1.1 Evolution of antibiotic resistance in microbes

Infections caused by bacteria but not treatable by common antibiotics, are one of the greatest healthcare concerns today. One of the most problematic microbes, alongside C. difficile, is the so-called hospital ‘superbug’ MRSA.

Methicillin is a type of antibiotic that was previously used to kill this bacterium, though it has now been superseded by more stable antibiotics such as flucloxacillin. In fact, the initials can equally well mean ‘multiple-resistant Staphylococcus aureus’, a reference to the bacterium’s resistance to a range of antibiotics. There are well over 30 different Staphylococcus species that grow in characteristic clumps, like tiny bunches of grapes (Figure 1). About 10 of these species are natural human commensals but can, under some conditions, develop into serious pathogens.

Figure 1 MRSA (methicillin-resistant Staphylococcus aureus). The individual microbes cluster together in colonies that look like miniature bunches of grapes.

Staph. aureus is of particular concern because it can cause a wide range of disease conditions from toxic shock syndrome (TSS) to skin infections, particularly infecting people with weakened immune systems or with wounds, making it a problem in hospitals. Although the bacterium can grow without oxygen, it grows and reproduces more quickly if oxygen is present. Such bacteria are called facultative anaerobes. In healthy people Staph. aureus naturally lives in the armpits and in the nose, but once it is inside the body it forms biofilms which protect it against the immune system and allow it to proliferate. Biofilms can form anywhere bacteria can find a surface to attach to, even including an apparently smooth surface such as plastic (Figure 2).

Figure 2 A biofilm of Staphylococcus which has formed on the inner surface of a plastic connector used to join medical tubing. The bacteria can be seen amongst filaments of polysaccharide produced by the bacteria. The background is the surface of the plastic tubing. Similar biofilms can form inside the human body.

Understanding how microbes develop resistance against antibiotics is one of the most important challenges in modern microbiology. It appears that antibiotic resistance genes arose by the process of mutation.

Although mutations occur naturally, they can also be induced by radiation and certain drugs. Most mutations are harmful to the organism possessing them, and so that individual dies. However, occasionally, by chance, a mutation can occur that is beneficial to the organism. In the case of microbes, this could be a mutation in a gene which allows the microbe to survive in the presence of antibiotics, i.e. to be resistant to antibiotics. Because microbes divide by binary fission, the mutant gene can spread very rapidly, as every offspring of the resistant microbe has an exact copy of the DNA and so will also carry the mutant gene. So, if one in 10 000 bacteria in a colony within a wound being treated with an antibiotic such as flucloxacillin, developed a mutation which made it resistant to the antibiotic, the other 9999 bacteria would die, but that one would survive and divide and within a few days, there could be a new colony of 10 000 resistant bacteria in the wound.

The antibiotic resistance genes may function in several ways. For example, they can cause the microbes to produce substances that change the structure of the cell wall to prevent the antibiotic entering or that break down the antibiotic before it can do any harm. Or they may change biochemical pathways (sets of reactions which produce important substances in the microbe), to by-pass the parts of the pathway which are normally sensitive to the antibiotic. The antibiotic resistance genes seem to be amongst those genes that are carried on plasmids.

Crucially, for the progress of antibiotic resistance, plasmids can be transferred from one microbe to another by a process called conjugation.

You will not be surprised to know that these small circles of DNA are also replicated and transferred into both of the new cells when one cell divides. There can be from one to more than a thousand copies of the same plasmid in any one microbe. In the process of conjugation, illustrated in Figure 3, one microbe can pass a plasmid to another of the same species. Two microbes come together and one of them extends an appendage called a pilus.

Maximise

Figure 3 The process of conjugation. (a) First, a hollow pilus is established between the two microbes leading to a connection. The open channel is then used to pass a copy of the plasmid which is transferred from one cell (on the left) to the cell on the right. (b) Two bacteria attached together with pili.

In conjugation the pilus is a hollow fibre a few micrometres long, that can connect the two microbes. One of the plasmids in one of the microbes opens up, is copied and is transferred along the length of the pilus into the attached microbe. Since the antibiotic-resistance genes are on the plasmids, this can result in the transfer of such resistance from one microbe to another. When that microbe divides (by binary fission) the resistance will be passed on to all of its offspring. This appears to be one of the reasons for the rapid spread of resistant microbes in hospitals. Conjugation does, of course, occur between microbes in other environments too and is a way in which genes that enable microbes to survive particular environmental stresses can be passed from one to another.

Yet another reason for the rapid spread of resistance genes and the adaptation to new antibiotics is the remarkable ability that microbes have to take up DNA, across the wall of their cells, from their environment without receiving it from another microbe. This process is known as transformation and is illustrated in Figure 4.

Figure 4 A schematic diagram showing a microbe taking up a DNA fragment from the environment by transformation.

Transformation allows genes which are completely new to that species to be taken up and incorporated into its DNA, where they may function correctly. Thus a microbe in which resistance to a particular antibiotic had never previously been seen, could take up a fragment of DNA which had been released into the environment on the death of another entirely different microbe. This fragment of DNA could contain a gene which confers resistance to that antibiotic if successfully integrated and, after several cycles of binary fission, a new resistant population could appear.

The spread of resistance to antibiotics can be surprisingly rapid. After the antibiotic penicillin was introduced for general use in 1947, Staphylococcus aureus became resistant to it in four years. In 1959, a new antibiotic, methicillin, was used to treat these resistant strains but the first methicillin-resistant strains (MRSA) were detected only two years later in 1961. These strains have recently spread to hospitals throughout the world. More recently, new strains are emerging that are resistant to the more modern antibiotic, vancomycin. VRSA (Vancomycin-resistant Staphylococcus aureus) appeared in 2002. The introduction of a new synthetic antibiotic linezolid in 2000, resulted in resistant strains appearing in 2003.

Antibiotic resistance can be thought of as an arms race between microbes and humans. Each time a new antimicrobial agent is developed, a microbe eventually undergoes a genetic alteration that allows it to acquire resistance which may spread through the microbial population. A major activity in microbiology is to find new antimicrobial agents to keep up with the ever-changing resistance of microbes. The spread of resistance has been made easier by two particular aspects of medical practice. The widespread use of antibiotics for minor infections increases the chance that a resistant strain of microbe might arise because more species of microbes come into contact with antibiotics. Resistance to antibiotics is also caused by patients taking incomplete antibiotic courses. This exposes microbes to concentrations of antibiotics which are not lethal, but are at sufficient concentrations preferentially to give an advantage to bacteria in the population that do have resistance. There is the risk that microbes with partial resistance may be more likely to survive and more quickly develop complete resistance. These resistant bacteria then multiply and dominate the population. By finishing a course of antibiotics one is more likely to kill the target microbes and so prevent them from developing resistance.