spots for infectious diseases, including those caused by antibiotic-resistant bacteria (Figure 2). Infections are spread through failures in hygiene, such as poor hand-washing technique, and because measures to prevent and control infections are inadequate or not followed consistently. Failure to wear protective clothing, to dispose of waste safely and to maintain a clean working environment all contribute to the spread of bacteria. You will learn more about the role of hygiene in preventing the spread of infections in Week 7. A high number of cases of infectious diseases not only increases the demand for antibiotics and drives resistance but also increases mortality and has a negative impact on quality of life (Figure 3).

In the next section, you will explore the link between the overuse of antibiotics and resistance. 4 Overuse of antibiotics Central to the growing problem of antibiotic resistance is the increasing demand for antibiotics. In the next activity, you will look at the relationship between antibiotic use and antibiotic resistance across Europe. Activity 3 Antibiotic use and antibiotic resistance in Europe Allow about 10 minutes First, look at Figure 4. The x-axis (horizontal) shows a measure of penicillin use in the year 2000 (given as the defined dose per 1000 inhabitants daily – DID. You do not need to know how this is calculated). The y-axis (vertical) shows the proportion of Streptococcus pneumoniae infections that were penicillin-resistant in the year 2001. Each point on the graph represents a country.

Now answer the following questions, based on the data in Figure 4. Which country had the lowest antibiotic use? Which country had the highest antibiotic resistance? The Netherlands (furthest to the left of the x-axis). France (highest on the y-axis). Does the graph show a correlation between antibiotic use and antibiotic resistance? A correlation simply means that there is a relationship between two sets of data (i.e. the antibiotic use on the x-axis and the antibiotic resistance on the y-axis). For example, as the value of X, increases, the value of Y also increases. Yes, there is a positive correlation between antibiotic use and antibiotic resistance. As antibiotic use increases, so does the proportion of antibiotic-resistant infections. Next, you will consider the factors that promote the overuse of antibiotics and encourage the development and spread of antibiotic resistance.

4.1 Factors leading to the overuse of antibiotics In the UK, antibiotics can only be obtained on prescription from a doctor. However, overprescription is a problem and online sales of antibiotics can circumvent regulation. In other countries, the unregulated, over-the-counter sales of cheap antibiotics allow people to self-medicate. Public attitudes and behaviours towards using antibiotics are a key factor in both the overuse and the misuse of antibiotics. In agriculture, antibiotics are mainly used to keep animals healthy and to promote growth (Figure 5). The rising global demand for a meat-based diet has led to more intensive, large-scale farming with animals reared in confined spaces where they are at greater risk of infection. Under these conditions, the demand for antibiotics is high. In some countries, more antibiotics are consumed by animals than by people. For example, in the USA, 70% of medically important antibiotics are consumed by animals.

In the next activity, you will consider how these factors might influence the consumption of antibiotics in different countries. Activity 4 Comparing antibiotic consumption in different countries Allow about 25 minutes In this activity, you will compare antibiotic consumption in two HICs – the UK and the USA – and two low-middle-income countries (LMICs) – China and India. Part A Figure 6 shows the consumption of antibiotics by country.

Complete the table of antibiotic use using the information given on the map in Figure 6. If you want to use the larger version of the figure, open it in a new browser tab or window so you can look at it alongside the activity. *A standard unit is defined as the equivalent of one pill, capsule or ampoule.

Country	Antibiotic use (all) /standard units^* per 1000 population in 2015
China
India
UK
USA

Which of these countries had the highest consumption of antibiotics per 1000 population in 2015 and which country had the lowest consumption? The UK used the most antibiotics at 21 632 standard units per 1000 population. China used the least antibiotics at 10 389 standard units per 1000 population.

Country	Antibiotic use (all) /standard units per 1000 population in 2015
China	10 389
India	10 554
UK	21 632
USA	18 389

Were you surprised by which of the four countries had the highest antibiotic consumption per 1000 population in 2015? Why or why not? Many different factors affect antibiotic consumption at a country level. For example, access to healthcare, availability and cost of drugs and public attitudes. In LMICs, poor access is probably a significant factor in antibiotic use. Although the UK and the USA have comparable levels of antibiotic use per 1000 population, the UK possibly has fewer barriers to health care and antibiotics are readily prescribed.Note that, although the UK has the highest antibiotic use per 1000 population, it has the lowest total antibiotic use. Both India and China used ten times more antibiotics in 2015 than the UK. This is due to their larger population size. Part B Figures 7a and 7b show the consumption of antibiotics in the four countries between 2000 and 2015.

Answer the following questions about antibiotic use over this time period. In which of these countries did antibiotic use rise? In which country did antibiotic use fall? Which country maintained similar levels of antibiotic use? India and China USA UK Can you make a general observation about the use of antibiotics in different countries? Antibiotic use varies between countries and within a country as demand rises or falls. Part C The table below shows projected change in antimicrobial consumption in China, India, the UK and the USA by 2030. Table 1 Projected change in antimicrobial consumption in China, India, the UK and the USA by 2030* The mg/PCU is a standard unit of measurement based on the animal population and the weight of the animal at the time of treatment with antibiotics. Data from CDDEP, 2013.

Country	Projected change in antimicrobial consumption by 2030 (%)
China	13
India	18
UK	1
USA	2

Which country is projected to have the greatest increase in antimicrobial consumption for animal use by 2030? What might be driving the increase? India is expected to increase its antimicrobial consumption by 18% over this period. Five LMICs, including India and China, are expected to account for one-third of increased antibiotic consumption in animals by 2030.This will be driven by consumer demand for meat products and hence more intensive farming practices which rely heavily on antibiotics. The potential consequences for antibiotic resistance are considerable (van Boeckel et al., 2015). Activity 4 revealed the extent to which antibiotics are used by people and for animals. The agricultural use of antibiotics is of particular concern because studies have shown that the sub-therapeutic doses of antibiotics used in intensive farming can encourage the development of resistant bacteria (O’Neill, 2015).

5 Misuse of antibiotics Begin this section by completing Activity 5 in which you reflect on your personal experience of using antibiotics. Activity 5 Taking antibiotics Allow about 5 minutes Answer the questions below, based on either a recent experience or according to how you would probably behave. The last time you felt ill, were you hoping or expecting the doctor to prescribe antibiotics? If you were given antibiotics, did you (a) take the correct number of tablets at the correct time and (b) complete the full course of treatment? Have you ever taken someone else’s ‘leftover’ antibiotics? Being prescribed unnecessary antibiotics, failing to complete the full antibiotic course or taking antibiotics that have not been prescribed to you all increase the risk of antibiotic resistance developing and spreading. The emergence and spread of antibiotic resistance is promoted when antibiotics are wrongly prescribed and/or taken, or when they are used indiscriminately. Self-medication is very common. For example, it accounts for over 30% of antibiotic use in LMICs (Ocan et al., 2015). The indiscriminate use of antibiotics leads directly to overuse. This is a global problem. It is common to use antibiotics to treat mild bacterial infections that could resolve spontaneously, and for viral or non-infectious diseases. There is an over-reliance on broad-spectrum antibiotics that target a wide range of bacteria because rapid diagnostics, which could identify the resistance profile of the pathogen, are lacking (Ventola, 2015). You will look more closely at rapid diagnostics in Week 7. Using antibiotics for non-therapeutic purposes, such as for animal husbandry (see Section 4), provides further opportunities to spread antibiotic resistance (Meek et al., 2015). You will look in more detail at some of these factors in the following sections.

5.1 Treatment of non-bacterial infections One-third of people in the UK believe that antibiotics will treat coughs and colds. But these conditions are caused by viruses which antibiotics are not effective against (Public Health England, 2015). Watch the following videos which explain why. Video 3 Bacteria and viruses are very different. HOST: Many of us already know that infectious illnesses are often caused by viruses or bacteria. But how many of us know actually what a difference that makes? You'd normally need a microscope to explore the difference between viruses and bacteria. But studying things in a lab is not really my scene. I find it easier to explain stuff when I can get my hands dirty and see things properly. That's why I've come here. The most obvious difference between viruses and bacteria is size. To us, a single bacterium might be pretty small, maybe a thousandth of a millimetre. But to a virus, they're looking fairly large. If we scale things up and took a typical virus to be the size of a suitcase, in which case a bacterium would be the size of a van. [HORN HONKS TWICE] And the comparison doesn't end there. Just like this van is a fully functioning machine with different working parts for specific jobs-- like wheels, engine, fuel pump, windscreen, etc. so too is a bacterium. It's a self-contained unit with a wall around it and all the biological machinery of a living cell. Whereas a virus just has a thin protein coat, inside it's practically empty-- no machinery of its own, just a string of genetic material, like DNA. Like, in fact, an instruction manual. Alone, it can do nothing. It has to hijack a living cell and turn it to its own purposes. It's only by using something else's biological machinery that a virus can repeatedly clone itself before bursting out and infecting countless more cells in a destructive chain reaction.

Video 4 Why different drugs are needed to treat bacterial and viral infections. HOST: These essential differences mean that we have to use very different weapons for fighting viruses and bacteria. Of course, one big weapon in a doctor's toolkit or medicine bag is their antibiotics. There are several different types of antibiotics, and because they work in subtly different ways, it means they're a tremendously versatile drug. What almost all antibiotics have in common is the ability to cripple a particular function of the bacterial cell. Now, there are many ways of doing this. [BUZZING] [ROCK MUSIC] With so many parts to attack, antibiotics can disable bacteria in many different ways. Whereas with a virus, there's nothing to disable. This is just the wrong tool for the job, which is why antibiotics are useless for viruses. So unless you have a bacterial infection, there is no point your doctor prescribing antibiotics. Nine times out of 10 with coughs and colds, it's a virus that's causing the problem. Drugs to combat viruses work in a totally different way. Most anti-viral drugs need to physically block the virus from getting into or out of the cell it needs in order to replicate. That should do it.

5.2 Wrong therapeutic use Antibiotic resistance is more likely to develop when antibiotics are used at too low a dose or taken for too short a time. In the next activity, you will explore the effect of treatment duration on gut bacteria. Activity 6 Effect of antibiotics on gut bacteria Allow about 20 minutes You might recall from Week 1 the typical growth pattern of a bacterial population (Figure 8 below). The death phase does not occur in the gut. This is because of the steady flow of material from mouth to anus, so that new food is always added and waste products are always removed. In the gut, the only time a population will decline like this is if something – for example, an antibiotic taken by mouth – kills it.

When a person swallows pathogenic bacteria, whether they become ill depends on the type of bacterium. For some types, only a few bacteria will cause illness. For other bacteria, millions must be taken in to cause any harm. The number of bacteria needed to cause illness is called the infectious dose. Now click on the image below to be taken to an interactive activity. There is a risk of resistance developing every time an antibiotic is used because only resistant bacteria can survive and reproduce in the presence of antibiotics. This process is called selection. The resistant bacteria can then pass their resistant genes to other bacteria. If the dose of antibiotic is too low, selection ensures that a few resistant bacteria will survive. Although the patient may start to feel better, the surviving bacteria will soon multiply, symptoms will return and the antibiotic will no longer be effective at the original dose used. The concentration of antibiotic within the body decreases with time. Why might failing to take an antibiotic regularly as prescribed make it less effective? Failure to maintain the antibiotic at a high enough level to kill all the bacteria allows an opportunity for resistant bacteria to be selected and resistance to develop So far this week you have focused on human behaviours that promote antibiotic resistance. You should by now appreciate the importance of using antibiotics correctly. In the next two sections, you will look at other facets of the problem. First, you will learn why we are running out of options to treat antibiotic-resistant infections.

6 The antibiotic discovery void The last class of antibiotic approved for clinical treatment was the lipopeptides, such as daptomycin, in 1987 (Figure 9).

A combination of factors is responsible for the ‘discovery void’. It can take between 12 and 15 years to develop a new drug and to clear regulatory hurdles, at a cost of around £1.5 billion (GSK, 2018). The number of novel antibiotics discovered through screening large numbers of soil bacteria has fallen considerably and synthetic approaches have been disappointing. Due to these practical and financial difficulties, many pharmaceutical companies closed their antibiotic development programmes after the 1980s. You will learn more about the barriers to developing new antibiotics in Week 6. Of the 51 potential new antibiotics currently in development, only ten are expected to be licensed for clinical use within ten years (WHO, 2017a). A lack of new drugs is not the only problem though, as you will discover next. 7 Inadequate diagnostics and global surveillance Antibiotic resistance is a global problem which requires a global collaborative approach to combat it. Well-equipped laboratories working in tandem with good surveillance systems can identify resistant isolates and reveal trends and outbreaks of infection. The information can then be used to inform treatment guidelines and clinical decision making, and so rationalise the use of antibiotics. Unfortunately, the laboratory capacity and surveillance systems of many countries, particularly low-income countries (LICs), are inadequate or non-existent (Figures 10 and 11).

Many of the themes discussed this week are brought together in the final section. 8 Case study: Neisseria gonorrhoeae In this week’s case study, you will look at how Neisseria gonorrhoeae, which causes gonorrhoea, is evolving into a superbug. Third-generation cephalosporins, also known as extended spectrum cephalosporins (ESCs), are the lynchpin of current therapy for gonorrhoea. But resistance to ESCs is emerging. The next activity will give you an overview of this topic. Activity 7 Antibiotic-resistant N. gonorrhoeae in the USA Allow about 10 minutes First, watch the following video about the emergence of antibiotic-resistant gonorrhoea in the USA. The video was produced by the USA’s foremost health protection agency – the Centers for Disease Control and Prevention (CDC). Video 5 Drug-resistant gonorrhoea: an urgent public health issue. NARRATOR: Gonorrhoea is a major public health concern in the United States. More than 800000 new infections occur each year. But because many people don't have symptoms, fewer than half are detected and reported to CDC. Untreated gonorrhoea can cause serious health problems. For women, it can increase their risk for a life-threatening ectopic pregnancy. And for men and women, the infection can cause conditions that can lead to infertility. It can also increase a person's risk of getting or giving HIV. Medication to treat gonorrhoea has been around for decades, but the bacteria has grown resistant to nearly every drug ever used to treat it. In the 1980s, resistance to penicillin and tetracycline grows, and they are no longer recommended to treat gonorrhoea. Fluoroquinolones are the leading drugs to treat gonorrhoea in the 1990s, but the bacteria was adapting to the drugs. By the 2000s, resistance to fluoroquinolones steadily takes hold. CDC modifies treatment recommendations throughout much of the decade to keep pace. In 2000, the drug is no longer recommended to treat people infected in Asia or the Pacific Islands. By 2002, this recommendation extends to California. By 2004, CDC no longer recommends it for men who have sex with men in the United States. By 2007, resistance is so widespread that CDC no longer recommends fluoroquinolones to anyone in the US to treat gonorrhoea. Only one class of antibiotics, known as cephalosporins, remains to treat the infection. There are two main cephalosporins to treat gonorrhoea-- the oral drugs Cefixime and the injection Ceftriaxone. In 2010, CDC takes additional measures to combat resistance, recommending dual treatment with either Cefixime or an increased dose of Ceftriaxone and Azithromycin or Doxycycline. But just two years later, in 2012, CDC updates treatment recommendations again in response to data suggesting the oral cephalosporin Cefixime is becoming less effective. And Gonorrhoea has become harder and harder to treat. Today, we are down to one last recommended treatment option-- dual treatment with an injection of Ceftriaxone and an oral dose of Azithromycin. Little now stands between us and untreatable Gonorrhoea. There are already troubling signs with the last recommended treatment. Abroad, infections resistant to Ceftriaxone have been detected in several countries. And within the US, while not common, resistance to Azithromycin has been found. The US hasn't seen treatment failure when using these two drugs together, but drug resistance is rising, and the pipeline for new drugs is shrinking. Our last treatment option won't last forever. We must keep drug-resistant Gonorrhoea as a leading priority. Learn more at cdc.gov/std/Gonorrhoea/arg.

Now answer the following questions, based on the video. Which two cephalosporins were introduced in the 2000s to treat gonorrhoea? When were cephalosporin-resistant N. gonorrhoeae first detected? In 2018, only one therapy was recommended for gonorrhoea. What was it? Why does the narrator predict that the last treatment option ‘won’t last forever’? Cefixime and ceftriaxone. In 2012. Dual treatment with ceftriaxone and azithromycin – a macrolide antibiotic. N. gonorrhoeae has developed resistance to every antibiotic used to treat gonorrhoea. Reports of isolates resistant to ceftriaxone or azithromycin are expected to become more frequent. ESC-resistant gonorrhoea has been reported in many other countries, as you will see in the next activity. Activity 8 ESC-resistant gonorrhoea Allow about 5 minutes WHO recommends not using an antibiotic in cases where resistance levels are 5% or higher and the bacterial pathogen is unknown. Would this apply to any countries in Figure 12?

Yes. ESC-resistance is at least 5% in over 20 countries. Using ESCs in these circumstances increases the likelihood of resistance developing and spreading. Although resistance to ESCs is low level and emerging, resistance to azithromycin is widespread. Gonorrhoea superbugs, which are resistant to ESCs and azithromycin, have already been isolated from Japan, France and Spain. (WHO, 2017c). In the final activity this week, you will look at the scale of the problem and the challenges faced in trying to bring the situation under control. Activity 9 The rise of antibiotic-resistant gonorrhoea Allow about 15 minutes First, read the article below about the global challenge of treating gonorrhoea effectively because of antibiotic resistance. Article 1: The world is running out of antibiotics, WHO report confirms (WHO, 2017c). Now answer the following questions, based on the article. How many people become infected with gonorrhoea each year? Why is this probably an underestimate? What percentage of N. gonorrhoeae isolates are ESC-resistant? How many countries have reported resistance to cefixime or ceftriaxone? Why might antibiotic resistance increase in the absence of suitable diagnostics? How many new drugs are currently being developed to treat gonorrhoea? About 78 million people worldwide. LICs, where gonorrhoea is thought to be more common, do not have reliable diagnostic and reporting systems. 66% Over 50 countries have reported ESC resistance. Asymptomatic cases are undiagnosed and untreated, and the infection spreads. Conversely, patients presenting with gonorrhoea-like symptoms may be presumed to have the disease and be prescribed unnecessary or incorrect antibiotics. Only three drugs are in clinical development. Globally, gonorrhoea is becoming increasingly difficult to treat as a result of antibiotic resistance. High infection rates result in high rates of antibiotic use. Inadequate diagnostics and poor monitoring and surveillance of AMR encourage the misuse and overuse of antibiotics. New antibiotics are urgently needed. The threat that gonorrhoea may again become impossible to treat is very real. 9 This week’s quiz Well done – you have reached the end of Week 5 and can now do the quiz to test your learning. Week 5 practice quiz Open the quiz in a new tab or window (by holding down Ctrl [or Cmd on a Mac] when you click the link). Return here when you have finished it. 10 Summary This week you learned how human behaviour has encouraged the spread of infections and antibiotic resistance. You should now be able to explain why the misuse and overuse of antibiotics leads to the selection of resistant bacteria and why resistant infections are becoming an increasingly serious global health problem. You should now be able to: describe the scale and nature of antibiotic resistance worldwide summarise how antibiotic resistance spreads explain how the overuse and misuse of antibiotics contribute to bacterial resistance list the factors which have prevented new antibiotics coming onto the market recognise how a lack of laboratory capacity and inadequate surveillance contribute to the development and spread of antibiotic resistance. Next week, you will look at how the problem of antibiotic resistance can be tackled by developing novel antibiotics or by making existing antibiotics more effective. You can now go to Week 6. Week 6: Restocking the antibiotic armoury Introduction In Week 5, you found out how and why antibiotic resistance has become a serious global, public health problem. You also learned about the types of behaviour that encourage the development and spread of antibiotic resistance. This week, you will look at one approach to tackling the crisis – replenishing the depleted stock of drugs used to treat antibiotic-resistant infections. There are two options: to make new types of antibiotic or to make existing antibiotics more effective. As you will discover this week, scientists and pharmaceutical companies must overcome many challenges in order to bring new drugs to the market. It is also worth bearing in mind the part that serendipity can play in this process. Start this week by watching the video below about the accidental discovery of penicillin. Video 1 Alexander Fleming’s discovery of penicillin. NARRATOR: Having been brought up on a farm in Scotland, scientist Alexander Fleming wasn't afraid of getting his hands dirty, examining nasty bacteria, like Staphylococcus aureus, which in humans as well as horses can cause death as well as vomiting and boils. One day in 1928, Fleming came back from his holidays, he found some cultures of the Staphylococcus aureus bacteria, which he'd meant to throw away, had died. But instead of throwing them away, he stopped to think what might have caused some of his sample to die and the rest to live. After a lot of time and effort in his lab, Fleming worked out that some of his sample had been contaminated by a particular fungus, which he then managed to grow himself. As an ex-soldier in World War I, he'd seen hundreds of soldiers die due to bacterial infection. And he figured that if the fungus could kill bacteria on his bench, it might also kill bacteria in wounded soldiers. And he was right. Having renamed his mould juice penicillin, it was ready for public consumption in time for the next war on D-Day. Penicillin has saved the lives of millions of people and horses.

By the end of this week, you should be able to: recall key events in the history of antibiotics explain how antibiotics are discovered and produced describe the current antibiotic armoury give reasons for the decline in the production of antibiotics outline approaches to make existing antibiotics more effective identify potential sources of novel antibiotics. 1 Origins of antibiotics You might recall from Week 1 that some antibiotics are produced naturally by bacteria or fungi (moulds) while others are semi-synthetic or fully synthetic. You will explore these terms further in the following sections.

1.1 Natural antibiotics Most natural antibiotics were discovered before the 1970s, using systematic non-target-based screening of soil samples. Relatively few antibiotics in use today are completely natural. Of these, about 20% are produced by fungi and 80% by a group of Gram-positive, filamentous soil bacteria called Streptomyces (Lo Grasso et al., 2016). Two types of fungi – the Penicilliums and the Cephalosporiums – have proved good sources of antibiotics. For example, Penicillium notatum (Figure 1) was the source of the original penicillin discovered by Alexander Fleming in 1928. Cephalosporium acremonium gave rise to the first-generation cephalosporins (Clegg, 2015) .

Natural antibiotics isolated from Streptomyces bacteria (Figure 2) include streptomycin, tetracycline, vancomycin, erythromycin and chloramphenicol (de Lima Procopio et al., 2012).

1.2 Synthetic and semi-synthetic antibiotics The very first antibiotics were discovered by screening large numbers of existing compounds from collections of chemical compounds known as chemical libraries. These were arsenic derivatives in the case of Salvarsan in 1909, and azo-dyes for sulfonamides in the 1930s. More targeted screening of chemical libraries later became the norm, such as looking for inhibitors of bacteria-specific metabolic pathways. This is how synthetic carbapenems were discovered (Silver, 2011). Semi-synthetic antibiotics are derivatives of natural antibiotics with slightly different but advantageous characteristics. For example, they can act against bacteria which are resistant to the original compound, have a greater spectrum of activity or cause fewer side effects. Semi-synthetic derivatives of penicillins and cephalosporins are known as generations. You will find out more about cephalosporin generations in this week’s case study. In the next section, you will find out how antibiotics are produced on an industrial scale.

2 The manufacturing process For antibiotics to help the masses of people who need them, production needs to be on an industrial scale. Different manufacturing processes are used for natural, synthetic and semi-synthetic antibiotics.

2.1 Producing natural antibiotics Natural antibiotics are complex chemicals which are synthesised stepwise by the bacteria or fungi that produce them in a series of enzyme-catalysed reactions. The starting compounds are usually products of the cell’s mtabolism – chemical reactions that allow a cell to obtain the energy and nutrients it needs to grow and survive. These essential compounds, or primary metabolites, are made in the exponential phase of growth. Antibiotics, however, are secondary metabolites, that is they are products of metabolism that are not essential for growth. You might recall from Week 1 that bacteria and fungi produce antibiotics to prevent competing organisms using nutrients and other resources. Why aren’t antibiotics produced during the exponential stage of growth? The exponential stage is when abundant resources allow rapid growth, so only primary metabolites are made. During the stationary phase, competition for nutrients increases and resources are diverted away from growth to make antibiotics. In manufacturing, pure cultures of antibiotic-producing bacteria and fungi are grown in huge bioreactors (containing thousands of litres) in a process known as batch fermentation. Batch fermentation favours antibiotic production by limiting the time that cells spend in the exponential growth phase. The antibiotic products are then harvested and purified. As you will discover in Activity 1, this process was not always so streamlined. Activity 1 Commercial production of penicillin – Part 1 Allow about 10 minutes When Alexander Fleming accidentally discovered penicillin in 1928, he had no idea what to do with it or how to reproduce it. This was left for other researchers to do. First, watch the video below about how penicillin was ‘rediscovered’ ten years later by Howard Florey and his Oxford-based research team. Video 2 Penicillin rediscovered. WOMAN: Behind this door is the original lab of the pioneering Oxford scientist Howard Florey. And it was in here that a team of brilliant minds turned Fleming's rather unexpected discovery into a miracle cure. This is the room where antibiotics were truly invented at the dawn of the Second World War. The team invented a way to purify the mould juice by combining it with ether and alkalines that drew away the harmful elements, creating an antibiotic pure enough for humans to take. Scientist and historian Dr. Eric Sidebottom was a pupil of the men involved. Clearly, if it was going to do any good at all to the masses it had to be mass produced. So were they able to develop penicillin here in Great Britain? DR. ERIC SIDEBOTTOM: Yes, to some extent they were, but Florey was always worried that he couldn't really persuade the British pharmaceutical industry to get involved. They were already committed to the war effort. So Florey made this difficult decision to take the problem to America. And the Americans did help. They increased production very considerably. They found a better strain of penicillium on a local melon in the market. They also managed to get it growing in a huge suspension tank, in a big tank. Whereas in Oxford, we'd grown it in bedpans.

Now answer the following questions, based on the video. What was the first key thing that Florey’s team achieved with penicillin? Why were British pharmaceutical companies reluctant to help develop penicillin? What did the American pharmaceutical companies achieve with penicillin? The penicillin was purified to a level that was safe for use in humans. In the late 1930s, British companies were prioritising the war effort. They increased production and isolated a more powerful Penicillium mould. In the UK, Florey’s team had managed to purify penicillin and treat a bacterial infection. This was a major achievement, but the team was hampered by a lack of funding and equipment, and yields of the drug remained poor. After the move to the USA, the research picked up pace. By 1943, the production of penicillin was under way with the new, more powerful strain growing in a different medium – corn syrup. However, the production process was still inefficient and yields of penicillin remained low. The second part of Activity 1 looks at the changes made to the manufacturing process that led to much higher yields of penicillin. Activity 1 Commercial production of penicillin – Part 2 Allow about 10 minutes The video below explains how the key to increased productivity was growing the Penicillium mould in a liquid, rather than as a layer at the bottom of a large flat-bottomed flask. As you watch the video, consider the following questions. What was the advantage of a liquid medium? Why was it critical to control the level of oxygenation? Video 3 Mass production of penicillin. [MUSIC PLAYING] MICHAEL MOSLEY: But despite these improvements, the inefficiency of the actual production process meant that by 1943 there were still only enough penicillin to treat a lucky few. MAN 1: The standard techniques were large white bottom flasks because you had a single layer of mould producing, the yields from each flask were minimal. MICHAEL MOSLEY: And the need for penicillin had never been more urgent. D-day, the greatest amphibious invasion in history, was only months away. Casualties were going to be horrific. MAN 2: Penicillin was the US military's second top research priority after the Manhattan Project, after nuclear weapons. MICHAEL MOSLEY: It was then that a small chemical company based in this building in Brooklyn, called Pfizer, got involved. Now, these days, Pfizer is better known for its anti-impotence drug, Viagra. But back then, they produced citric acid used in fizzy drinks. Now, they realised, as everyone else had, that if you just grow penicillin on the surface of a liquid then that is going to be really inefficient. What you want to do is grow it throughout the liquid. The problem is that penicillin needs oxygen to grow. So they came up with a solution which they hoped would work. MAN 1: The oxygenation came with a tube introduced into the medium into which oxygen was pumped. But you couldn't put too much, and you couldn't put too little. So learning how much was right was key. MICHAEL MOSLEY: There was absolutely no guarantee the technique would work. But the company took a gamble. Because of wartime shortages, they had to convert an old Brooklyn ice factory, scrounging a boiler from Indiana and a lift from Long Island. With just two months to go before D-day, they installed 14 giant fermentation tanks. Then they added the corn syrup and the cantaloupe mould before turning on the air. The results were spectacular. They soon began producing five times as much penicillin as originally planned. By June 1944, the D-day invasions, there was enough penicillin for every injured soldier. And most of it was produced right here in the Pfizer plant in Brooklyn.

The liquid culture allowed more Penicillium to be grown and more penicillin to be produced. If the oxygen level is too low, Penicillium will die because it needs oxygen to grow. Too much oxygen can be toxic to fungi and bacteria – as it can be to humans.

2.2 Producing synthetic and semi-synthetic antibiotics Antibiotics are very complex molecules. Synthetic compounds that resemble or mimic a natural antibiotic are rarely made for this reason. An exception is chloramphenicol (Figure 3). Novel synthetic antibiotics can be made in laboratories from scratch using a multi-step process that starts with the requisite chemical building blocks and ends with the pure compound. However, this process involves considerable development time and production costs.

Semi-synthetic antibiotics represent a half-way house. They are made by chemically modifying the active part of a natural antibiotic to create a single new molecule. A large amount of natural antibiotic is produced by batch fermentation. Then it is purified and chemically modified to create new antibiotics with enhanced therapeutic activity. Promising compounds identified by screening chemical libraries can similarly be modified to enhance activity and safety. Next, you will find out how many existing antibiotics are still effective against bacterial infection.

3 Current status of antibiotics The heyday of antibiotics was from the 1940s to the 1960s and 1970s. This was a time when new antibiotic classes were identified and approved for clinical use, and antibiotic resistance was under control. Since then, the stock of antibiotics able to cure bacterial infections has been seriously depleted. Antibiotic resistance has soared and the development of new drugs has not kept pace with the need to replace those which no longer work (Figure 4).

In recent years, two potential new classes of antibiotic have been discovered from soil bacteria: teixobactin in 2015 (Ling at al., 2015) and the malacidins in 2018 (Hover, 2018). In the next activity, you will explore how quickly bacteria develop resistance to newly introduced antibiotics. Activity 2 Antibiotic activity and resistance timeline Allow about 10 minutes Review Figure 5 and then answer the questions below.

Which antibiotic class had the longest interval between its introduction and resistance appearing? What can you say in general about how long it takes resistance to develop once an antibiotic is brought into clinical use? In some cases, such as penicilin, resistance to the antibiotic is observed before use of the drug becomes widespread. Why is this? It took 13 years before resistance to imipenem, the first carbapenem, to be observed. Resistance can develop quickly once an antibiotic becomes widely used. In most cases, resistance develops within two years of the antibiotic being introduced. As you learned in Week 3, bacteria may have an intrinsic resistance to an antibiotic. It is not too surprising then that resistant bacteria are observed during the period between the initial discovery of an antibiotic and the point at which it is approved for clinical use. Research and development into new drugs is an essential component of strategies to tackle the antibiotic resistance crisis. Unfortunately, as you will see in Section 4, this is not straightforward. 4 Barriers to new antibiotics – and possible solutions In Week 5, you were introduced to some of the challenges involved in creating new antibiotics. Complete Activity 3 to see how many you can remember. Activity 3 Factors which account for the antibiotic discovery void Allow about 5 minutes In Week 5, you briefly considered the factors which account for the antibiotic discovery void. Which factors can you remember? You might have recalled that new drugs are rarely now discovered by screening soil samples or chemical libraries. Pharmaceutical companies are deterred by the financial costs and regulatory hurdles involved and many no longer have the research capacity to develop new antibiotics. There is belated recognition by governments, public health agencies, medical communities and others that the barriers to antibiotic development must be removed and the antibiotic pipeline rebuilt. New regulatory policies and financial incentives to address this problem are being proposed (O’Neill, 2016), but barriers still remain.

4.1 Discovery barriers After initial successes, non-target-based screening of soil samples soon became unviable. Known natural antibiotics were continually being ‘rediscovered’, making it difficult to identify promising new compounds. Meanwhile, the screening of chemical libraries faltered because their random contents did not lead to compounds with desirable characteristics. However, new technologies may revive interest in both types of screening. Transcription profiling is a technique that identifies which genes are being expressed by bacteria. Profiling samples from soil or other microbe-rich environments is a quick way of distinguishing between rediscovered and novel antibiotics. Chemical libraries could also be created which are predisposed to generate compounds with antibiotic-like attributes.

4.2 Scientific barriers The biggest barrier to new antibiotics may be a lack of investment in the basic, multidisciplinary research that underpins drug discovery and development (Figure 6).

Research has focused on identifying compounds that inhibit essential bacterial processes, for example cell wall synthesis. However, other key attributes of antibiotics are poorly understood, hampering efforts to discover new types of drug. The lack of new antibiotics to target infections caused by Gram-negative bacteria is particularly troubling because these are very difficult to treat. Can you recall from Weeks 2 and 3 the features of Gram-negative bacteria that make them intrinsically resistant to antibiotics? The outer membrane of Gram-negative bacteria is impermeable to many antibiotics. Inside the cell, efflux pumps transport toxic substances, including antibiotics, out of the cell. Understanding the unique characteristics of antibiotics that allow them to penetrate cells, and to accumulate within in high concentrations, will enable focused chemical libraries to be created. It will also facilitate targeted screening programmes. An alternative to discovering new antibiotics is to make existing drugs more effective. You will find out more about this in the next section.

5 Making existing antibiotics more effective Given the challenges in bringing new antibiotics to the market, it makes sense to make existing antibiotics work again. It may also be cheaper and quicker. For example, simply using a combination of three antibiotics can overcome resistance, even if each drug is no longer effective when used on its own (Graham, 2016). In this section, you will look at other ways in which antibiotics can be made more effective.

5.1 Resistance breakers Existing antibiotics can be made more effective by co-administering antibiotics with resistance breakers – drugs that do not kill bacteria themselves but instead help an antibiotic to overcome resistance. One advantage of using failing antibiotics is that they have already been safety-tested, so development time and costs will be lower. However, this may only be a short-term fix because resistance is likely to develop to these drug combinations too (Garner, 2016).

5.2 Nano-encapsulation A new but promising area of research is to encapsulate an antibiotic in a polyester polymer to create a nanoparticle, between 1 and 100 nanometres in size. (A nanometre (nm) is a unit of length equal to one billionth of a metre.) Nano-sized antibiotic carriers can kill bacteria more effectively than unencapsulated antibiotics. This may be because the transportation of nanoparticles to the site of infection is more efficient, allowing higher concentrations of antibiotic to build up. Another possibility is that the nanoparticles somehow protect the antibiotic from bacterial resistance mechanisms (Friedrich-Schiller-Universitaet Jena, 2017).

5.3 Chemical modification For many years, scientists have chemically modified antibiotics incrementally, broadening the spectrum of activity and increasing effectiveness and usability. This usually reverses antibiotic resistance at the same time. This happens with the cephalosporins, which are discussed in Section 6. In the next activity, you will learn about a single, specific modification that could potentially reverse resistance by making the antibiotic more powerful. Activity 4 Making antibiotics more powerful Allow about 15 minutes First, read the short article below about a promising new way of overcoming antibiotic resistance. Then complete the table comparing the characteristics of the two antibiotics vancomycin and oritavancin. Article 1: ‘Brute force’ can overcome antibiotic resistance (UCL, 2017).

Characteristic	Vancomycin	Oritavancin
Therapeutic use
Killing mechanism
Time taken to kill a bacterial cell

Characteristic	Vancomycin	Oritavancin
Therapeutic use	Last resort treatment for MRSA	Complex skin infections
Killing mechanism	Disrupts vital cellular processes	Clusters latch onto bacterial surface and then push apart, generating a strong force that ruptures the cell.
Time taken to kill a bacterial cell	6–24 hours	15 minutes

Now answer the following questions, based on the article. Which is the more powerful antibiotic and why? How do the scientists plan to use their findings in future research? Oritavancin is more powerful. The force used by oritavancin to push into a bacterial cell is 11 000 times more powerful than that of vancomycin. To help inform the design of new antibiotics, and to make similar modifications to existing antibiotics, in order to make them more powerful Next, you will look at the modifications that have been made to different generations of cephalosporins.

6 Case study: cephalosporin antibiotics In this week’s case study you will learn about the history and development of cephalosporin antibiotics. You might recall from previous weeks that these are broad-spectrum, bactericidal, ß-lactam antibiotics. The story starts in Italy where the first cephalosporin, cephalosporin C, was discovered in cultures of C. acremonium found growing in a sewer near the Sardinian coast. You can find out more in Activity 5. Activity 5 The history of cephalosporins Allow about 15 minutes First, listen to the following audio recording about the discovery and development of cephalosporins. Audio 1 Discovery and development of cephalosporins. NARRATOR It may not surprise you to hear that a story which starts knee-deep in sewage will end with the spread of drug-resistant infections like MRSA and c-dif. But this story doesn't take an obvious path, meandering as it does through the history of medicine, along paths paved with the best of intentions, setting the scene for what may be the biggest health challenge mankind has faced since the dawn of medicine. This is the story of cephalosporins, a diverse class of structurally similar antibiotics. But back to the sewage. As you might imagine, a sewer system is a rich microbial ecosystem, fed with human waste and plenty of water. This gives rise to interesting interactions, where many species fight among the faeces, vying for dominance and continually evolving new ways to stay on top. As a result, the chemical mecanisms developed by some of these species make ideal drug candidates. In 1948, Italian scientist Giuseppe Brotzu was examining fungal cultures extracted from a sewer in Sardinia. Although penicilin had been discovered twenty years earlier, it only became regularly used to treat infections in 1942. So the idea that fungi could provide useful antibacterial drugs was still relatively new and exciting. He noticed that cultures of the fungus Cephalosporium acremonium supressed the growth of Salmonella typhi, the infectious agent responsible for typhoid, and that a crude filtrate from the fungus could also inhibit the growth of Staphylococcus aureus. In Oxford, Guy Newton and Edward Abraham, the latter best known for his contribution to the development of penicillin, took the cephalosporin story to the next chapter. They isolated and refined compounds from the culture, removing side chains until they fell upon 7-aninocephalosporic acid, or 7-ACA, the nucleus of cephalosporins. This, much like 6-aminopenicillanic acid with penicilin acts as the starting block for a host of derivatives, the mother to several generations of drugs. Cefolotin, the first of the cephalosporins to reach the market, was first released in 1964 and continues to be used today. Along with penicillin, monobactams and carbapenems, cephalosporins sit in a class known as the beta-lactam antibiotics. They share a similar mode of action, inhibiting cell wall development and leading to cell disruption and lysis. First-generation cephalosporins in common with other early drugs in their class were only really effective against gram-positive bacteria, which includes the bugs modified to increase oral availability and plasma stability, as well as to improve activity against gram-negative bacteria. There are just two drugs in the fifth generation: Ceftobiprole and Ceftaroline. But they account for the only drugs of their type that are effective against methicillin-resistant Staphylococcus-aureus, or MRSA. So cephalosporins are the drugs that could, but for a score of years, have been penicillin, and Brotzu's name may have been as well known as Flemming's. They've certainly attracted wide use having become a major part of the antibiotic arsenal of most hospitals in developed nations, both as a treatment and a prophylactic to prevent infection post-surgery. But instead of the fame and glory, cephalosporins have attracted critism and are considered by some to be the driving force behind the development of drug-resistant bacteria. In a 2001 article in the Journal of Antimicrobial Chemotherapy, Stephanie Dancer of the Vale of Leven District Hospital in Dunbartonshire examined the evidence that 'Cephalosporin usage is the most important factor in the selection and propogation of microorganisms, such as Clostridium difficile, methicillin-resistant Staphylococcus aureus, penicillin-resistant pneumococci, multiply resistant coliforms and vancomycin-resistant enterococci, the continuing increase of which threatens the future of antimicrobial therapy.' Dunbarton goes on to explain that because cephalosporins are effective against a broad spectrum of pathogens, they are often deployed early before lab tests have confirmed the specific cause of an infection. But they're not effective against all common infectious bacteria, and those unaffected therefore have opportunity to overgrow. For known pathogens, this obviously leads to secondary infections, but it can also alter the balance of your microbiota so much that low risk or even friendly bacteria can become a problem - when allowed to overgrow Candida albicans causes yeast infections and Clostridium difficcile, which can exist without issue in the gut for years, can become a killer. Dunbarton goes on to conclude 'the selection pressure created by heavy usage of cephalosporin antibiotics over the last twenty years has generated a pleathora of multiply-resistant organisms. The risks posed by overuse of cephalosporins remain only speculative, unless specific proof is forthcoming. By then though we may be contemplating the post-antibiotic era.' So a drug that started its story in a sewer and went on to find glory treating and preventing infection may be one of the causes of two major changes in the history of medicine. Now answer the following questions, based on the audio recording. Who discovered the first cephalosporin? What antibacterial activity did a crude extract derived from C. acremonium demonstrate? Which chemical structure is described as the ‘nucleus’ of cephalosporins and why is it significant? What is the difference between the first and later generations of cephalosporins in terms of their spectrum of activity? The Italian scientist Giuseppe Brotzu (1895–1976). The extract could suppress the growth of Salmonella typhi and Staphylococcus aureus. The starting point for all cephalosporin derivatives (generations) is 7-aminocephalosporic acid, or 7-ACA. First-generation cephalosporins are only effective against Gram-positive bacteria. The later generations have increasing activity against Gram-negative bacteria. Guy Newton and Edward Abraham were interested in cephalosporin C because, although it was a weak antibiotic, it was resistant to ß-lactamase.This is the bacterial enzyme and resistance factor that can inactivate ß-lactam antibiotics. The drive behind the experiments that resulted in 7-aminocephalosporic acid (7-ACA) was to create a chemically modified derivative of cephalosporin C with enhanced antibacterial activity and intact ß-lactamase resistance. Newton and Abraham found that modifying cephalosporin C could effect the desired change, as long as the 7-ACA ‘nucleus’ containing the ß-lactam ring remained intact (Figure 7).

To this day, all cephalosporins are semi-synthetic and are derived from cephalosporin C via 7-ACA. Batch fermentation of the natural antibiotic produces vast quantities of the drug which is converted to the 7-ACA intermediate before being further modified to produce the range of cephalosporins on the market (Wright et al., 2014).

6.1 Different generations of cephalosporins There are five generations of cephalosporins. Figure 8 shows how two chemical groups of 7-ACA, the acetyl group and the acylamino side chain can be modified, leaving the ‘nucleus’ intact. Do not worry if you are not familiar with these chemical structures. For this course you should just be aware that these modifications give rise to different generations of cephalosporins with a different spectrum of activity. If you would like to know more about chemical reactions you might like to try our free OpenLearn course Discovering chemistry. For examples of each generation, see Figure 9.

Each successive cephalosporin generation has improvements in the spectrum of activity and in some pharmacological properties. This greatly expands the clinical uses of these drugs. The later generations are sometimes called ‘extended spectrum cephalosporins’ (ESCs). In Activity 6, you can compare the characteristics of different generations of cephalosporins. Activity 6 Characteristics of cephalosporin generations Allow about 10 minutes Table 1 below summarises the characteristics of different cephalosporin generations. Table 1 Spectrum of activity of different cephalosporin generations(Based on Reygaert, 2011; Wright et al., 2014)

Cephalosporin generation	Activity against:			Resistance to:		Examples
Cephalosporin generation	Gram-positive	Gram-negative	MRSA	ß-lactamase	ESBLs	Examples
1	++++	+	no	+	no	cephalothin cefazolin
2	+++	++	no	++	no	cefamandolecefaclor
3	++	+++	no	++	no	cefiximeceftriaxone
4	++++	++++	no	+++	no	cefepimecefclidine
5	++++	++++	yes	+++	no	ceftobiprole

Key: + = trace amount; ++ = small amount; +++ = moderate amount; ++++ = large amount. Review the table and then answer the following questions. Which generation has the lowest activity against Gram-negative bacteria and which has the highest? With each successive generation, what do you notice about the activity against Gram-positive and Gram-negative bacteria? Which generation(s) have the greatest resistance to ß-lactamases? Are any cephalosporins resistant to ESBLs? Which cephalosporin has activity against MRSA? The first-generation drugs have the lowest activity against Gram-negative bacteria; the fourth and fifth generations have the greatest. The first-generation cephalosporins had good activity against Gram-positive bacteria but poor activity against Gram-negative bacteria. Activity against Gram-negative bacteria improved with second and third generation drugs, but at the expense of activity against Gram-positive bacteria. The last two generations of cephalosporins have good activity against both Gram-positive and Gram-negative bacteria. The fourth and fifth generations No. Resistance to ESBLs, particularly those produced by Gram-negative bacteria, is becoming a serious problem. Fifth generation cephalosporins, like ceftobiprole, are active against MRSA. The chemical evolution of cephalosporin C via 7-ACA into over 30 new broad-spectrum antibiotics was a breakthrough in the fight against antibiotic resistance. Unfortunately, the widespread practice of using cephalosporins for empiric treatment, that is treatment without a definitive diagnosis, may have selected for multi-drug-resistant bacteria and encouraged the spread of resistance (Clegg, 2015). The need for new antibiotics is urgent and, as you will see in the final section this week, scientists are looking in some unlikely places for them.

7 ‘Bioprospecting’ for new antibiotics Our planet is a rich, largely untapped source of antibiotics and other drugs. Most antibiotics in use today can be traced back to bacteria. But only a tiny proportion of bacteria have been screened for antibiotics and nearly all of them were isolated from soil. There is now renewed interest in a systematic search for natural antibiotics in every conceivable location worldwide. This is known as bioprospecting.

7.1 Back to the soil Recent technological advances and innovations have allowed a much wider range of microbes to be cultured and novel species and new metabolites to be identified. For example, Activity 7 reveals how teixobactin was discovered in 2015 by a team of scientists in the USA who managed to isolate and culture a previously unidentified soil bacterium. Teixobactin is a new class of antibiotic which is active against Gram-negative but not Gram-positive bacteria (Ling et al., 2015). Activity 7 Discovering teixobactin Allow about 15 minutes First, listen to the interview with Dr Kim Lewis, leader of the research team who discovered teixobactin. Audio 2 Discovery of teixobactin. SCIENTIST This is a very old problem in microbiology on cultured microorganisms. It's more than 100 years old. And people have tried to replicate the natural environment in the laboratory, and that actually didn't work very well. So we decided to do the exact opposite and simply grow them in their natural environment. And so the way we do that is we have a simple gadget, which we call a diffusion chamber. So we take a sample from soil, for example, dilute it, mix it up with agar, and instead of pouring it in a Petri dish, we sandwiched it between two semi-permeable membranes. And then this contraption, which we call diffusion chamber, that goes back into the soil where we took bacteria from. And so essentially, what that does, that tricks bacteria. Now, they don't know that something happened to them. Everything diffuses through that chamber. They get all the nutrients or growth factors from soil. And once they grow into colonies, then what we found is that with a high probability, they will then grow in a Petri dish. And now you can screen these organisms for their ability to make antibiotics. We've been collaborating with NovoBiotic, a startup company that does a fairly massive screening using our methods. And one of their typical sources for soil is the backyard of Lucy Ling, VP of biology. And she lives in Lexington, Massachusetts. We got a very large number of anti-microbial compounds, about 30% of soil bacteria will make anti-microbials. And so the next important step is to try to figure out which are the ones that are potentially interesting and useful. And so this new compound, teixobactin, came out of that effort. Now put the steps below in the correct order to match the culturing technique described by Dr Lewis. First Second Third Fourth Fifth Sixth Seventh Eighth What are the advantages of this new technique? It re-creates the normal growing conditions of the bacteria, allowing them to be successfully cultivated. The recovery rate by this method is 50% compared with only 1% of cells from soil samples cultured on a Petri dish (Ling et al., 2015).

7.2 Antibiotics from leafcutter ants The close relationship between South American leafcutter ants and the fungus Leucoagaricus gonglophorus, which the ants farm for food, has also opened up potential avenues of research. Watch the video below to find out more. Video 4 South American leafcutter ants. NARRATOR: Antibiotics are under threat. But salvation may, again, lie with a team of British scientists who are working to invent a brand new antibiotic using South American ants. These ants live in underground nests. For food, they grow a fungus garden made from rotting leaves and flowers. To keep their food clean, they use an antibiotic to kill any germs. DR. IAN BEDFORD: Sometimes you get a contaminant. And, of course, the ants need to get rid of that or else it will just start destroying their food source. NARRATOR: So what is it that you've discovered about what they're doing that's so exciting? DR. IAN BEDFORD: What's being found is that the worker ants actually have a bacteria that they grow on their backs. And this bacteria has very, very powerful antimicrobial properties. So when the ants discover there's a contaminant in their fungus garden, they can actually smear some of this bacteria onto the unwanted bacteria and kill it off. So by isolating that bacteria that's on their backs, this has potential for controlling all sorts of problems that we now face. NARRATOR: For instance? DR. IAN BEDFORD: The bacteria can control MRSA. NARRATOR: So are we looking here at something that could give us the first new antibiotic in, well, the best part of 40 years? DR. IAN BEDFORD: Quite possibly. And it also opens up a lot of new avenues for research.

7.3 Antibiotics from extreme environments Scientists are looking further and further afield to discover new bacterial types and antibiotics, including some extreme locations like the Atacama Desert and under the sea. The Atacama Desert (Figure 10) is one of the driest places on Earth. Some areas have just 1 mm of rain per year. This inhospitable place is home to a recently discovered bacterium – Streptomyces leeuwenhoekii. It produces metabolites called chaxamycins which have antimicrobial activity (UEA, 2016).

Bacteria living in a symbiotic relationship with marine sponges (Figure 11) produce various metabolites which are thought to protect their sponge host from predators and pathogens. Marine Streptomyces and the Salinispora, which were only discovered in 1989, are two groups of bacteria currently attracting the attention of scientists (UEA, 2016).

7.4 Looking closer to home Scientists are also bioprospecting closer to home and looking at samples taken from everyday objects and places. A bioprospecting project which recruited thousands of people to help is described in Activity 8. Activity 8 Citizen Science ‘Swab and Send’ project Allow about 10 minutes First, listen to the following interview with Dr Adam Roberts about the ‘Swab and Send’ project. Listen from 13:35 to 21:00. Now answer the following questions. What is ‘Swab and Send’? What sort of samples have the swabs come from? Have any new antibiotics been identified through this initiative? It is a crowd-funded Citizen Science project to get members of the public involved in the discovery of new antibiotics. Anything and everything, including dead bald eagles, workplace objects, faeces and train tickets. Yes. Twenty candidates have been discovered that can kill multidrug resistant E. coli and the yeast Candida albicans or MRSA.

8 This week’s quiz Well done – you have reached the end of Week 6 and can now do the quiz to test your learning. Week 6 practice quiz Open the quiz in a new tab or window by holding down Ctrl (or Cmd on a Mac) when you click on the link. Return here when you have finished it. 9 Summary This week, you learned that we have few working antibiotics left to treat bacterial infections. More and better treatment options are urgently needed, either in the form of new antibiotics or by modifying existing drugs. Unfortunately, as you should now realise, scientific, financial and regulatory barriers to the discovery and development of new antibiotics means that this is not an easy option. You should now be able to: recall key events in the history of antibiotics explain how antibiotics are discovered and produced describe the current antibiotic armoury give reasons for the decline in the production of antibiotics outline approaches to make existing antibiotics more effective identify potential sources of novel antibiotics. Next week, you will learn about another approach to tackling the antibiotic resistance crisis – reducing the use of antibiotics. You can now go to Week 7. Week 8: Alternatives to antibiotics Introduction As you should now appreciate, if we are to preserve antibiotics for the future, we need to make sure that they are used carefully and not wasted. In Week 7, you looked at two ways of reducing antibiotic use. This week, you will look at some alternatives to antibiotics. Begin this week by watching the following video, which describes how vaccines can help to tackle antibiotic resistance. Video 1 How vaccines help to beat superbugs The wider use of vaccines can help to combat antibiotic resistance because they prevent infections in humans and animals, reducing the need for antibiotics (Figure 1).

In addition to vaccines, there are many new areas of scientific research that could lead to the development of future alternatives to antibiotics. In this week, you will look at some of this research. You will focus on alternative treatments rather than alternative strategies that could be used to prevent infection. Consequently, you will not look at approaches such as the use of probiotics. Currently, none of these alternatives could replace antibiotics as a treatment for infections. However, using these alternatives in combination with antibiotics to treat minor infections could preserve antibiotics for treating life-threatening cases in the future. By the end of this week, you should be able to: identify some alternatives to antibiotics explain how inhibiting quorum sensing decreases bacterial virulence describe the advantages and disadvantages of using phage therapy to kill bacteria understand how predatory bacteria can be used to tackle infections give examples of how traditional remedies can be used to treat infections. 1 Disrupting bacterial communication Bacteria are single-cell organisms. For many years, they were thought to act as individuals and not be influenced by the bacteria around them. However, bacteria can communicate with each other in a process called quorum sensing. Activity 1 What is quorum sensing? Allow about 10 minutes Watch part of the video at the following link in which Bonnie Bassler from Princeton University describes the discovery of quorum sensing. The discovery of quorum sensing. Watch from 51:24 until 53:57. Now answer the following questions, based on the video. When do the fluorescent bacteria Vibrio fischeri fluoresce (emit light)? a) When they detect other bacteria b) When they are on their own c) Always d) Never The correct answer is (a) when they detect other bacteria. Vibrio fischeri use quorum sensing to detect the presence of other bacteria and alter their behaviour so that they fluoresce. How do the bacteria detect the presence of other bacteria? a) Using chemical messengers b) By touching each other c) Both of the above The correct answer is (a) chemical messengers. Bacteria release chemical messengers that build up as the number of bacteria increases. Above a critical level, receptors on the surface of bacteria detect the chemical messenger and change their behaviour. Which of the following statements about quorum sensing are true? (a) Quorum sensing is the mechanism that bacteria use to communicate. (b) Quorum sensing allows bacteria to synchronise changes in their behaviour. (c) Quorum sensing allows bacteria to detect the presence of other bacteria. (d) All of the above The correct answer is (d) all of the above. Quorum sensing is the process by which bacteria use chemical messengers to detect and communicate with other bacteria, in order to synchronise changes in their behaviour. As you saw in Activity 1, bacteria release chemical messengers which can be used to detect the presence of other bacteria. When the number of bacteria reaches a critical level, these chemical messengers cause bacteria to alter their behaviour (Figure 2).

1.1 Using quorum sensing to treat infections On its own, a single bacterium cannot cause an infection but, just like the bacteria in Activity 1, pathogenic bacteria use quorum sensing to coordinate their behaviour and attack together. Disrupting this coordinated response by blocking quorum sensing could help to treat infections, as you will see next. Activity 2 Preventing infections by disrupting quorum sensing Allow about 15 minutes Watch part of the video in the following link in which Bonnie Bassler explains how quorum sensing antagonists (drugs that disrupt quorum sensing) can prevent infections by Vibrio cholerae. Preventing infections by disrupting quorum sensing. Watch from 54:50 until 57:05. Now answer the following questions, based on the video. What effect would a quorum-sensing antagonist have on bacteria? a) It kills them b) It blocks their ability to communicate with each other c) It helps them to communicate with each other d) It makes them grow faster The correct answer is (b) it blocks their ability to communicate with each other. What effect does the quorum-sensing antagonist have on the fluorescence of Vibrio fischeri bacteria? a) It stops them fluorescing b) It makes them fluoresce more c) It does not have any effect The correct answer is (a) it stops them fluorescing. How many people die from Vibrio cholerae infections? a) Less than 100 000 per year b) 100 000 per week c) Over 100 000 per year The correct answer is (c) over 100 000 people per year. What is the first step in a Vibrio cholerae infection? a) Expression of a protein that causes the bacteria to fluoresce b) Expression of a virulence protein that allows the bacteria to stick to the gut c) Expression of a protein that makes the bacteria move faster The correct answer is (b) expression of a virulence protein that allows the bacteria to stick to the gut. How does blocking quorum sensing affect the expression of this protein and the Vibrio cholerae infection? a) It increases protein expression and prevents the infection b) It decreases protein expression and prevents the infection c) It increases protein expression and increases the likelihood of infection d) It decreases protein expression and increases the likelihood of infection e) It has no effect on protein expression or infection The correct answer is (b) it decreases protein expression and prevents the infection.

1.2 Using quorum sensing to reduce antibiotic resistance Vibrio cholerae are not the only pathogenic bacteria that use quorum sensing to control the expression of virulence genes. Quorum sensing in MRSA promotes the formation of biofilms which are aggregates of bacteria attached to a surface or tissue. Biofilm formation on medical devices such as catheters or respiration tubes can be a major source of infection in intensive care units (ICUs) (Figure 3). Preventing biofilm formation by blocking quorum sensing could reduce the rate of antibiotic-resistant healthcare-associated infections (HCAIs).

2 Other ways to kill bacteria Unlike quorum sensing, some antibiotic alternatives are bactericidal. Just like the bactericidal antibiotics you looked at in Week 2, these treatments work by killing the bacteria that cause infections. In Week 4, you learned about the horizontal gene transfer mechanism of transduction. In this process, DNA is transferred between bacteria via infection with viruses known as bacteriophages. Bacteriophages can also be exploited to treat infections. When bacteriophages infect bacteria, they replicate and assemble new virus particles, before lysing (bursting) and killing the bacteria, releasing the new bacteriophage particles (Figure 4).

2.1 Phage therapy Phage therapy to treat bacterial infections exploits the bactericidal lysis step of bacteriophage infection to kill bacteria. It was first developed more than 90 years ago by researchers in the former Soviet Union and is routinely used to treat chronic infections in former Soviet states such as Georgia. In the next activity, you will look at some of the advantages and disadvantages of this treatment. Activity 3 The advantages and disadvantages of phage therapy Allow about 10 minutes Listen to the following interview with Martha Clokie, a phage researcher, who discusses the advantages and disadvantages of phage therapy. While you are listening, note down any advantages and disadvantages of phage therapy in Table 1 below. You may also like to think about any ways in which phage therapy is similar to and different from antibiotic treatment. Audio 1 Interview with Martha Clokie on phage therapy. MARTHA CLOKIE There's much more of an awareness that we need to develop alternatives to antibiotics, such as bacteriophages. So there's a lot of interest there from doctors who previously were not as interested in this approach. INTERVIEWER And what about their use in Georgia? It's been pretty successful, from the sound of things. But why are we still tinkering around the edges in this country? MARTHA CLOKIE Well, that's exactly what the Georgians ask. They've used them historically, so they have a long history of using them. They have doctors who are trained to use them, and they use them in specific ways, very often. So, for example, if somebody presents with a respiratory condition that's chronic, they will be given phages. And, whereas, if they have an acute condition, they'll be given antibiotics. So they use phages to prevent antibiotic resistance, and they use them in specific ways. In the West, we still don't have an appropriate regulatory pathway for phages to be used. So there are no regulated phage products that we can use. In order for something to be regulated, you need to go through appropriate clinical trials. So we are getting there, because there are several clinical trials going on at the moment for different conditions. So that's new since we spoke last. INTERVIEWER I want to ask a historical question – why hasn't phage therapy been developed more in this country and in the States? We've known about phages for 100 years, but we've really focused on antibiotics rather than phages. Why is that? MARTHA CLOKIE Well, I think bacteriophages were just thought to be unnecessary. Antibiotics were thought to be the silver bullet. They worked. In a way, to use an antibiotic, you have to know much less about the bacteria that you're trying to treat. With phages, they have this exquisitely specific relationship between the phage and the pathogen. So if you want to treat an E. coli infection, you need to make sure your virus will target that particular E. coli strain. And I think because it was a more complicated product to develop, from the specificity point of view and also in terms of making a standard product, it is more complicated to make up a phage mixture than it is to make a pure antibiotic. That was thought that it wasn't a necessary thing. INTERVIEWER And, in Georgia, is what you're suggesting that, although they are using it and relatively effectively using phages to treat actual bacterial infections, they haven't necessarily gone through the same procedures that we would have done in terms of clinical trials and assessments? MARTHA CLOKIE That's right. And it's partly because they've always used them. So their argument has been, well, we've used them and we know they work, therefore, why do we need to do these other assessments? INTERVIEWER Which is sort of fair enough, I suppose. MARTHA CLOKIE I think we can learn, probably, in the West, we can learn a lot from the way that the Georgians use phages. And, probably, we can combine that with the way in which we want to develop antimicrobials. So I think it's good to take a real collaboration between phage scientists – people who are used to drug development – and doctors at the front end who are seeing patients that they can't treat. So it's going to be these sort of collaborations, so we can figure out how, what conditions are going to be most suitable for using phages for, how will phages be used, how will they be combined, how will we do our dosing, our formulation, our delivery. All these bits of work need to be sort of done as it were in parallel to come together. INTERVIEWER So probably in the future, most effective as part of the combined therapy for complex infections. Is there any chance that bacteria will develop resistance to phages in the same way that they have done to antibiotics? MARTHA CLOKIE Well, bacteria are really good at evolving resistance to anything. So if you chuck anything in a bacteria, it will become resistant to it. Now the advantage of phages is that, very often, the thing that the bacteriophage actually needs to hook onto is really important for that bacteria. So, for example, if it loses the surface protein on the outside to stop the bacteria from attaching to it, then it can't colonise anymore. So there's been plenty of evidence to show that often when bacteria become resistant to phages, they become less virulent pathogens. So they can't colonise and cause infection. So, at the moment, I think the first generation of bacteriophage products that will be developed will be when we have mixtures of different bacteriophages that kill in different ways. Now, if you were exposing a bacteria to phages that kill using different mechanisms, then it's much, much harder for them to become resistant. So these types of resistance studies are really important, but we can circumvent the problems that we've had using antibiotics by using them correctly and in the correct combinations to actually slow down these resistance rates.

Advantages	Disadvantages

Similarities to antibiotic treatment	Differences from antibiotic treatment

Advantages	Disadvantages
Resistance is less likely to develop (bacteria that are resistant to phage therapy are also often less virulent).	Require more information about the bacteria causing the infection because they are often very specific for a particular bacterial typesMore difficult to make than antibioticsNo regulatory pathway and very few clinical trials in the UK or the west
Similarities to antibiotic treatment	Differences from antibiotic treatment
Bactericidal	Phage therapy is specific for each bacterial strainBroad-spectrum antibiotic treatments can target many different pathogens

Historically, phage therapy has relied on identifying bacteriophages that target the infection-causing bacteria and then administering these bacteriophages to infect and lyse the bacterial cells. However, more recently, the isolation of phage-derived enzymes known as lysins has opened the possibility of developing new phage-based pharmaceuticals.

2.2 Lysin treatment Lysin treatment is similar to phage therapy because it uses the ability of phages, and enzymes derived from them, to kill bacteria by cell lysis. Phage lysins (also known as endolysins) are bacteriophage enzymes that destroy the peptidoglycan cell wall of target bacteria. This causes them to burst and release new bacteriophage particles. Like the bacteriophage they are derived from, lysins are specific for certain bacteria and can target different peptidoglycan types. Would you expect Gram-negative or Gram-positive bacteria to be more susceptible to phage lysins? You may need to revisit Week 2 Section 4.1 to remind yourself of the differences between Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, the peptidoglycan layer is protected by an outer membrane. In contrast, Gram-positive bacteria do not have an outer membrane, so disrupting the peptidoglycan layer is more likely to cause these bacteria to burst. In the next activity, you will look at data from an experiment measuring the effect of phage lysin treatment on bacteria. Activity 4 Measuring the effect of phage lysins on bacteria Allow about 15 minutes In this experiment, the presence of intact bacteria is measured using light. Turbidity is a measure of how well light passes through a liquid. If a sample contains lots of suspended particles, it will appear turbid or cloudy and light will not easily pass through it. In contrast, light will pass straight through pure water which does not contain any particles. As a result, the water will appear clear, not turbid. Turbidity can be used as a measure of the density of bacterial cells in a sample (Figure 5).

Microbiologists use spectrophotometers to shine light through bacterial samples to determine their turbidities. Samples with a high bacterial cell density will appear more turbid than samples with a low bacterial cell density. In this experiment, the turbidity of two bacterial samples was measured over 5 minutes (300 seconds) using a spectrophotometer. Sample 1 (orange line in Figure 6) was treated with a phage lysin while sample 2 (blue line in Figure 6) was not. Figure 6 shows the results of the experiment (Schmelcher et al., 2012). The turbidity of the sample is plotted on the vertical y-axis (labelled ‘normalised OD (optical density)’), while the time in seconds is plotted on the horizontal x-axis.

Study the graph carefully and then answer the following questions. What happens to the turbidity of the lysin-treated sample over time? At the start of the experiment, the sample has a turbidity of 1.0. As the time progresses, the turbidity of the sample decreases until it reaches almost 0 at around 200 seconds. What happens to the density of the bacterial cells in this sample during the experiment? At the start of the experiment, the bacterial cell density is high but, as the experiment continues, the cell density decreases Why? The phage lysin lyses the bacteria, killing them so that the sample no longer appears turbid. Does the turbidity of the sample without lysin change during the experiment? No. The turbidity of the untreated sample does not change. Why do you think this sample has been included in the experiment? This sample shows that the decrease in turbidity in the sample during the experiment is caused by the presence of lysin in the sample and not by another factor. In this section, you looked at one alternative to antibiotics that works by killing bacteria. Next you will look at research into the natural defences of a bacterium called Bdellovibrio bacteriovorus which has the potential to be exploited as another bactericidal alternative to antibiotics.

3 Exploiting the natural defences of bacteria In Weeks 3 and 4, you saw how antibiotic resistance naturally evolved to protect bacteria as they compete for limited resources in the wild. But antibiotics are just one weapon in bacteria’s defence arsenal. Some bacteria prey on other microbes, attacking and killing them. These predatory bacteria could be exploited to kill infectious pathogens as another alternative to antibiotics. The best known type of predatory bacteria is Bdellovibrio bacteriovorus (Figure 7).

Bdellovibrio bacteriovorous attaches to the surface of its prey. Once attached, it penetrates the bacterial cell membrane and replicates. Finally, the prey bacterium is lysed, releasing new B. bacteriovorous particles into the environment (Figure 8).

3.1 Treating infections with Bdellovibrio bacteriovorus In the next activity you will look at some recent research that uses Bdellovibrio bacteriovorus to treat infections in an experimental model. Activity 5 Treating infections with Bdellovibrio bacteriovorus Allow about 15 minutes Listen to the following audio clip from the BBC’s Inside Science programme. Liz Sockett from the University of Nottingham talks about research using Bdellovibrio bacteriovorus to treat Shigella infections in zebrafish. You may also like to watch Video 2 which shows a Bdellovibrio bacteriovorus bacterium (labelled in red) preying on a Shigella bacterium (labelled in green) inside a zebrafish larva. Audio 2 Interview with Liz Sockett about research with Bdellovibrio bacteriovorus. INTERVIEWER: Using one organism to kill another might not seem like such a great idea, but cats do make very good ratters. Scale things down a bit, and we might just be able to use one bacterium to kill another. Shigella is a family of bacteria that causes dysentery, and hundreds of thousands of deaths, mostly children, every year. Now good sanitation prevents Shigella infections, but there is no vaccine that stops it. Enter Bdellovibrio bacteriovorus. It's another type of bacteria, but crucially, it's a predatory one. It inserts itself inside its victims and then destroys them from within. And a new paper just published shows that Bdellovibrio can be used to cure a Shigella infection, admittedly in zebrafish, but with no apparent side effects. And because these bacteria attack a wide range of other microbes, this might just be a way of treating the impending antibiotic resistance crisis. Elizabeth Sockett is the woman behind this study. And I asked her how the experiment worked. ELIZABETH SOCKETT: The experiment's a real synergy between two labs. So Serge Mostowy's lab could set up experimental infections into zebrafish with the Shigella. And in our experiments, the Shigella were green fluorescent. They're injected into the hind brain of the zebrafish. Normally, they cause a lethal infection and the zebrafish die. What we did was we injected red Bdellovibrio into the same compartment. And we tested whether those red Bdellovibrio or a buffer control could change the infection. So the great thing about this experiment was we could see live the numbers of red and green bacteria changing inside the patient, who's the zebrafish. We could also measure the survival of the zebrafish 48 hours later, because they normally die of a Shigella infection of that level. And what we saw was that the red Bdellovibrio started to kill the green Shigella inside the zebrafish, and the survival of the zebrafish improved a huge amount. INTERVIEWER: So you inject, first of all, you infect the zebrafish with Shigella, which would kill them. ELIZABETH SOCKETT: Yes. INTERVIEWER: Then you inject-- ELIZABETH SOCKETT: With the Bdellovibrio. INTERVIEWER: And that kills the Shigella. ELIZABETH SOCKETT: Yes. And interestingly, in this experiment the Bdellovibrio start killing first the Shigella, but then the immune system of the fish that wasn't coping with the Shigella wakes up and gets a stimulus from this predation. And it comes in and clears up the rest of the Shigella from the infection. So what we think is that the Bdellovibrio is actually releasing some components of the Shigella as it's killing them early on and the immune system is being awakened to the presence of the Shigella. And also the numbers are dropping due to the direct killing by the Bdellovibrio. Video 2 Predation of Shigella by Bdellovibrio bacteriovorus inside a zebrafish larva (Willis et al., 2016).

Using the information in the interview, complete the following statements about the experiment described by Professor Sockett. The missing words are given below to help you. Bdellovibrio bacteriovorus Shigella Zebrafish (a) _________ were used as the host for the infection. (b) _______ infections are normally lethal for zebrafish. (c) ________ preys on __________ killing them and stopping the infection. (d) As well as killing the _______ bacteria directly, _____ stimulate the host immune system to help clear the infection. a) Zebrafish were used as the host for the infection. (b) Shigella infections are normally lethal for zebrafish. (c) Bdellovibrio bacteriovorus preys on Shigella, killing them and stopping the infection. (d) As well as killing the Shigella bacteria directly, Bdellovibrio bacteriovorus stimulate the host immune system to help clear the infection. Now listen to another clip from the same interview in which Professor Sockett discusses how Bdellovibrio bacteriovorus could be used as a treatment and the advantages that this treatment might have. Audio 3 Interview with Liz Sockett about Bdellovibrio bacteriovorus. INTERVIEWER Some of the press have picked up on this study and this paper and reported it as being this might be a way to address the big problem with antibiotic resistance. Do you think that that is a plausible channel? PROFESSOR SOCKETT So I think this could be good for injected antibiotic resistance clearance at sites in the body. At a surgical site- so if there's an infected wound site- we might inject the Bdellovibrio into that position in one site in the body, rather than necessarily take it into the gut. This won't be like a drug where you take it orally and it spreads throughout the whole of your body and removes bacteria at all different sites. But because these Bdellovibrio are alive, and because they do tackle antibiotic-resistant bacteria naturally, they can change as the antibiotic-resistant bacteria change. So it's a living therapy. INTERVIEWER And does that get around the possibility that the bacteria they're attacking, such as Shigella, might develop resistance to Bdellovibrio in the first place? PROFESSOR SOCKETT Yes, because if, say, there is a resistant strain, you could always co-culture it in the lab with Bdellovibrio and select for Bdellovibrio that get around the resistance it develops. But the Bdellovibrio themselves use hundreds of enzymes to degrade the bacteria. So it's not like a drug with a single target, it's a system of lots of different methods. It's like having lots of weapons and using them all at once on the pathogen. So that's a good thing. Note down in the table below any differences between a potential Bdellovibrio bacteriovorus treatment and antibiotics that Professor Sockett mentions in the audio clip.

	Bdellovibrio bacteriovorus	Antibiotics
Type of treatment
Likelihood of resistance arising
Tackles antibiotic-resistant bacteria

	Bdellovibrio bacteriovorus	Antibiotics
Type of treatment	Local injection at site of infection, e.g. a wound	Systemic – taken orally and spreads throughout the body to treat infections at many sites
Likelihood of resistance arising	Low – living treatment so can adapt as the infectious pathogen changesUses many enzymes to kill the bacteria, rather than one specific target, so resistance would require many changes in the infectious bacteria	High – one mechanism of action so may require a single change in the bacteria for resistance arise
Tackles antibiotic-resistant bacteria	Yes	No (depending on whether the bacteria are resistant to the prescribed bacteria)

4 A lesson from history Before antibiotics were discovered, people developed their own approaches to treating infections. These traditional remedies were often developed through trial and error and were passed on by word of mouth. Their effectiveness was very unlikely to have been rigorously tested by a clinical trial. In the light of the antibiotic era, old remedies can seem bizarre. However, some of them were effective at treating infections and scientists have uncovered some interesting potential antibiotic alternatives among them. You can read about one historical example in the next activity. Activity 6 An Anglo-Saxon remedy for MRSA Allow about 10 minutes Eat leeks in March and wild garlic in May, and all the year after the physicians may play. (Traditional Welsh rhyme) Is there any truth in this rhyme? Read the following short article from New Scientist magazine to help you decide! Article 1: ‘Anglo-Saxon remedy kills hospital superbug MRSA’ As the article in Activity 6 mentions, the challenge in developing traditional remedies as antibiotic alternatives is to understand how they work. In the following sections, you will look at the scientific mechanisms underlying the antibacterial activity of two traditional remedies that have attracted interest as antibiotic alternatives – natural honey and metals.

4.1 Natural honey Honey is a natural product that has been widely used in traditional medicine for centuries and is still used in modern medicine. The antibacterial properties of honey were first reported by the Dutch scientist van Ketel in 1892 (Dustmann, 1979) and it is active against up to 60 types of bacteria. Table 1 summarises some of the clinically important bacteria mentioned in this course that honey has antibacterial activity against. Table 1 Antibacterial activity of honey against clinically important infections.

Bacterial type	Clinical importance
Staphylococcus aureus	Hospital and community acquired infections
Vibrio cholerae	Cholera
Escherichia coli	Urinary tract infections, septicaemia, wound infections
Pseudomonas aeruginosa	Wound and urinary infections

Honey can be both bacteriostatic and bactericidal, depending on the concentration used. Its antibacterial activity is related to the following four properties. High hydroscopicityHoney has a high sugar content and is hydroscopic; that is, it absorbs moisture from its environment. This causes bacteria to dehydrate in the presence of honey. AcidityHoney is acidic, with a pH between 3.2 and 4.5. At this acidic pH, many bacteria cannot grow. Hydrogen peroxide contentWhen it is diluted, honey produces the chemical hydrogen peroxide (H₂O₂) from glucose. This chemical reaction requires the enzyme glucose oxidase (Figure 10). H₂O₂ can kill bacterial cells.

Phytochemical factors Honey contains a large number of phytochemicals which are chemicals produced by plants. Many phytochemicals have antibacterial properties. For example, allicin (Figure 10), produced by crushing garlic, has antibacterial activity against several bacterial pathogens, including MRSA and P. aeruginosa.

4.2 Metals In the fourth century BCE, the Greek physician Hippocrates used metals such as copper and silver to treat wounds (Alexander, 2009). Thus metals could be the oldest antimicrobial agents. More recently, dressings impregnated with silver have been used to improve wound healing. However, we are only just starting to understand how these metals exert these effects. Metals can: disrupt biofilms act togther with other antibacterial agents inhibit bacterial metabolism kill bacteria. The mechanisms that metals use to kill bacteria depend on the chemistry of the metal but they can include: producing chemicals that damage DNA damaging bacterial proteins disrupting the bacterial cell membrane preventing bacteria from acquiring the nutrients they need to grow. It is important to remember that metals can also be toxic to humans, which may limit their effectiveness as antibiotic alternatives. 4.2.1 Modern antibacterial applications of metals Many modern applications of metals have focused on the use of nanoparticles (particles between 1 and 100 nanometres in size) (Figure 11).

Activity 7 Using silver nanoparticles to improve the effectiveness of antibiotics Allow about 10 minutes Silver nanoparticles can be used to improve the effectiveness of antibiotics. Read the following short excerpt from an article in the Guardian newspaper and then answer the questions below. At Boston University, a team of biomedical engineers found that conventional antibiotics could kill between 10 and 1,000 times as many bacteria, including many previously resistant strains, when boosted with silver ions. This ancient remedy for infection – described by the Greeks in 400 BC – works in two ways: first by disrupting bacterial metabolism, causing bacteria to self-destruct; and second by making their cell membranes more permeable to the antibiotic. However, while the research is promising, these drugs still have to pass safety testing, as ingesting too much silver can be toxic for humans. (Cox, 2017) The researchers showed that silver nanoparticles enhanced the ability of antibiotics to treat Gram-negative bacterial infections. Why do you think this might be? (Hint: you may want to revisit some material in Weeks 2 and 3 to help you answer this question.) Gram-negative bacteria are resistant to many antibiotics because the antibiotics cannot cross their impermeable membrane. By disrupting the cell membrane of Gram-negative bacteria, silver nanoparticles make it easier for the antibiotic to enter the cell and reach its target. How would improving the effectiveness of antibiotics in treating Gram-negative infections help to tackle antibiotic resistance? Improving the effectiveness of an antibiotic could reduce either the dose of antibiotic required or the amount of time needed to treat the infection. Both of these will help to reduce antibiotic demand. The ability to treat infections caused by Gram-negative bacteria with new antibiotics may provide alternatives that could be used when the infection is resistant to routinely used antibiotic treatments. Metals can also be used as antibacterial surfaces. Copper surfaces can kill microbes, including HCAIs such as MRSA, within minutes to hours. The use of antibacterial copper surfaces in hospitals has been suggested to reduce the spread of HCAIs. Activity 8 Reducing the spread of HCAIs by using copper surfaces Allow about 10 minutes Read the following article which describes a trial using copper surfaces to reduce infection transmission in a Birmingham hospital. Article 2 Copper fittings ‘all but eliminate superbugs’ Where else do you think copper surfaces could be used to reduce infection transmission? Copper could be used to make medical devices. It could also be used in care homes, schools and other public places; in fact, anywhere that stainless steel is currently used. For example, one of South America’s largest theme parks – Fantasilandia in Chile – has recently replaced many of its most frequently touched surfaces with copper to try to reduce infection transmission (Keevil, 2017). You have now looked at several different alternatives to antibiotics. Many of these alternatives are still a long way from being used routinely to treat infections. It is likely that a combination of these alternatives, together with new and existing antibiotics, will be required to treat infections in the future. You should now complete this week’s quiz which covers material from the last four weeks of this course.

5 This week’s quiz It’s now time to complete the Week 8 badged quiz. It is similar to the previous quizzes but this time, instead of answering 5 questions, there will be 15, covering Weeks 5 to 8. Week 8 compulsory badge quiz Remember that the quiz counts towards your badge. If you’re not successful the first time, you can attempt the quiz again in 24 hours. Open the quiz in a new tab or window by holding down Ctrl (or Cmd on a Mac) when you click on the link. 6 Summary This week you learned about some alternatives to antibiotics. You should now be able to identify some antibiotic alternatives and understand some of the science underlying how they work. You have now reached the end of this course. You should have a better understanding of: how antibiotics treat infections how bacteria become resistant to antibiotics how our use of antibiotics has influenced resistance to them the approaches that are being used to tackle resistance. Finally, you might like to reflect on what you have learned by completing the antibiotic quiz which you first did in Week 1. Activity 9 Has your opinion changed? Allow about 5 minutes In Activity 9 in Week 1, you used the four questions below to help you form your own opinion on antibiotic resistance. Now answer these questions again and compare your answers with those in Week 1. Has what you have learned in this course changed your opinion? (a) On a scale of 1 (low) to 10 (high), how serious a problem is antibiotic resistance? (b) What, if anything, can be done about antibiotic resistance? (c) Whose responsibility is it to address this problem? You might like to think about: whether individuals should take some responsibility or whether it is up to the medical profession, governments, etc. the extent to which different countries and regions should work together to address this problem. (d) How urgent a problem is it? How soon should action(s) be taken? Where next? If you’ve enjoyed this course you can find more free resources and courses on OpenLearn. New to University study? You may be interested in our courses on science. Making the decision to study can be a big step and The Open University has over 40 years of experience supporting its students through their chosen learning paths. You can find out more about studying with us by visiting our online prospectus. Week 7: Reducing antibiotic use Introduction At this point in the course, you should appreciate that our use of antibiotics is contributing to the growing problem of antibiotic resistance. The Review on Antimicrobial Resistance led by Jim O’Neill (2016) proposed a ten-point plan to tackle the problem (Figure 1).

Begin this week by watching the following video which introduces the ten-point plan from the O’Neill report. Video 1 Ten steps to reducing antimicrobial resistance. TEXT ON SCREEN The 10 steps to reducting AMR according to the Review on Antimicrobial Resistance. 1 Public awareness. Improving global awareness of AMR to reduce use of antibiotics when theyre not needed. 2 Improved hygiene. Focus on improving basic access to water and sanitation, and reduce infectious diseases in hospital and care settings. 3 Reducing antibiotics in agriculture and their dissemination in the environment, as well as use simply to promote growth. 4 Global surveillance of AMR. From levels of drug resistance to consumption of antimicrobials in both humans and animals. 5 Develop rapid diagnostics. Investing in the Global Innovation Fund to revolutionise the way antimicrobials are used. 6 New vaccines and alternatives. Lowering the demand for reactive treatments and renew early research for vaccines. 7 Recognition of hard work. Increasing the numbers of people working in infectious disease, the recognition of those already doing so, and improving pay for important work. 8 Creating a Global Innovations Fund. Supporting early-stage development in a field where public and private R&D is lacking. 9 Incentives for investment. Creating a commercial viable market for antibiotics that will incentivise investment in R&D spending. 10 Collaboration on a global scale. Bringing together global partners including the G20 and the UN to build worldwide support against AMR.

In Weeks 5 and 6, you looked at two of these ten points – surveillance and drugs. You saw how antibiotic-resistant infections can be tracked and how new antibiotics are being developed. This week, you will look at two other points mentioned in the O’Neill report – sanitation and hygiene, and rapid diagnostics. By the end of this week, you should be able to: reflect on how antibiotic use can be reduced understand how infections are transmitted describe the role of good hygiene in reducing the spread of infectious diseases give examples of how the diagnosis of antibiotic-resistant infections can be improved to reduce antibiotic use. 1 Why do we need to reduce antibiotic use? Antibiotics have revolutionised modern medicine and demand for them is high. However, as you saw in Week 5, there is a high correlation between the use of antibiotics and antibiotic resistance. Antibiotic use is often unnecessary, so strategies to lower the demand for antibiotics or reduce their unnecessary use are crucial to tackling resistance. Several points in O’Neill’s ten-point plan contribute to lowering demand and reducing antibiotic use (Figure 2).

Public awareness – increasing public awareness of when and how to take antibiotics correctly will reduce unnecessary use and lower demand. Sanitation and hygiene – reducing the spread of infectious diseases (including antibiotic-resistant ones) by improving sanitation and hygiene lowers the demand for antibiotics. Antibiotics in agriculture and the environment – reducing the unnecessary use of antibiotics in healthy animals to prevent infections in large-scale farming. Vaccines and alternatives – vaccines and alternatives to antibiotics that prevent and treat infections will lower the demand for antibiotics. You will learn more about this in Week 8. Rapid diagnostics – broad-spectrum antibiotics are often given before the infection is identified, just in case. Tools to rapidly diagnose infections will reduce the unnecessary prescribing of broad-spectrum antibiotics. Human capital – increasing the number of trained microbiologists and infection control specialists, while increasing awareness of antibiotic resistance among healthcare professionals, will reduce the unnecessary use of antibiotics by reducing infection rates and altering prescribing habits. For the rest of this week you are going to look at two of these strategies – sanitation and hygiene, and rapid diagnostics. 2 A simple way to reduce the spread of infections You might recall from Week 1 that, before the discovery of antibiotics, the best way to treat infections was to prevent them. Activity 1 Preventing infections: a lesson from history Allow about 10 minutes Watch the video below in which you will see how, even before we knew bacteria existed, the Hungarian physician Ignaz Semmelweis (Figure 3) demonstrated the importance of hygiene in controlling the spread of infections.

Video: Ignaz Semmelweis and the birth of infection control We all now know how important it is to wash our hands but Semmelweis’s colleagues refused to accept his findings. Thinking back to Week 1 of this course, what scientific discoveries might have led to a wider acceptance of Semmelweis’s hypothesis that hand washing could prevent the spread of infection? When Semmelweis proposed his theory that hand washing could control the spread of infections, many people believed that miasmas – bad components of the air – were the cause of disease. Once Louis Pasteur and Robert Koch provided the scientific proof for germ theory, the value of hand washing was appreciated and Semmelweis was given credit for his work. Sanitation and hygiene have improved since the 19th century but 2.3 billion people in low-middle-income countries (LMICs) do not have basic sanitation facilities (WHO and UNICEF, 2017). Even in high-income countries (HICs), with access to good sanitation, hygienic behaviours are often poorly carried out. Improving sanitation and hygiene can prevent infection, reducing the need for treatment and limiting the opportunities for antibiotic resistance to develop. To understand how improving sanitation and hygiene can reduce the spread of infectious diseases, you first need to look at how infections are transmitted.

2.1 How infections are transmitted Bacteria can be transmitted (passed from one individual to another) either directly between people, or indirectly through air, water, food or other objects in the environment. In addition, bacteria can be transmitted from animals to humans via infected meat or water contaminated with animal faeces. How might transmission from animals to humans lead to antibiotic-resistant infections in humans? As you saw in Week 5, healthy farm animals are sometimes fed antibiotics to prevent infections. This increases the chance of resistance to these antibiotics developing. Transmission from animals to humans will increase the risk of resistant bacteria being transmitted to humans. 2.1.1 Direct person-to-person transmission Bacteria can be transmitted directly between people. This can occur by touching, during unprotected sex, or from a mother to her child during birth or breastfeeding. 2.1.2 Indirect transmission of pathogens Indirect transmission occurs when an infected person sheds bacteria into the air, water, food or onto other objects in the environment (known collectively as fomites), which can then infect someone else. Figure 4 summarises these indirect transmission routes.

Fomites are objects in the environment, such as door handles, cups and pens, that are routinely touched and can transmit infections. Healthcare-associated infections (HCAIs) can easily be indirectly transmitted to susceptible patients via fomites. A quarter of HCAIs are caused by antibiotic-resistant bacteria such as MRSA. In the next activity, you will look at fomites that might transmit HCAIs. Activity 2 Fomites and healthcare-associated infections Allow about 5 minutes Identify the fomites in this picture of hospital staff in 2001 that might transmit HCAIs.

Neck ties, stethoscopes, long-sleeved clothing, hospital badges worn at waist height and wrist watches could all brush against patients with infections and act as fomites, transmitting the infection to a susceptible individual. Consequently, in UK hospitals today, all staff must have their arms bare below the elbows and wrist watches and neck ties are banned. Sanitation and hygiene both play an important role in preventing indirect transmission via the routes summarised in Figure 5. Next, you will look at how sanitation and hygiene prevent faecal–oral transmission and the effect that this could have on antibiotic use.

2.2 The role of sanitation and hygiene As you saw in the previous section, pathogens can be transmitted indirectly by the faecal–oral route. Faecal–oral transmission occurs when unclean hands, food or other objects contaminated by faeces enter the mouth. These different faecal–oral transmission routes are illustrated by the ‘F-diagram’ (Figure 6).

Transmission by these routes can be stopped (or reduced) by sanitation and hygiene barriers (Figure 6). A primary barrier prevents the initial contact with faeces. This includes improving access to toilets to separate faeces from the environment and washing hands after going to the toilet. A secondary barrier prevents infectious pathogens being eaten by the new host. This includes using a clean water source, washing hands before preparing or consuming food, and covering food. Next, you will look at how these hygiene barriers can be used to lower antibiotic demand and reduce unnecessary use.

2.3 The role of hand washing in reducing the spread of bacteria Unwashed hands transmit bacteria from hand to mouth and by the faecal–oral route (see Video 2). Video 2 Bacteria on our hands. NARRATOR How many bacteria live on our hands? Here, a man briefly places his unwashed hand on a dish of nutrient gel. You can see a faint hand print where the surface of the gel has been disturbed. Watch what happens when the plate is kept warm at body temperature. Colonies of bacteria develop in just a few hours most of these bacteria are harmless organisms commonly found on our skin, but unwashed hands readily transmit pathogenic bacteria and viruses from person to person and from hand to mouth.

Hygienic behaviours such as hand washing are an important public health measure that can prevent transmission by the faecal–oral route. Hand washing with soap may be ‘the single most cost-effective way of reducing the global burden of infectious disease’ (Curtis et al., 2011). Is hand washing with soap an example of a primary or a secondary hygiene barrier? Washing hands after going to the toilet is a primary barrier; washing hands before preparing or consuming food is a secondary barrier. In the next activity, you will look at the effectiveness of hand washing to remove bacteria. Activity 3 Investigating the effectiveness of hand washing with soap Allow about 30 minutes In this activity you carry out a very simple experiment to look at the effectiveness of hand washing to remove bacteria. Since bacteria are too small to see, you will use glitter to represent the infectious pathogens. Materials Glitter (environmentally friendly glitter can be found online) Hand lotion Soap and hand-washing facilities Paper kitchen towel Method Put a small amount of hand lotion on your hands and rub it in so that it is spread out evenly. Place a pea sized pile of glitter in the palm of one hand and rub your hands together to spread the glitter over both palms. Note down where the glitter is spread over your hands. You may like to take a photograph or draw a sketch of the areas of your hands with glitter on them. Record your observations in Table 1. Wipe your hands with a dry piece of kitchen towel. Now consider the following questions: How much of the glitter is still on your hands? Has the paper towel effectively removed all of the ‘glitter bacteria’? Record your observations in Table 1. Now repeat the experiment but at the end of Step c wash your hands in cold water. Record your observations in Table 1. Finally repeat the experiment once more, washing your hands in warm water with soap at the end of Step c. Record your observations in Table 1 and then answer the questions below. Table 1 Experimental results

Hand washing intervention	Observations
No hand washing
Dry kitchen towel
Cold water
Warm water with soap

1. Which hand-washing method was the most effective at removing the ‘glitter bacteria’? Hand washing with soap and warm water is more effective than a paper towel or cold water at removing bacteria. 2. Were there any areas where the ‘glitter bacteria’ remained on your hands after using all of the hand-washing techniques? You may have found ‘glitter bacteria’ between your fingers or on the backs of your hands even after hand washing with soap. These places are frequently missed when washing hands, allowing bacteria to be transmitted. 3. What hand-washing advice would you give to healthcare workers hoping to reduce the spread of antibiotic-resistant bacteria? Many hospitals and other healthcare settings now provide training and guidance on effective hand washing as part of their infection control procedures (see Video 3). Posters with this guidance are often displayed near handwashing stations. Video 3 Effective hand washing. NARRATOR Hand-washing should take you about one minute. Use a timer or count from one to 10 in each of the following steps. Wet hands with water and apply enough soap to cover all surfaces of the hands. Let the water run smoothly to avoid touching the tap later on. Rub hands, palm to palm, to obtain a good quantity of foam. Then rub right palm over the back of left hand with interlaced fingers and vise versa. Rub again, palm to palm with fingers interlaced. Rub the back of your fingers to opposing palms with fingers interlocked, repeating this action for each hand. Rub rotationally left thumb clasped in right palm and vise versa. To clean the tips of the fingers, rub rotationally backwards and forwards with clasped fingers of right hand in left palm and vise versa. [MUSIC PLAYING] Rinse hands thoroughly with running water. Dry hands thoroughly with a single use towel. If the tap is not elbow operated, use this towel to turn off the tap without touching it directly. Your hands are now clean and safe.

In LMICs, the lack of access to clean water and soap can make sustaining effective hand washing difficult. However, even when clean water and soap are freely available, many people, including healthcare professionals, still do not wash their hands thoroughly (Judah et al., 2010) (Figure 7).

On average, healthcare workers adhere to recommended hand hygiene procedure only 40% of the time (WHO, 2009) but, as you will see in the next section, improving hand washing in hospitals and other healthcare settings can be an effective way to reduce the spread of antibiotic resistance.

3 Case study: reducing antibiotic resistance by improving hand washing There have been many campaigns to improve hand washing, particularly in healthcare settings. In the next activity, you will look at how effective these campaigns have been in reducing the transmission of antibiotic-resistant bacteria and, in particular, bacteria that are resistant to cephalosporins. Activity 4 Improving hygiene to reduce antibiotic-resistance – Part 1 Allow about 15 minutes Reducing HCAIs

Read the following short article about the ‘Clean Your Hands’ campaign (Figure 8) and then answer the questions below. Article 1: Hand Hygiene campaign ‘cut superbug infections’. How was hand washing measured in the campaign? Hand washing was measured by looking at the amount of soap and alcohol-based hand gel being purchased by hospitals. Was hand washing altered during the campaign? Yes, the amount of alcohol gel and soap purchased by hospitals during the campaign trebled from 22 ml to 60 ml per patient per day. What happened to infection rates during the campaign? Rates of MRSA infection more than halved while rates of C. difficile infection decreased by more than 40%. Is there a correlation between hand washing and infection rates? A correlation simply means that there is a relationship between the two sets of data. For example, there is a positive correlation between antibiotic use and antibiotic resistance: as antibiotic use increases, antibiotic resistance also increases. Yes, there is a negative correlation between hand washing and the rate of antibiotic-resistant infections: as hand washing increased, rates of infection decreased. Activity 4 Improving hygiene to reduce antibiotic-resistance – Part 2 Allow about 15 minutes Reducing the spread of cephalosporin-resistant infections You might remember from Weeks 3 and 4 that ESBL-producing bacteria are resistant to cephalosporins. In the second part of this activity, you will look at how hand hygiene in intensive care units (ICUs, also known as intensive therapy units (ITUs)) can affect the rate of cephalosporin-resistant infections. Hospital ICUs must work to reduce the emergence and spread of antibiotic-resistant infections because patients are frequently treated with broad-spectrum antibiotics. They are also at high risk of infection from the use of invasive medical devices such as respiration tubes and catheters. In 2006, a study aimed to assess the effect of a hand hygiene intervention on the number of ESBL-producing Klebsiella pneumoniae infections in ICUs (Prospero et al., 2010). The intervention consisted of a training course and the introduction of alcohol-based hand gels. Two ICUs took part in the study. ICUb continued its normal hand hygiene practices. ICUa introduced the use of alcohol-based hand gel in addition to its previous hand-washing measures. The number of cases of ESBL-producing K. pneumoniae infection was recorded before and after the intervention was introduced. Table 2 shows the findings of the study. The infection rate is recorded as the number of cases of infection per 1000 days of patient hospitalisation. Table 2 Cases of infection in two ICUs before and after the introduction of a hand hygiene intervention.

	ICUa (with intervention)	ICUb (no intervention)
Pre-intervention cases (no. per 1000 days hospitalisation)	4.50	4.02
Post-intervention cases (no. per 1000 days hospitalisation)	1.68	8.31

Now use the data in the table to answer the following questions. What happened to the number of infection cases in the ICU where the intervention was introduced (ICUa)? The number of infection cases decreased during the intervention period from 4.50 cases per 1000 days hospitalisation to 1.68 cases per 1000 days hospitalisation. What happened to the number of infection cases in the ICU without the intervention (ICUb)? The number of infection cases increased during the intervention period from 4.02 cases per 1000 days hospitalisation to 8.31 cases per 1000 days hospitalisation. Was the hand-washing intervention successful? Yes, the number of infection cases decreased in the ICU with the intervention but increased in the ICU without the intervention. This suggests that using the alcohol-based hand gel was effective at reducing infections. What effect would you expect the campaigns in this activity to have on the demand for antibiotics? Improving hand washing and decreasing the transmission of antibiotic-resistant infections should decrease the demand for antibiotics by lowering the number of infections requiring treatment. In Sections 2 and 3 you saw how improving hygiene can act as a barrier to pathogen transmission, reducing the unnecessary use of, and lowering the demand for, antibiotics. Next, you will look at another way to reduce antibiotic use – using rapid diagnostics to reduce the unnecessary prescribing of broad-spectrum antibiotics. 4 Rapid infection diagnostics Prescribing unnecessary antibiotics increases the chances of antibiotic resistance developing. This is often because broad-spectrum antibiotics are prescribed before the infection is diagnosed. Reducing the amount of time taken to diagnose an infection will help to reduce the unnecessary prescribing of antibiotics (Figure 9). This is the goal of rapid diagnostics.

4.1 Traditional approaches to infection diagnosis Your family doctor often relies on empirical diagnosis to decide what kind of infection you have. This means that they use their clinical experience to diagnose the infection based on your symptoms. Why might an empirical diagnosis lead to unnecessary antibiotics being prescribed? Empirical diagnoses rely on symptoms to diagnose an infection. For example, a persistent cough and fever could be symptoms of a chest infection. However, infections can be bacterial, viral or fungal and an empirical diagnosis cannot determine the cause of the infection. Antibiotics will not treat infections caused by viruses or fungi, therefore a prescription for antibiotics would be unnecessary in these cases. In many cases a family doctor will send a sample of the infection for laboratory diagnostic testing. In the next activity, you reflect on your personal experience of being treated for an infection. Activity 5 Being prescribed antibiotics Allow about 5 minutes Think about a time when you, or someone you know, was prescribed antibiotics. Then answer the questions below, based on your experience. Did the doctor send a sample for testing? How long did you have to wait for the results? If your family doctor suspects that you have a bacterial infection, they will often send samples for traditional laboratory diagnostic tests. However, as you will see below, it can take up to a week to get the results of these tests. Meanwhile, you might be prescribed broad-spectrum antibiotics to try to treat the infection and prevent it from becoming worse. Traditional laboratory diagnostic tests rely on culturing the bacteria for at least 36 hours to determine the type of infection and the drugs that it is susceptible to (see Video 4). This can delay the prescription of narrow-spectrum antibiotics that specifically target the infection. Video 4 Culturing bacteria to test for antibiotic susceptibility. INSTRUCTOR When a bacterial culture grows to cover the entire surface of a plate, it is called a lawn. To grow good lawn, we can use a swab. We immerse a pre-sterilised swab in a broth culture and drain the excess liquid against the side. We make a streak down the centre of the plate and then rub the swab across the surface of the plate. First in one direction and then in another to ensure maximum coverage. After overnight incubation, the lawn is visible. Lawns can be used to test the sensitivity of the bacteria to different substances, such as antibiotics. Here, specially prepared paper discs impregnated with a known quantity of antibiotic are used. The discs are placed on the surface of the plate after inoculation. After overnight incubation, clear zones-- indicating inhibition of bacterial growth-- can be seen around some or all of the discs. In this example, we can see that the culture contains at least two populations of bacteria. One that forms small, yellowish colonies and is sensitive to the antibiotic. And one that forms larger white colonies and is not sensitive to the antibiotic. This technique is very useful for testing antibiotic sensitivity and resistance patterns of pathogens.

While the results of a traditional laboratory diagnostic test are being processed, broad-spectrum antibiotics are often prescribed to try to treat the infection before diagnosis. In some cases, the treatment will be effective. However, if the infection is not caused by pathogenic bacteria, or if the infection-causing bacteria are resistant to the prescribed antibiotic, another prescription may be required. Rapid diagnostic tests do not rely on culturing bacteria so they can reduce the time taken to diagnose the infection (Figure 10). This means that doctors can quickly prescribe a treatment that effectively and specifically targets the infection. This helps to reduce the unnecessary prescribing of broad-spectrum antibiotics.

In the next section, you will look at rapid diagnostic tests for infection more closely.

4.2 The perfect rapid diagnostic test The perfect diagnostic test for bacterial infection should answer four questions: Is the infection bacterial or viral? If the infection is bacterial, what type of bacteria is causing the infection? Are the bacteria resistant to a particular drug? Which drugs are the bacteria susceptible to? Traditional diagnostic tests – such as those shown in Video 4 – can answer all four questions but not quickly. The challenge for rapid diagnostics is to answer these questions within minutes so that doctors and other healthcare professionals can decide which antibiotics are needed.

4.3 What rapid diagnostic tests detect There are many rapid diagnostic tests in development and clinical use. In this course, it is impossible for you to look at all of them. Broadly speaking, they are designed to detect either the pathogen or the patient’s response to pathogen infection. 4.3.1 Detecting the patient infection response Some tests detect chemicals produced by the patient in response to infection – these are known as biomarkers. One example is the chemical procalcitonin (PCT) which is made in response to bacterial, but not viral, infections. It can easily be detected in a blood sample taken from the patient. In the following video you will see how PCT tests can be used in clinical practice to reduce the unnecessary use of antibiotics. You can then practise interpreting PCT levels in Activity 6. Video 5 How rapid diagnostic testing for PCT reduces unnecessary antibiotic prescribing. MAGGIE When you take into account farm use, GPs, and hospitals, it's estimated that 2/3 of antibiotic use is either highly questionable or totally unnecessary. And that's a tragic waste. So the most recent developments are in methods to cut down that waste, and that could ultimately save many lives. One of those methods is being introduced here at the Royal Hampshire hospital. DR MATTHEW DRYDEN Hello, Mrs. Preston. MRS PRESTON Hello. DR MATTHEW DRYDEN I'm Dr Dryden. We've popped by today to see how you are. Can you tell us what brought you into hospital? MRS PRESTON Yeah, I have difficulty breathing. I get very, very breathless. DR MATTHEW DRYDEN One of the reasons we've come along as the infection team is to try and decide if you need any antibiotics. MRS PRESTON I see, yes. DR MATTHEW DRYDEN We've got a special blood test, which is a relatively new development, which can distinguish between bacterial infection and viral infection or no infection at all. So I think we will do that on your samples, which will help us make a decision as to whether you need antibiotics or not. MRS PRESTON Right. Right. Okey doke. MAGGIE Yeah, yeah, lovely to have met you. Cheers. DR MATTHEW DRYDEN Maggie, this is the virology and immunology lab. This is the lab that we would bring Gloria's blood to. Downstairs, Gloria's blood is being cultured. Now, the interesting thing is that the blood cultures are going to take up to five days to grow bacteria. They may come up in 24 hours if we're lucky, if they're there. But this test we can do in two hours, which helps us decide whether she's got a bacterial infection or not, and whether we have to give her antibiotics. MAGGIE The test is looking for levels of a blood protein called procalcitonin, which rises during a bacterial infection, but not during a viral infection. DR MATTHEW DRYDEN This is a machine that does procalcitonin tests. And we've got a result coming out here now. So here's the readout for the test. And it gives us a very accurate representation of the procalcitonin concentration in the blood. It is less than 0.05. And that indicates, for this particular patient, there's no necessity to get antibiotics now. MAGGIE Dr Dryden closely monitors patients like Mrs Preston to make sure not giving them antibiotics is the right decision. And since introducing the system, this unit has cut down antibiotic use by half. DR MATTHEW DRYDEN To have a test like procalcitonin, which enables us with our clinical diagnosis to be sure that the patient has or has not got a bacterial infection is really useful. MAGGIE It's only being used in a handful of places so far. But the ultimate goal is to make this test economical enough to use in GP surgeries where 80% of antibiotics are prescribed.

Activity 6 Interpreting PCT levels Allow about 10 minutes Figure 11 shows the levels of PCT – in micrograms (μg) of PCT per litre of serum – in a patient with an infection and receiving treatment over 3 days (72 hours). Study Figure 11 carefully and then answer the questions below.

How do the patient’s PCT levels change over the 72-hour period? The patient’s PCT levels rise from 0 at infection onset to 5 micrograms per litre after approximately 16 hours. When antibiotic treatment starts, PCT levels decline from 5 micrograms per litre to less than 1 microgram per litre after 72 hours. Does the patient have a bacterial infection? How can you tell? Yes, the patient’s PCT levels rise from 0 at infection onset to 5 micrograms per litre after approximately 16 hours. Elevated PCT levels indicate that the patient has a bacterial, rather than a viral, infection. Is the antibiotic treatment effective? How can you tell? Yes. Once, antibiotic treatment has started, the patient’s PCT levels decrease from 5 micrograms per litre to less than 1 microgram per litre, indicating that the patient’s infection has cleared and the antibiotic treatment is effective. How could measuring the PCT levels in a patient be used to reduce unnecessary antibiotic use? Decreasing PCT levels show that the infection is being effectively treated by the prescribed antibiotics. If PCT levels remain high after treatment, doctors could quickly prescribe an alternative treatment, reducing the need to take ineffective antibiotics for an extended period. 4.3.2 Detecting the pathogen Many rapid diagnostic tests detect the presence of infectious bacteria directly. Some tests can detect the presence of bacterial DNA, or antibiotic resistance genes in the sample, by a laboratory technique called polymerase chain reaction (PCR). This is known as molecular diagnostics which can be an extremely powerful diagnostic tool. Other tests rely on chemical reactions that cause a detectable colour change when bacteria (or a bacterial enzyme or product) are present in the sample. The information these tests provide can vary. Some, such as the urinary tract infection (UTI) dipstick test, detect the presence of bacteria in a sample. Others, such as the Nordmann/Doret/Poirel test, can provide information on the bacteria’s susceptibility to antibiotics. 4.3.3 The UTI dipstick test

Several Gram-negative bacteria cause urinary infections, for example E. coli and Klebsiella. They produce an enzyme called nitrate reductase and can convert the nitrates in urine to nitrites. You do not need to know the details of this reaction, just that these nitrites can be detected using the Greiss reaction in which nitrites react to produce a pink dye. The presence of pink on the dipstick test can therefore indicate a bacterial urinary infection, although this test is not completely reliable. 4.3.4 The Nordmann/Dortet/Poirel test The Nordmann/Dortet/Poirel (NDP) test can detect the presence of ESBL-producing bacteria in blood samples in less than two hours (Nordmann et al., 2012). As you might remember from Week 4, ESBLs are a major determinant of resistance to cephalosporin antibiotics. The presence of ESBLs in a bacterial strain can be used to diagnose a cephalosporin-resistant infection. The NDP test detects the enzyme activity of ESBLs in the sample. What enzymatic reaction do ESBLs catalyse? Recall from Week 3 that β-lactamases destroy β-lactam antibiotics by hydrolysing the β-lactam ring. When cefotaxime (a third-generation cephalosporin) is hydrolysed by ESBLs, it results in acidification; that is, the sample becomes more acidic. In the NDP test, this acidification can be measured as a colour change in the sample using a pH indicator. The colour of a sample gives a visible indication of the presence of ESBL-producing bacteria (Figure 13).

4.4 The future for rapid diagnostics Although rapid diagnostic tests are faster than traditional diagnostic tests at diagnosing an infection, they still often need to be carried out in a laboratory by highly trained staff. Since antibiotics are often given in non-hospital settings, particularly in LMICs, delivering diagnostic information at the point-of-care (POC) is key to reducing unnecessary antibiotic use. The POC is where a patient presents with the illness, for example a doctor’s surgery, hospital, care home, pharmacy or mobile clinic. In the next activity you will look at the factors that are important when designing a POC test. Activity 7 Designing the perfect point-of-care diagnostic test Allow about 10 minutes The Longitude prize is a competition to design a POC diagnostic test. Figure 14 is taken from the Longitude prize website and illustrates some of the factors that need to be considered when designing a POC diagnostic test.

Imagine that you are part of the design team developing a new POC diagnostic test for use in mobile clinics in rural Africa. How would the factors above influence the design of your device? You may have noted down several factors, some that we considered include: Mobile clinics in LMICs may not have access to reliable refrigeration or mains power therefore the POC test should be able to be stored at ambient temperature and should operate without the need for external power sources. Clinics may not be staffed by highly-trained laboratory technicians so the test should be easy to use and interpret with minimal training. Clinics may be in remote locations and patients may have to travel long distances to attend therefore the results should be rapidly obtained so that patients can be diagnosed and prescribed appropriate treatment without needing to return to the clinic.

5 This week’s quiz Well done – you have reached the end of Week 7 and can now do the quiz to test your learning. Week 7 practice quiz Open the quiz in a new tab or window (by holding down Ctrl [or Cmd on a Mac] when you click the link). Return here when you have finished it. 6 Summary This week, you learned more about the relationship between antibiotic use and antibiotic resistance. You should now appreciate the role of hygiene and sanitation and rapid diagnostics in reducing demand and preventing the unnecessary use of antibiotics. You should also understand how pathogens are transmitted and the role of good hygiene in blocking this transmission. You also learned that traditional diagnostic methods can contribute to unnecessary antibiotic use. You should now be able to give some examples of rapid diagnostic tests and appreciate their role in tackling antibiotic resistance. 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(2012) ‘Rapid detection of extended spectrum beta-lactamase producing Enterobacteriaceae’, Journal of Clinical Microbiology, vol. 50, no. 9, pp. 3016–22 [Online]. Available at http://jcm.asm.org/content/50/9/3016.full (Accessed 24 February 2018). O’Neill, J. (2016) Tackling Drug-resistant Infections Globally: final report and recommendations [Online]. Available at https://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf (Accessed 11 January 2018). The Open University (2015) ‘1.4.2 Indirect person-to-person transmission’, SDK100 Science and health [Online]. Available at https://learn2.open.ac.uk/mod/oucontent/view.php?id=993001&section=1.4.2 (Accessed 6 February 2018). Prospero, E., Barbadoro, P., Esposto, E., Manso, E., Martini, E., Savini, S., Scaccia, F., Tantucci, L., Pelaia, P. and D’Errico, M. 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Available at www.liebertpub.com/doi/abs/10.1089/sur.2008.9941 (Accessed 9 March 2018). Cox, D. (2017) ‘The “superantibiotics” that could save us from a bacterial apocalypse’, The Guardian, 23 October [Online]. Available at www.theguardian.com/lifeandstyle/2017/oct/23/the-superantibiotics-that-could-save-us-from-bacteria-apocalypse (Accessed 9 March 2018). Dustmann, J. H. (1979) ‘Antibacterial effect of honey’, Apiacta, vol. 14, pp. 7–11. Keevil, B. (2017) ‘Copper is great at killing superbugs – so why don’t hospitals use it?’, The Conversation, 24 February [Online]. Available at https://theconversation.com/copper-is-great-at-killing-superbugs-so-why-dont-hospitals-use-it-73103 (Accessed 9 March 2018). O’Neill, J. (2016) ‘Tackling Drug-resistant Infections Globally: final report and recommendations’ [Online]. Available at https://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf (Accessed 11 January 2018). Schmelcher, M., Donovan, D. M. and Loessner, M. J. (2012) ‘Bacteriophage endolysins as novel antimicrobials’, Future Microbiology, vol. 7, no. 10, pp. 1147–71 [Online]. Available at www.ncbi.nlm.nih.gov/pmc/articles/PMC3563964/ (Accessed 8 March 2018). Willis, A., Moore, C., Mazon-Moya, M., Krokowski, S., Lambert, C., Till, R., Mostowy, S. and Sockett, L. (2016) ‘Injections of predatory bacteria works alongside host immune cells to treat Shigella infection in zebrafish larvae’, Current Biology, vol. 26, no. 24, pp. 3343–51 [Online]. Available at www.cell.com/current-biology/fulltext/S0960-9822(16)31152-6 (Accessed 8 March 2018). This free course was written by Rachel McMullan and Sarah Palmer. It was first published in October 2018. Except for third party materials and otherwise stated (see terms and conditions), this content is made available under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Licence. The material acknowledged below is Proprietary and used under licence (not subject to Creative Commons Licence). Grateful acknowledgement is made to the following sources for permission to reproduce material in this free course: Introduction Images Course Image: © smartboy10/Getty Images. Week 1 Figures Figure 1 © Heiti Paves in WikiMedia https://creativecommons.org/licenses/by-sa/3.0/ Figure 2a © Eye of Science/Science Photo Library Figure 2b © Steve Gschmeissner/Science Photo Library Figure 2c © NIAID Figure 2d © NIAID Figure 2e © David Dorward/NIAID Figure 2f © NIAID Figure 4 from Gell=band, H, et al (2015) The State of the Worlds Antibiotics 2015 https://www.cddep.org/publications/state_worlds_antibiotics_2015/ Figure 5 from Gell=band, H, et al (2015) The State of the Worlds Antibiotics 2015 https://www.cddep.org/publications/state_worlds_antibiotics_2015/ Audio-visual Video 2 BBC Learning Zone © BBC Video 4 What is a superbug?, Pain, Pus and Poison, episode 2 © BBC Video 5 A very serious issue, Sally Davis talking in 2013 © BBC Audio 1 Scientists’ perspective on the antibiotic resistance threat, Inside Science, BBC Radio 4 Fighting Antimicrobial Resistance (last broadcast 9 June 2016) © BBC Text Article 1: The History of Germ Theory, Big Picture by the Educational and Editorial Teams at Wellcome Trust Article 2: extract from Bacteria that resist 'last antibiotic' found in UK, James Gallagher Health Editor, BBC News website 21.12.2015, © BBC Week 2 Audio-visual Video 1 In pursuit of ‘magic bullets; the seminal work of Paul Ehrlich, Pain, Pus and Poison (2013) © BBC Video 2 How do antibiotics work?, courtesy e-bug http://www.e-bug.eu/ Video 3 Penicillin and beyond, Pain, Pus and Poison © BBC Week 3 Text Article 1: © BBC News Images Figure 5: Created by the Open University using https://www.rcsb.org/pdb/ngl/ngl.do?pdbid=1VQQ HYPERLINK; https://www.rcsb.org/pdb/ngl/ngl.do?pdbid=1VQQ&bionumber=1"& HYPERLINK; https://www.rcsb.org/pdb/ngl/ngl.do?pdbid=1VQQ&bionumber=1"bionumber=1 Figure 6: adapted from:Pfizer ATLAS https://atlas-surveillance.com/ Figure 7: adapted from BSAC Data © The Open University Week 4 Images Figure 4 © Nick Kim, CartoonStock Figure 7 © Dr L Caro/Science Photo Library Figure 13: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011202 Copyright: © 2010 Smet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/2.0/ Figure 14: AS Rose and PW Hildebrand. NGL Viewer: a web application for molecular visualization. Nucl Acids Res (1 July 2015) 43 (W1): W576-W579 first published online April 29, 2015. doi:10.1093/nar/gkv402 http://www.rcsb.org/pdb/ngl/ngl.do?pdbid=1YLJ&bionumbe Audio-visual Video 1: An introduction to the acquisition and spread of antibiotic resistance, Bacteria Vs Antibiotics by Kevin Wu https://ed.ted.com/on/x5MqTCry https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en Video 3: HHMI Week 5 Images Figure 2 Review on antimicrobial resistance, https://amr-review.org/sites/default/files/HI%26S%20Infographic_1_v4%20white.jpg https://creatiand 5vecommons.org/licenses/by/4.0/ Figure 3 Review on antimicrobial resistance, https://amr-review.org/sites/default/files/HI%26S%20Infographic_1_v4%20white.jpg https://creatiand 5vecommons.org/licenses/by/4.0/ Figure 4 Review on antimicrobial resistance, https://amr-review.org/sites/default/files/HI%26S%20Infographic_1_v4%20white.jpg https://creatiand 5vecommons.org/licenses/by/4.0/ Figure 5 Review on antimicrobial resistance, https://amr-review.org/sites/default/files/HI%26S%20Infographic_1_v4%20white.jpg https://creatiand 5vecommons.org/licenses/by/4.0/ Figure 6 from Center for Disease Dynamics, Economics and Policy (CDDEP) cdde.org Figure 9: © ReAct group 2015 Figure 10 Global antimicrobial resistance surveillance system (GLASS) report: early implementation 2016-2017. Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO Figure 11 Global antimicrobial resistance surveillance system (GLASS) report: early implementation 2016-2017. Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO Figure 12: from Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action; © 2017 World Health Organization. Licensee Public Library of Science http://creativecommons.org/licenses/by/3.0/igo/ Audio-visual Video 2: © European Centre for Disease Control https://ecdc.europa.eu/ Videos 3 and 4: Bang Goes the Theory, Series 7, Bugs © BBC 2013 Video 5: Drug-resistant gonorrhoea: an urgent public health issue.Centers for disease control and prevention CDC Text Article 1: The world is running out of antibiotics, courtesy World Health Organisation http://www.who.int/ Week 6 Images Figure 1: AJC1 in Flickr https://creativecommons.org/licenses/by-sa/2.0/ Figure 2: Docwarhol https://creativecommons.org/licenses/by-sa/4.0/deed.en https://commons.wikimedia.org/wiki/File:Streptomyces_griseus_color_enhanced_scanning_electron_micrograph..jpg Figure 3: adapted from bas scheme 8 in this paper Wright, P. M., Seiple, I. B., and Myers, A. G. (2014). The Evolving Role of Chemical Synthesis in Antibacterial Drug Figure 4: adapted from http://www.pewtrusts.org/en/research-and-analysis/reports/2016/05/a-scientific-roadmap-for-antibiotic-discovery © Pew Charitable Trust Figure 6: http://www.pewtrusts.org/en/research-and-analysis/reports/2016/05/a-scientific-roadmap-for-antibiotic-discovery © Pew Charitable Trust Figure 7: adapted from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536949/ Figure 8: adapted from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536949/ Figure 9: taken from: PharmaFactz; Medicinal Chemistry of Beta-Lactam Antibiotics; © PharmaFactz 2018 Audio-visual Video 2: Penicillin rediscovered, clip from Britains Greatest Invention (broadcast on BBC) courtesy Thoroughly Modern Media Video 3: Mass production of penicillin, Pain, Pus and Poison, Episode 2, © BBC. Video 4: South American leafcutter ants, clip from BBC Britains Greatest Invention (2017) courtesy of Thoroughly Modern Media Audio 1: Cephalosporins-Chemistry in its element, Chemistry World; https://www.chemistryworld.com. Audio 2 Inside Science, Radio 4, International Year of Soil, broadcast 15/1/2015. © BBC Text Article 1: Brute force can overcome antibiotic resistance (UCL 2017), courtesy of Joseph Ndieyira; UCL Week 7 Images Figures 1 and 2: from ‘Review on Antimicrobial Resistance.’ https://amr-review.org/sites/default/files/HI%26S%20Infographic_1_v4%20white.jpg https://creatiand 5vecommons.org/licenses/by/4.0/ Figure 3: Ignaz Semmelweis / Etching by Dob; akg-images Figure 6: Unicef; https://www.unicef.org.uk Figure 7: Royal Pharmaceutical Society Figure 8: Crown Copyright http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Figure 9: ‘Tackling drug-resistant infections globally: Final report and recommendations’; HM Government and Wellcome Trust; https://creativecommons.org/licenses/by/4.0/ Figure10: https://amr-review.org/infographics.html https://creativecommons.org/licenses/by/4.0/ Figure 11: Safely Reduce Antiobiotic Exposure; BRAHMS Procalcitonin (PCT): ThermoFisher Scientific Figure 12: By J3D3 [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)], from Wikimedia Commons Figure 13: taken from Journal of Clinical Microbiology; Rapid Detection of Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae Patrice Nordmann, Laurent Dortet and Laurent Poirel Figure 14: Nesta; https://creativecommons.org/licenses/by-nc-sa/4.0/ Audio-visual Video 1: Ten steps to reducing antimicrobial resistance.The Association of the British Pharmaceutical Industry; 10 Steps to Reducing AMR - The Review on Antimicrobial Resistance Video 5: Bang Goes the Theory, Series 7, broadcast 11 March 2013, © BBC Week 8 Images Figure 1: from ‘Review on Antimicrobial Resistance.’ https://amr-review.org/sites/default/files/HI%26S%20Infographic_1_v4%20white.jpg https://creatiand 5vecommons.org/licenses/by/4.0/ Figure 3: Janice Carr (2005) https://phil.cdc.gov/Details.aspx?pid=7483 Figure 5: Activity 4; courtesy Rachel McMullan Figure 6: Activity 4; adapted from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563964/figure/F3/ Figure 7: Science Photo Library/UIG Figure 8: Estevez https://creativecommons.org/licenses/by-sa/3.0/deed.en https://commons.wikimedia.org/wiki/File:Bellovibrio-cycle.svg Figure 9: courtesy Rachel McMullan Figure 10: stevepb/Pixabay.com; https://creativecommons.org/publicdomain/zero/1.0/deed.en Figure 11: Kateryna Kon/Science Photo Library Audio-visual Video 1: How vaccines help to beat superbugs; Vaccines Today Video 2; Predation of Shigella by Bdellovibrio bacteriovorus inside a zebrafish larva (Willis et al., 2016). Predation of GFP - Shigella by mCherry Audio 1: Interview with Martha Clokie on phage therapy, Inside Science, Radio 4, © BBC 2016 Audio 2: Interview with Liz Sockett about research with Bdellovibrio bacteriovorus, Inside Science; Radio 4 © BBC 2016 Audio 3: Interview with Liz Sockett about research with Bdellovibrio bacteriovorus, Inside Science; Radio 4 © BBC 2016 Text Article 1: courtesy © New Scientist Ltd (2015) https://www.newscientist.com/article/dn27263-anglo-saxon-remedy-kills-hospital-superbug-mrsa/ Every effort has been made to contact copyright owners. If any have been inadvertently overlooked, the publishers will be pleased to make the necessary arrangements at the first opportunity. Don't miss out If reading this text has inspired you to learn more, you may be interested in joining the millions of people who discover our free learning resources and qualifications by visiting The Open University – www.open.edu/openlearn/free-courses.