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Isolating and identifying bacteria (human health)

Introduction

In this module you will learn about microbiology techniques for identifying bacteria, focusing on WHO priority pathogens which are the focus of the Global Antimicrobial Resistance Surveillance System (GLASS). You will start by looking at the diverse types of clinical samples sent to microbiology laboratories and factors that can impact the testing of samples and reporting of test results. Basic tests used routinely in laboratories to isolate and identify bacteria are discussed next, followed by newer and/or more advanced methods usually performed by reference laboratories. Finally, you will be introduced to Quality Control (QC) measures that should be implemented in the clinical microbiology laboratory to ensure that test results are reliable and accurate. You will learn more about quality measures in the Quality assurance and AMR surveillance module.

Basic knowledge of concepts such as bacterial growth and bacterial structure is assumed. If you are unfamiliar with these concepts or would like a refresher course, you might want to look back at the Introducing antimicrobial resistance module or this Microbioloby textbook (OpenStax, 2021). Information about the GLASS programme can be found in the Introducing AMR surveillance systems module.

After completing this module, you will be able to:

  • know where samples are obtained from and reflect on the process by which bacterial samples are processed in your workplace
  • describe the principles of laboratory tests used to isolate and identify key bacterial pathogens in human health, which are the focus of the GLASS programme
  • know when and why advanced testing such as mass spectrometry and automated systems are used
  • know the importance of procedures designed to ensure the quality of laboratory work relating to isolating and identifying bacteria in your workplace.

Activity 1: Assessing your skills and knowledge

Timing: Allow about 10 minutes

Before you begin this module, you should take a moment to think about the learning outcomes and how confident you feel about your knowledge and skills in these areas. Do not worry if you do not feel very confident in some skills – they may be areas that you are hoping to develop by studying these modules.

Now use the interactive tool to rate your confidence in these areas using the following scale:

  • 5 Very confident
  • 4 Confident
  • 3 Neither confident nor not confident
  • 2 Not very confident
  • 1 Not at all confident

This is for you to reflect on your own knowledge and skills you already have.

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1 Principles of sampling and specimen collection

In this section we introduce the basics of sampling and how to collect and process clinical specimens.

1.1 Origins of samples

Clinical samples come from a range of different settings, such as:

  • secondary and tertiary care patients in hospital
  • community patients seen in clinics
  • specialist community clinics and outpatient departments, for example tuberculosis (TB) or sexual health services.

Activity 2: Types of sample

Timing: Allow about 10 minutes

Make notes on the types of samples that are sent to your microbiology laboratory. Compare your response with the sample answer.

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Discussion

Microbiology samples include the following:

  • Swabs, for example from wounds, throats, eyes and ears
  • Genital (cervical and urethral) samples
  • Urine
  • Blood
  • Other normally sterile fluids, such as cerebrospinal fluid (CSF), synovial fluid
  • Stools
  • Sputum – for TB and sometimes other respiratory pathogens
  • Pus and tissue from sterile and non-sterile sites.
Described image
Figure 1 Clinical swabs.

Clinical samples are generally sent to microbiology laboratories to guide antimicrobial therapy (Figure 1). This is done by identifying which pathogens are present in the sample by culturing them. These pathogens can then additionally be tested for antimicrobial resistance (AMR).

  • What factors can prompt the sending of samples?

  • Factors that trigger a request for microbiological tests on a sample include the following:

    • The physician wants to determine if infection is present.
    • Standard antibiotics are not working (treatment failure).
    • A very severe or unusual infection.
    • Local policies or guidelines on sampling.
    • Financial or other external incentives. For example, if a contract means the physician or healthcare organisation gets paid more if extra samples are sent, more are likely to be analysed, some perhaps unnecessarily, leading to a waste of resources. Ideally, the decision to send a sample is made without financial considerations in mind.
    • Local culture or peer pressure, since people are likely to send, or not send, samples for testing according to what their colleagues are doing.
  • What factors can deter the sending of samples, even if clinically necessary?

  • Factors that can deter physicians from sending clinical samples for testing include:

    • difficulty in accessing a laboratory, perhaps due to logistical or transport problems
    • a lack of equipment to obtain samples, for example sterile swabs or blood culture bottles
    • a lack of trust in laboratory test results
    • turnaround times which are too long to be useful
    • cost pressures, for example the patient cannot afford the test or there is only a small or no budget available for testing
    • no local culture of doing tests
    • not understanding the benefits of testing.

Clinical specimens are also routinely used as a source of data for AMR surveillance. However, a significant proportion of patients who could have a microbiology test performed never get sampled and so no results are available for them. This introduces an element of bias into the data. As a consequence, more severe cases and treatment failures could be over-represented. Tertiary care cases could also have more samples sent than those in more remote settings which could affect the overall results if resistance patterns differ between the settings. For long term, sustainable AMR surveillance, however, it is much cheaper and simpler to use clinical samples and the associated data than establishing a study to sample more randomly. The data are also always available whereas a surveillance study might be a one-off event.

Whilst it is not possible to avoid all potential bias when using clinical samples for surveillance purposes, it helps to be aware that reported rates of AMR may not be representative of the country or region as a whole.

1.2 Impact of sample quality on testing and reporting of results

Not all samples received by a laboratory will be of high quality, that is, contaminant free and in sufficient quantity. Prior to starting the testing process, laboratory staff should be aware of potential quality issues relating to specific sample types that could lead to false results.

Blood and CSF are normally sterile; therefore any microorganisms found in samples taken from these parts of the body are likely to be the cause of an infection. However, if there is contamination of the sample during the process of collection, commensal bacteria may be isolated as a contaminant. Similarly, urine samples should also be sterile but are easily contaminated at the time of collection. These contaminants can grow in the sample, making interpretation of the culture results difficult.

It is important to recognise that when samples are taken from most other sites in the body the environment is not sterile. This means that a mix of the normal commensal flora, colonising organisms and contaminants will all be present. For example, normal flora are likely to be found on superficial swabs, genital swabs and in respiratory specimens.

Clinical staff can take several measures to improve the quality of the specimens they send to laboratories, either by reducing contamination and/or increasing the yield of true pathogens. For example, sterile containers and equipment should be used for sample collection, to avoid the introduction of contaminating organisms.

In general, ‘the larger the better’ is a good rule to follow for specimens. Surface swabs just dipped in material make much poorer specimens than a larger, deeper sample because there is less chance of finding the infectious agent amongst the contaminants. A spoonful of faeces is better than a swab dipped in faeces, which is likely to pick up many orders of magnitude more commensals than pathogens. This is likely to make subsequent isolation of the pathogen more difficult.

Some types of sample may require special treatment or have other considerations.

  • Sputum samples, for instance are very useful for diagnosing TB and are frequently sent to diagnose other respiratory tract infections. However, the results have to be considered carefully as the sample is likely to be contaminated with upper respiratory tract flora. Identifying all the organisms in a sputum sample will not help the diagnosis and takes up a lot of laboratory time and resources.
  • Blood cultures, which are important in diagnosing severe and/or bloodstream infections, are another sample type that is easily contaminated. Even though the blood is taken carefully to minimise contamination and inoculated directly into blood culture media by the person taking the samples, contaminants will still sometimes be found. Prof Koch’s guide to perfect blood cultures (2010), shows how careful decontamination of the skin and bottles, plus aseptic technique can minimise sample contamination.
  • Urine samples need to be collected carefully in midstream. The sample should be refrigerated if it cannot be processed immediately. Boric acid can be used as a preservative if this is not possible. Microscopy helps determine whether a urinary tract infection (UTI) is present before culturing by looking for pus cells which indicate inflammation/infection.

Laboratory staff also need to take care not to introduce contaminants into cultures. Good laboratory practice should be followed at all times.

After testing it is important to avoid reporting any additional organisms and instead focus only on the causative pathogen, if found. Correct reporting is needed to guide accurate treatment decisions and for surveillance purposes.

  • What might be the consequence if commensal organisms are reported in test results?

  • The reporting of commensal organisms in microbiology tests can lead to unnecessary or inappropriate antimicrobial treatment. For surveillance, it could make the results inaccurate.

Activity 3: Thinking about contamination in your workplace

Timing: Allow 15 minutes

A Standard Operating Procedure (SOP) is a written step-by-step instruction. SOPs are needed for all the specimen types a laboratory processes to ensure only the relevant pathogens for a specific anatomical site are reported and not normal flora. This ensures that even if contaminated specimens are received, this will not lead to incorrect reporting.

If your laboratory has a SOP for sample processing and reporting, look at this now.

  1. Which organisms should be reported as normal flora and which are likely to be pathogens?
  2. What other types of information does your SOP have about clinical sample processing?

If you do not have access to a relevant SOP you can download a SOP for the processing of skin, superficial and non-surgical wound swabs (Adapted from PHE, 2018).

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Discussion
  1. You should have noted that cultures may contain a mix of pathogens, commensal flora, and sometimes contaminants.
  2. SOPs for sample processing and reporting should include the following:
    • A statement that normal flora are likely to contaminate samples from the body site/sample type under consideration, unless the site is normally sterile.
    • A list of pathogens important for that particular site. Other commensal organisms commonly isolated from the specified site, to be reported as ‘Regional flora’, ‘Normal flora only’ or ‘Likely colonisation’.
    • Guidance for semi-quantitative reporting is helpful e.g. +-; +; ++; +++. This can be used to give the clinician an idea as to whether an organism which could either be colonising or causing an infection, is present in large numbers. For example, if a wound has a few colonies of S. aureus mixed with other skin flora it is less likely to represent an infection than heavy, pure growth of the same organism.
    • Suggested comments to add to reports, for instance, ‘Normal flora only’ or ‘No antibiotics indicated unless there are clear signs of infection present’.
    • Your blood culture SOP is likely to state that everything that grows in blood cultures should be reported, as what appears to be a contaminant may be significant if it grows on several occasions. Organisms which are unlikely to be causing a bacteraemia e.g. coagulase negative staphylococcus in a patient without intravenous (IV) lines, might have an additional comment such as ‘doubtful clinical significance’ or ‘significance uncertain’ to discourage unnecessary antibiotic use. These organisms usually do not need sensitivity testing as this only encourages unnecessary antibiotic use. If your laboratory has a Microbiology or Infectious Diseases physician, they will probably be the one deciding if the isolates are significant or not.

If your laboratory SOP did not include all this information, look at the example provided.

1.3 Transport, labelling and processing of samples

Specimens should be sent to the laboratory as quickly as possible so that the causative agent is still viable (able to replicate and divide) when it gets there, and is not overwhelmed by the growth of commensal organisms or contaminants. A variety of techniques are used to achieve this aim and to minimise the effects of any delay. Refrigeration and carefully designed transport media can keep the organisms viable for longer but add to costs.

Each sample should be uniquely labelled and registered in a database so that it can be attributed to a specific patient, and its analysis prioritised according to the urgency with which the result is required. It is important to make sure results are stored securely to protect the patient’s confidentiality.

Activity 4: Processing specimens in your workplace

Use the question prompts below to think how clinical specimens arriving in your laboratory are processed.

  1. How are samples recorded? Do you have an electronic system or database or are samples recorded in a book? What steps do you take to protect patient confidentiality?
  2. Is there anything that can be improved in your system?
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Discussion

Things to consider are listed below; you may have thought of other points.

Patient confidentiality is important so that personal information in relation to the diagnosis including microbiology requests and results is kept safe – whether this is electronic or paper based. If the laboratory is small, it may be safer and easier to write the results in a book rather than setting up a database. For large numbers of specimens an electronic system is faster, but data must be kept secure and with safeguards in place to prevent data loss if the system crashes.

It is important to understand how the physicians will be using the results and work with them to make sure what you are giving them is useful to them. A close working relationship also helps them understand what the laboratory can and cannot do. Physicians need results as soon as possible to guide treatment. It is useful to monitor ‘turnaround times’ – how long it takes between receiving the specimen and issuing the results. Sometimes issuing interim results, such as the Gram stain result, may also be helpful.

Not all testing of isolates can be carried out in a hospital laboratory and specimens/cultures may need to be sent to a reference laboratory for further processing. For example, some organisms may be difficult to culture or may need more advanced techniques to identify to species level (see Section 3.3).

Activity 5: Preparing samples for transfer to a reference laboratory

Timing: Allow 5 Minutes

What factors do you need to consider when sending isolates to a reference laboratory?

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Discussion

Factors you may have considered are listed below; you may have thought of others.

  • The basic growth requirements for most pathogens are catered for by culture on a nutrient, blood or a chocolate agar slope and stored at room temperature.
  • Fastidious organisms have specific requirements and may also need special transport conditions otherwise they may not survive the journey. It may not always be possible to send these.
  • Some isolates may require refrigeration or freezing, this may not be practical in your laboratory. Refrigerated transport may be required for such samples.
  • If freezing of cultures is essential then it must be done properly, for example with beads and glycerol, so as not to kill the organisms. Once frozen, cultures should not be thawed until use as repeated freeze-thaw cycles lead to deterioration.
  • Safe transport must be arranged for hazardous organisms or samples.
  • It is important to consider how long cultures remain viable. It may be necessary to keep some in reserve and send batches for analysis.

Accurate labelling that ensures patient confidentiality is important. Generally, isolates are given a number that can then be cross-referenced back for full identification.

2 Basic tests used to isolate and identify bacterial pathogens

We turn now to a description of the laboratory tests used to identify pathogens.

2.1 Culture media

Growing isolates from clinical samples in the laboratory is a useful part of diagnosing the cause of an infectious disease and is an essential step for determining the antibiotic sensitivities of bacteria.

All artificial media comprise a mineral base, a nitrogen source, a carbon source and an energy source. Supplements of organic compounds may be added to encourage or suppress the growth requirements of particular microorganisms. Media may be in liquid (broth) or solid form.

Most clinical samples are inoculated directly onto solid, agar-based culture media on a plate. When grown at optimal temperatures in suitable conditions, this process enables the laboratory technologist to view individual colonies and select them for additional work according to their appearance (colonial morphology) and relative abundance. Table 1 provides details of the main media types used routinely in microbiology laboratories. Note that media may fall into more than one of the categories listed, for example it may be both selective and indicator.

Table 1 Media types used in diagnostic microbiology
Media Use
Basic Standard nutritional content for non-fastidious organisms. Also used as a base for other media.
Selective Contains substances (antibiotics, bile salts etc.) which suppress certain organisms. Used for specimens where a lot of competing commensal flora are likely to be found.
Enriched Contains extra nutrients (blood, vitamins etc.) for fastidious organisms which have special growth requirements.
Indicator Contains substances which change colour, for example when carbohydrate fermentation leads to acid production by certain organisms.
Chromogenic Contains chromogenic substrates that change colour in response to the presence of bacterial enzymes. The colour change is species specific, therefore organisms can be identified by colony colour. Allows rapid, basic identification but is relatively expensive and requires well-controlled storage conditions.

Activity 6: Culture media in your workplace

Timing: Allow 10 minutes

Use Table 2 to list the types of agar-based media you have in your laboratory and what you use them for. Note which category of media described in Table 1 each comes into. What considerations govern your laboratory’s choice of media?

Table 2 Media used routinely in your workplace
Name of medium Use Category
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Discussion

You may use many of the media in the example provided or use others not on the list. The media available to your laboratory will depend on factors such as local suppliers, transport and logistics, preferences in your country/region, and cost.

There may be more up-to-date media than the ones you are using as the technology continues to advance. These media save a lot of time, but may be too costly or complicated to use for many district laboratories or for those processing small numbers of specimens. Ready-made media save time in pouring, sterilising, and QC, but are not always available or practical outside of big cities.

Name of agar medium Use Category
Nutrient General purpose; supports growth of a wide range of non-fastidious organisms Basic
Blood For fastidious and non-fastidious organisms, most swabs, subculture of blood cultures Enriched
Chocolate Growing the most fastidious organisms Enriched
CHROMagarTM Orientation urines Identify common pathogens quickly Chromogenic
CLED (cysteine- lactose- electrolyte-deficient) Urine samples: only culture those samples with positive microscopy. This medium has characteristic colony appearance and colour for the common pathogens. It also stops Proteus from swarming over the plate. Selective Indicator
MacConkey Isolate Gram-negatives Selective Indicator
Modified New York City (MNYC) Isolate Neisseria gonorrhoeae Selective Enriched
Mueller-Hinton (M-H) Antimicrobial Susceptibility Testing (AST) – can add supplements for some species Enriched
Xylose Lysine Deoxycholate (XLD) agar Isolate Salmonella and Shigella species Selective Indicator

2.1.1 Processing blood cultures

Blood cultures are inoculated directly from the patient into sterile liquid media. These media may be locally made, or commercial culture bottles can be purchased. The bottles are incubated in the laboratory, observed and sub-cultured. They are plated out onto agar plates for further work to determine if the blood culture is positive. Sub-culturing on a mix of media types is necessary to isolate most of the organisms likely to cause bloodstream infections.

  • Which types of organisms would you expect to isolate when sub-culturing onto blood agar in atmospheric oxygen, MacConkey in atmospheric oxygen, and chocolate agar in CO2.

  • With these culture media and conditions you should be able to isolate the main pathogens causing bloodstream infection. The chocolate agar and the CO2 will allow fastidious organisms like Streptococcus pneumoniae and Neisseria species to grow. Most other organisms will grow well on blood agar in atmospheric conditions. Colonies can be taken from this for the next stages of testing. MacConkey is used to help in the initial identification of Gram-negative organisms (see Section 4.3).

There are also a number of automated systems in common use, such as BD BACTECTM, or BacT/ALERT® (Biomerieux) which use pre-prepared bottles (Figure 2). These can monitor the samples for growth and indicate when it is time to do the sub-culture. Although the equipment is expensive, the risk of contaminants being introduced is reduced and turnaround times are usually faster too, as the subculture can be set up as soon as the equipment indicates positive growth, instead of once daily as is usual for manual blood cultures.

Figure 2 Removing a positive bottle from an automated blood culture machine

2.2 Gram stain

The Gram stain test allows you to see basic bacterial morphology and group bacteria into two main types; Gram-positive and Gram-negative.

Watch Video 1 to see how the test is performed. If you are unable to watch this video, you can view a transcript of the content by clicking on ‘Show transcript’ below.

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Video 1 How to perform a Gram stain
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Note that laboratories without a gas supply can still perform Gram stain tests by using single use sterile plastic loops which are disposed of into disinfectant.

Activity 7: Troubleshooting

The Gram stain may seem like a simple test, but things can still go wrong. Think about your own laboratory practice – why might Gram-positive organisms appear Gram-negative? What measures can you take to prevent this happening?

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Discussion

Factors that can lead to incorrect results for the Gram stain include:

  • excessive heat fixation damaging the cell wall
  • over-decolourisation, so that the stain is washed out
  • old or badly stored iodine
  • using an old culture to do the smear – organisms are damaged.

Measures that can be taken to prevent errors include:

  • following laboratory SOPs exactly
  • making sure reagents are dated and renewed regularly – if reagents are stored long enough bacteria can grow in them and appear on the slides!
  • only using fresh cultures.

3 Commercially available and more advanced tests

Once the specimens have been cultured and single colonies are available growing on a plate, additional testing is done to determine the bacterial species. Many laboratories rely on standard biochemical testing. Biochemical test strips are available which enable multiple tests to be performed at the same time.

3.1 Biochemical test strips

Multiple biochemical tests in a single kit form, come as a strip with micro wells and reagent. The testing process is straightforward; inoculate, incubate, add some more reagents then read and compare the result to a standard. Examples of testing kits include Analytical Profile Index (API, Biomerieux) and Microbact (Thermofisher).

Activity 8: API identification of Gram-negative pathogens

Timing: Allow 15 minutes

In this activity you will first watch a video showing how to set up an API test for the identification of Gram-negative pathogens, and then answer some related questions.

First, watch the video Setting up an API20E (2011).

Note that in the video Enterbacterales are referred to by the old term ‘Enterobacteriaceae’.

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Now, answer the following questions:

  1. Why do you need to pick off individual colonies for the API test?
  2. Would the same API kit be suitable for all of the following: E. coli, Shigella, Salmonella, Klebsiella, Acinetobacter?
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Discussion
  1. Individual colonies are used so you can be sure it is a pure culture. No identification method works on a mixture. This applies to all methods, not just the test strips
  2. No. ‘E’ in API20E stands for Enterobacterales (formerly known as Enterobacteriaceae), so all of these organisms are identified by API 20E except Acinetobacter. Acinetobacter, as a non-glucose fermenter, is not classified as Enterobacterales. A different kit (API 20NE – ‘Non-Enterobacterales’) would be needed to identify Acinetobacter.

Activity 9: Individual biochemical tests versus test kits

Timing: Allow 10 minutes

What are the pros and cons of using individual biochemical tests rather than test kits such as API or similar?

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Discussion
Test type Advantages Disadvantages
Individual
  • Lower cost
  • Only need to stock essential tests
  • Gives enough information to identify most isolates to guide treatment
  • Interesting for laboratory technologist as requires skill
  • Hard to get an accurate identification unless you have all of the required reagents
  • Time consuming
  • Quality control of all reagents is required. Different reagents may need different storage conditions.
  • Requires a high level of expertise
Test strip kits
  • Easier and less time-consuming to perform
  • Gives a numerical answer which can be used to give a relatively accurate identification
  • Rapid 4-hour versions available for some kits
  • Higher cost
  • Most need 24-hour incubation so may take longer to get a result
  • Refrigerated storage required
  • Online access to a Standards database is desirable, though not essential
  • Leads to a loss of laboratory interpretive skills and expertise

3.2 Chromogenic media

Chromogenic media, first mentioned in Section 2.1, are useful in certain situations, for example to identify:

  • organisms in potentially mixed samples – if you have a lot of samples to test (see Figure 3)
  • specific species with a reasonable level of accuracy without the need for additional testing
  • resistant strains/species.

Whilst expensive, this relatively new media type saves a lot of time in laboratories that deal with large numbers of routine urine samples.

Described image
Figure 3 Examples of a chromogenic test plate.

Activity 10: Species identification

Timing: Allow 5 minutes

In Figure 3B, Klebsiella and Enterobacter look the same (metallic blue) and for clinical purposes are usually reported as ‘coliform species – non-E. coli’. However, for management and for surveillance purposes, it may be necessary to identify K. pneumoniae from other coliforms, particularly if there is concern about MDR strains of K. pneumoniae.

What options would you have to differentiate the Klebsiella species from other non-E. coli coliforms?

If you are not able to answer this question now, you can return to it after having studied other sections of this module.

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Discussion
  • API20E, MALDI-TOF (see Section 3.3.1), and automated systems would all be able to differentiate these bacteria. However, if none of these is available, motility and biochemical tests are performed. Although these two organisms are very similar Enterobacter is motile whereas Klebsiella is not so the motility test is a useful way of discriminating between them.
  • The additional tests might be too time-consuming and/or expensive to do on every isolate just for surveillance. However, if the tests are not done then this could potentially be a source of bias and error in AMR data.
  • One option is to send the sample to a reference laboratory for testing, but if only the more resistant isolates are sent this would again be a potential source of bias and error in AMR data.
  • A better option might be to implement a sampling framework where a proportion of isolates received by the laboratory are identified for AMR surveillance purposes.

3.3 Advanced testing

Some testing methods are beyond the scope or resources of the hospital laboratory and are more likely to be found in public health and reference laboratories. MALDI-TOF and automated systems are two methods found increasingly in larger clinical laboratories. Most molecular methods (e.g. Whole Genome sequencing, 16S rDNA PCR, molecular typing) are currently only used widely for research and in reference laboratories. We describe some of these methods below.

3.3.1 MALDI-TOF

Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (Croxatto et al., 2012) gives a result faster than most standard biochemical test methods and at a lower cost per sample. It is both accurate and reliable, and gives identification to species level from a pure culture within minutes. In recent years, MALDI-TOF has revolutionised microbiology practice in laboratories where it is used, having largely replaced most biochemical identification methods.

Disadvantages of MALDI-TOF are that the equipment is expensive and often requires expensive maintenance contracts. It is also not effective for all organisms. For example, it cannot distinguish well between E. coli and Shigella, nor between S.pneumoniae and viridans type Streptococci. For these, additional biochemical testing is still needed for confirmation.

The rapid throughput means that hundreds of isolates can be processed daily using very little technologist time, meaning that it is most cost-effective for laboratories with large sample throughputs (identifying several thousand isolates per year). For these laboratories, the cost of the instrument and the maintenance contracts is very rapidly offset in savings on reagents and staff time.

It is possible to use MALDI-TOF to identify a small number of resistance mechanisms (e.g. for MRSA) but it is still necessary to perform separate antibiotic susceptibility tests (AST).

If you are not familiar with MALDI-TOF, watch the following short video about this method (Theory of MALDI-TOF Mass Spectometry, 2016).

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3.3.2 Automated systems

Automated systems, such as Vitek II (Biomerieux), BD Phoenix (Beckton Dickinson) and Beckman-Coulter-MicroScan are used in some clinical laboratories. Effectively these are an automated version of the biochemical test strips, with more reagents and analytic software. The cost of such systems is high, but less work is needed to complete the tests. A further advantage is that AST can be done at the same time.

3.3.3 Commercial PCR

Commercial PCR is often used for example, with enteric pathogens or for Methicillin-resistant Staphylococcus aureus (MRSA) detection. Advantages are the reduced work involved, as PCR tests can run on an automated ‘plug-in and play’ platform, and the rapid turnaround time for results – the same day. However, commercial PCR tests are expensive, and isolates must still be cultured if AST is needed. Most kits are specific for one or two bacterial species so are more useful for screening or confirmatory testing for specific target organisms, rather than testing for all the clinical pathogens that might be in a sample.

4 Identifying the key pathogens in global AMR surveillance

4.1 The eight key pathogens

Eight key bacterial pathogens in humans have been identified by the World Health Organization (WHO) as the focus of the GLASS surveillance programme for AMR (Table 3) (WHO, 2015). These are all pathogens that a hospital laboratory would normally be able to identify from one or more of four specimen types.

Table 3 WHO key pathogens for global AMR surveillance
Pathogen Gram reaction
Staphylococcus aureus Gram positive
Streptococcus pneumoniae
Acinetobacter species Gram negative
Escherichia coli
Klebsiella pneumoniae
Neisseria gonorrhoeae
Salmonella species
Shigella species

E. coli, K. pneumoniae and Acinetobacter species have been selected by WHO for AMR surveillance as all have globally widespread strains which are multi-drug resistant (MDR), even pan-resistant to antibiotics. Resistance can spread readily between the species and these organisms have proven capacity for outbreaks and spread between institutions. Such resistant strains are harder and more expensive to treat, wasting resources and posing a threat to modern medicine. For example, patients on chemotherapy need frequent antibiotics both for treatment and prophylaxis. If they encounter resistant organisms, then these patients have a high risk of dying from infection even if their cancer was curable.

Enteric pathogens like Salmonella and Shigella are included in the GLASS surveillance programme due to increasing AMR and public health importance. For Salmonella there are broadly two areas of concern: zoonotic infections causing diarrhoea, and sometimes invasive infection in patients with other infections such as HIV or malaria, and S. typhi and paratyphi causing enteric fever. For Shigella, these organisms are highly transmissible by the faeco-oral route, and AMR is a problem in areas with poor sanitation.

  • Why has N. gonorrhoeae been chosen out of all the pathogens causing sexually transmitted infection (STI) as a focus for surveillance?

  • Infection with N. gonorrhoeae leads to a lot of morbidity including pelvic inflammatory disease and infertility. Resistance is a big problem – especially to oral antibiotics. Some strains are already only sensitive to IV antibiotics, and untreatable strains are a becoming a possibility. N. gonorrhoeae can be cultured in a clinical laboratory whereas it is not possible to test the organisms that cause Chlamydia, Syphilis etc. for AMR. Resistance is, fortunately, less of an issue so far with these organisms.

Activity 11: Usual isolation site of key pathogens

For each of the images below, can you identify the site you would expect them to be isolated from? Note: for some pathogens, there may be more than one site.

Click 'reveal discussion' to see the answer.

S. aureus tends to form grape-like clusters with each bacterium being up to 1 μm in diameter.

Which site or sites in the body is S. aureus isolated from?

Discussion

Answer: S. aureus is a common cause of infection in the community and of healthcare associated infection (HCAI). Sepsis or bloodstream infection mostly starts from an infective focus such as a wound, infected IV line or osteomyelitis. S. aureus can also cause skin and soft tissue infections such as boils and abscesses. MRSA strains of S. aureus are a particular threat in healthcare settings. A significant percentage of people carry S. aureus asymptomatically in their nose and on the skin. It can lead to infection if the body’s defences are breached, for example by a wound or medical procedure.

For GLASS, S. aureus isolated from bloodstream samples are the focus of interest.

S. pneumoniae usually forms pairs (diplococci) or occasionally short chains.

Which site or sites in the body is S. pneumoniae isolated from?

Discussion

S. pneumoniae is a common cause of pneumonia, meningitis and ear infections, as well as infections in HIV patients. It is typically isolated from bloodstream samples in severe infections. It can also be found in upper respiratory and surface swabs, sputum and CSF cultures.

GLASS surveillance focuses on bloodstream infection isolates.

A. baumannii

Acinetobacter are gram-negative, coccobacillary-shaped bacteria. A. baumannii accounts for most Acinetobacter infections in humans and is the species of concern. However, identification to species level is technically challenging so many sites report as Acinetobacter species only.

Which site or sites in the body are Acinetobacter species isolated from?

Discussion

Acinetobacter are classic HCAI pathogens, for example affecting patients in intensive care, particularly with open wounds or on a ventilator. Acinetobacter are typically isolated from the bloodstream, wound swabs, sputum and rarely from urine.

For GLASS, bloodstream isolates are the focus of interest.

E. coli is a typical rod-shaped, Gram-negative bacterium, about 2 µm long.

Which site or sites in the body is E. coli isolated from?

Discussion

E. coli is the most common cause of UTI and a leading cause of bloodstream infection. Some strains have specific virulence factors allowing them to infect the urinary tract (Sarowski et al., 2019). Different pathogenic strains of E. coli are a common cause of diarrhoeal disease, for example as a result of food poisoning. E. coli are also a common cause of intra-abdominal infections such as Cholecystitis.

E. coli is typically isolated from bloodstream and urine. They can also be isolated from stools, but this requires special techniques to distinguish the pathogenic strains from commensal E. coli which is prevalent in the gut.

For GLASS, isolates from bloodstream and urine are the focus of interest.

K. pneumoniae is a typical rod-shaped, Gram-negative bacterium about 2 µm long.

Which site or sites in the body is K. pneumoniae isolated from?

Discussion

K. pneumoniae is a common cause of HCAI including UTI, pneumonia, and bloodstream and wound infections.

K. pneumoniae is typically isolated from bloodstream and urine samples and GLASS focuses on these. It can also be found in swabs and sputum samples, but it is harder to be sure it is acting as a pathogen rather than colonising these sites.

N. gonorrhoeae forms Gram-negative diplococci, 0.6–1 μm in diameter.

Which site or sites in the body is N. gonorrhoeae isolated from?

Discussion

N. gonorrhoeae causes the STI gonorrhoea and is commonly isolated from genital samples, and occasionally bloodstream samples in cases of disseminated infection.

For GLASS, N. gonorrhoeae isolated from genital samples are the focus.

S. typhimurium is an example of Salmonella species. Salmonella are Gram-negative, rod-shaped bacteria about 1–3 µm in length.

Which site or sites in the body are Salmonella species isolated from?

Discussion

Enteric fever is caused by S. typhi/paratyphi with the other non-typhoidal strains causing diarrhoeal disease. Enteric fever strains only affect humans, but the other strains can also be transmitted via animals and animal products. This is important in the One Health context.

For GLASS, Salmonella isolated from both stool and blood samples are the focus.

Shigella are Gram-negative, rod-shaped bacteria typically 1–3 μm in length.

Which site or sites in the body are Shigella species isolated from?

Discussion

Shigella species cause diarrhoeal disease and cause significant morbidity in young children.

For GLASS, Shigella isolated from stool samples are the focus.

Your laboratory is almost certainly identifying other organisms than the ones listed in Activity 11. However, the focus of this module is on some of the issues around identifying the eight key GLASS pathogens under global AMR surveillance. Your laboratory should have comprehensive SOPs for isolating and identifying these organisms. In the section that follows we discuss some important considerations about the identification of each of them.

4.2 Identifying Gram-positive cocci

Your laboratory needs to be able to reliably confirm the identification of S. aureus and S. pneumoniae (see Figure 4).

Described image
Figure 4 How to identify S. aureus and S. pneumoniae.

Enterococci can show any of the three patterns of haemolysis. They are generally Lancefield Group D can be confirmed using biochemical tests such as aesculin hydrolysis or litmus milk reduction. They ferment a range of sugars including lactose.

For S. aureus, a presumptive identification is done by the characteristic appearance of colonies on agar and a positive catalase test. A minimum of two tests from the following must be used to confirm identification: tube coagulase, slide coagulase, or DNAse. Alternatively, MALDI-TOF can be used.

  • Why is more than one test needed to confirm S. aureus?

  • Bacteria, being living organisms, will occasionally not test the way the book says they should, so more than one test is needed to confirm. Some ‘coagulase negative’ Staphylococci will test positive on one of the coagulase tests. This is important for clinical reasons, to give the right treatment, and also for AMR surveillance. For example, methicillin-resistant, coagulase-negative Staphylococcus could be mis-reported as MRSA leading to inappropriate and unnecessary antibiotics and providing inaccurate surveillance data.

  • What system of controls for tests should be in place?

  • For any identification, not just for S. aureus, it is important to use positive and negative control organisms to make sure your reagents are working correctly. Ideally these organisms are reference strains obtained from the UK National Collection of Type Cultures (NCTC) or the American Type Culture Collection (ATCC). For these tests the controls are set up in parallel with the test organism.

S. pneumoniae is fastidious and hard to grow (Figure 5). For cultures grown in conditions that should support its growth, such as on blood agar under CO2, the organism is suspected by its typical appearance on solid media. This, plus a positive optochin sensitivity or bile solubility test, is usually sufficient to confirm species. Latex agglutination tests are also available. If using MALDI-TOF, optochin or bile solubility testing must also be performed, as MALDI-TOF is, at present, unable to distinguish between S. pneumoniae and viridans Streptococci.

Described image
Figure 5 Appearance of S. pneumoniae colonies on blood agar.

Activity 12: Testing for S. pneumoniae in your workplace

Timing: Allow 10 minutes

Think about your workplace activities and consider these questions.

  1. If you are able to culture and identify S. pneumoniae reliably in your laboratory, how are you differentiating it from other streptococcal species?
  2. If isolation of this organism is not reliable, or you do not usually work with this organism, what might be practical barriers to its identification?
  3. How might laboratory practice, for example the ease of isolating S. pneumoniae, affect AMR surveillance for this organism?
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Discussion
  1. Differentiation from other streptococcal species can be by appearance, haemolysis, specific confirmatory tests and by the fact that it only grows on blood-containing media under CO2.
  2. CO2 for incubation, and blood-containing media are both necessary for growth so these are limiting factors. Autolysis can occur in blood cultures. This is when the organism grows so fast in some media that it outstrips its nutrient supply, and no viable organism is detected on subculture. So you will need to have access to the right culture media and conditions to isolate this organism.
  3. Under-reporting from some laboratories who struggle to isolate the organism versus better equipped laboratories, could lead to bias if antibiotic resistance rates varied between the settings.

4.3 Identifying Gram-negative organisms

Gram-negatives are found in a range of clinical samples. As they are common causes of UTI, urine culture media are designed to isolate them. They are also an important cause of bloodstream infection – either Gram-negative sepsis, for example from a urinary source, biliary sepsis or an IV line infection, or as enteric fever caused by Salmonella typhi or paratyphi so can be isolated from blood cultures. When found in superficial swabs they are often growing as contaminants or colonising an existing wound.

The approach taken to identify Gram-negative organisms depends on several factors.

  1. The site of the body where the sample was taken. In Activity 11, for example, you learned which type of sample would be most likely to yield which key pathogen.
  2. The likelihood of the sample from this site containing a mixture of organisms – commensals or contaminants
  3. Whether there is already a presumptive identification. For example, stool samples are usually cultured on media which selects for enteric pathogens. When S. typhi and S. paratyphi are suspected, blood cultures may also be taken to look for these organisms. The identification process of these organisms from blood cultures will be longer than from stool samples as you do not have the benefit of seeing the appearance on the selective medium.

In practice, identification of Gram-negative organisms often proceeds by a process of elimination; one positive test result dictates the choice of a secondary test and so on. This process requires laboratory technologists to have the appropriate knowledge and skills to perform this screening process.

The following sections outline how the six Gram-negative key GLASS surveillance pathogens are identified for laboratories without access to MALDI-TOF/automated systems.

4.3.1 Enterobacterales species

E. coli and K. pneumoniae are two amongst a variety of coliforms (Enterobacterales).

Your laboratory is likely to be using a differential, indicator medium based on lactose fermentation – usually either CLED or MacConkey – to isolate these organisms from urine samples (Figure 6). For blood samples you will most likely be identifying all Gram negatives.

Figure 6 (A) Yellow, lactose-fermenting colonies of E. coli on CLED. (B) A non-lactose fermenter (NLF) on CLED showing no significant change to the greenish-blue colour of the medium.
Activity 13: Identifying Enterobacterales in your laboratory
Timing: Allow 10 minutes

How do you identify the different Enterobacterales species (also known as Enterobacteriaceae or coliforms) in your laboratory? Make some notes and then compare with the sample answer.

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Discussion

There are a number of ways of identifying Enterobacterales species depending on budget, number of specimens handled, and relative cost of staffing time versus reagents, available equipment etc.

Methods you may have listed include:

  • Indicator media.
  • Motility test, biochemical tests (for example, indole test, Voges–Proskauer test etc.)
  • Commercial options such as disk forms of the biochemical tests, for example Rosco DiatabsTM or biochemical strip tests, for example API or Microbact™
  • Automated systems or MALDI-TOF.

The UK Standards for Microbiology Investigations (SMI) Collection (PHE, n.d.) provides guidance for a wide range of appropriate tests used by laboratories in well-resourced settings. Alternative guidance for laboratories in resource poor settings is provided by Cheesbrough (2006).

Figure 7 How to identify Enterobacterales for laboratories with access to basic tests only (adapted from Yong Ng et al., 2010).
  • Why is it useful to identify Enterobacterales to species level for GLASS?

  • If the species is known, it can make it easier to spot when an organism has deviated from expected patterns of behaviour or developed mechanisms of resistance. For example, a plasmid may have jumped species or the organism acquired new resistance genes. It then becomes possible to focus on tracking and controlling the organism. On a local scale you can identify and control outbreaks quicker.

Resistant organisms of some species are more important pathogens from the Public Health point of view than others. For example, K. pneumoniae can cause big HCAI outbreaks, and there are widespread transmissible MDR uropathogenicE. coli strains (Pitout and DeVinney, 2017). Other Enterobacterales species, however, tend to be found more sporadically.

K. pneumoniae is the only Klebsiella species which is a current focus for GLASS, because it is responsible for the majority of Klebsiella infections, including drug-resistant infections. Other Klebsiella species can still cause severe infections but have not been responsible for such extensive outbreaks. In practice, identifying as far as Klebsiella is enough for clinical purposes and many laboratories will not have the facilities for identification to species level. In this case, further identification of Klebsiella isolates, for example MDR strains or from outbreaks, will have to be done at a reference laboratory.

As discussed in Section 3.2 though, only sending the resistant ones to the reference laboratory for species identification could lead to bias in the surveillance figures. This would make it hard to tell the prevalence of the resistant strains in the area accurately. However, it has the major benefit of allowing you to be aware of which resistant Klebsiella species and strains are present in your hospital.

4.3.2 Enteric pathogens

Your laboratory will almost certainly identify a wide variety of enteric pathogens from stool samples. For GLASS, the focus is on Salmonella and Shigella.

The first step in identification is the appearance of colonies on solid media. For example, on XLDShigella colonies are red, while Salmonella are pink with a black middle (Figure 8). Both organisms have pale colonies on MacConkey agar as they are non-lactose fermenters (NLF). They are also urease negative.

Described image
Figure 8 Salmonella colonies in a mixed culture on XLD agar.
  • Why is it important to do a urease test?

  • NLF enteric organisms, such as Proteus and related species (Providentia, Serratia and Morganella) have a similar appearance on solid media. It is therefore important to do a urease test to prevent mis-identifying these as enteric pathogens.

    Confirmatory tests for Salmonella and Shigella include:

    • a negative oxidase test – to differentiate from other NLF organisms like Pseudomonas
    • Kligler’s Iron Agar (KIA) test
    • biochemical tests
    • antisera agglutination tests

    For details of all tests, see the UK SMI guidance for Salmonella (PHE, 2015a) and Shigella (PHE, 2015b)

4.3.3 Acinetobacter

Acinetobacter are non-fermenting organisms which can be differentiated from other non-fermenters such as Pseudomonas species because they are oxidase negative (Figure 9). Biochemical tests like API would be needed to confirm if necessary, but phenotypic tests to identify at species level are not very reliable and identification to species level requires specialist molecular testing. MDR isolates are best confirmed at the reference lab (Vijayakumar et al., 2019).

Figure 9 Oxidase test – used to distinguish Acinetobacter from Pseudomonas species.

4.3.4 Neisseria gonorrhoeae

N. gonorrhoeae is a fastidious and delicate organism and requires careful handling. It doesn’t survive drying or temperatures much below body temperature. It is cultured on special media such as Modified New York City (MNYC) or Thayer Martin media, and grown in a CO2 enriched atmosphere at 35–36 ºC, otherwise it will not grow at all.

Initial identification from culture media is by Gram stain (Gram-negative cocci) and oxidase test (positive). Confirmatory tests include:

  • individual biochemical tests or test strips (e.g. API-NH, Biomerieux)
  • immunological tests, for example a slide-based co-agglutination test of which there are many commercial kits available (e.g. Phadebact® Monoclonal GC Test, MKL diagnostics).
  • Why is it necessary to confirm identification of N. gonorrhoeae with more than one test?

  • A lot of commensal Neisseria species can be found in the genital tract and it is important to distinguish between these and the pathogenic N. gonorrhoeae. Getting this wrong can have negative repercussions for the patient and their relationships as well as for AMR surveillance. This particularly applies if the patient is not known to be at high risk of STI.

5 Fundamentals of quality control for isolating and identifying bacteria

In a clinical microbiology laboratory several factors can affect the accuracy and reliability of test results. Quality control (QC) measures are therefore put in place to monitor whether tests perform as expected and do so reliably. You will learn more about QC and other Quality procedures in the Quality assurance and AMR surveillance module.

Activity 14: QC in clinical microbiology laboratories

Timing: Allow 5 minutes

Think about what you have learned in this module. Why is QC important when identifying pathogens from clinical samples?

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Discussion

QC ensures the following:

  • You can actually grow the organisms you’re looking for.
  • Selective media are inhibiting the appropriate organisms and allowing the growth of others.
  • You are identifying organisms correctly.
  • Your media and test reagents are working correctly, for example if they have deteriorated during storage or there is something wrong with what the manufacturer has supplied, the QC tests will pick this up.
  • You are performing the tests under optimal conditions, for example the incubator is at the right temperature.
  • Organisms are correctly identified before AST, which is species-specific, is performed.
  • The results from the laboratory can be relied on as accurate for both clinical and surveillance use.
  • Ultimately patients get better treatment as a result of timely and accurate diagnosis.

6 Identifying pathogens in your workplace

You should now have a better understanding of the microbiology techniques used in a variety of settings for identifying bacteria, and in particular the GLASS priority pathogens. Before you finish the module, complete this final activity, which will help you think about your laboratory’s diagnostic capacity.

Activity 15: Identifying pathogens in your workplace

Timing: Allow 20 minutes

Think about the eight GLASS priority pathogens and then reflect and make notes in response to the following questions.

  1. Are you identifying all of these pathogens to the required level in your laboratory?
  2. Are you confident that the identification is accurate?
  3. Are there areas where you feel your laboratory may not be reliably identifying pathogens? Which organisms does this apply to?
  4. How could the diagnostic capacity of your laboratory be improved and what are the barriers to achieving this? For example, what extra equipment and reagents might you need? Are the correct controls being used? Do staff need more training?

Finally, think about how you would make a case to your Laboratory Manager to convince them to support you in improving your laboratory’s diagnostic capacity.

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Discussion

You may find it helpful to discuss your thoughts with colleagues before talking to your Laboratory Manager.

7 End-of-module quiz

Well done – you have reached the end of this module and can now do the quiz to test your learning.

This quiz is an opportunity for you to reflect on what you have learned rather than a test, and you can revisit it as many times as you like. 

Open the quiz in a new tab or window by holding down ‘Ctrl’ (or ‘Cmd’ on a Mac) when you click on the link.

8 Summary

In this module you have learned about microbiology tests used to isolate and identify key human pathogens which are the focus of the WHOGLASS programme. Some of these tests are routinely performed in a hospital microbiology laboratory while other, more advanced techniques are used only in reference laboratories. You have also been introduced to factors related to sample quality and sample handling which can negatively impact testing, and to QC measures which can help ensure the accuracy and reliability of test results.

You should now be able to:

  • know where samples are obtained from and reflect on the process by which bacterial samples are processed in your workplace
  • describe the principles of laboratory tests used to isolate and identify key bacterial pathogens in human health, that are the focus of the GLASS programme
  • know when and why advanced testing such as mass spectrometry and automated systems are used
  • know the importance of procedures designed to ensure the quality of laboratory work relating to isolating and identifying bacteria in your workplace.

Now that you have completed this module, consider the following questions:

  • What is the single most important lesson that you have taken away from this module?
  • How relevant is it to your work?
  • Can you suggest ways in which this new knowledge can benefit your practice?

When you have reflected on these, go to your reflective blog  and note down your thoughts.

Activity 16: Reflecting on your progress

Timing: Allow 10 minutes

Do you remember at the beginning of this module you were asked to take a moment to think about these learning outcomes and how confident you felt about your knowledge and skills in these areas?

Now that you have completed this module, take some time to reflect on your progress and use the interactive tool to rate your confidence in these areas using the following scale:

  • 5 Very confident
  • 4 Confident
  • 3 Neither confident nor not confident
  • 2 Not very confident
  • 1 Not at all confident

Try to use the full range of ratings shown above to rate yourself. 

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When you have reflected on your answers and your progress on this module, go to your reflective blog and note down your thoughts.

9 Your experience of this module

Now that you have completed this module, take a few moments to reflect on your experience of working through it. Please complete a survey to tell us about your reflections. Your responses will allow us to gauge how useful you have found this module and how effectively you have engaged with the content. We will also use your feedback on this pathway to better inform the design of future online experiences for our learners.

Many thanks for your help.

Now go to the survey.

References

Cheesbrough, M. (2006) District laboratory practice in tropical countries: Part 2 [Online]. Available from https://all-med.net/ pdf/ district-laboratory-practice-in-tropical-countries/ (Accessed 19 February, 2021).
Croxatto, A., Prod’hom, G. and Greub, G. (2012) Applications of MALDI-TOFF mass spectrometry in clinical diagnotstic microbiology [Online]. DOI: https://doi.org/ 10.1111/ j.1574-6976.2011.00298.x (Accessed 22 February, 2021)
Ng, L.S.Y., Thean Yen Tan, T. Y. and Yeow, S.C.S. (2010) A cost-effective method for the presumptive identification of Enterobacteriaceae for diagnostic microbiology laboratories [Online] DOI: https://doi.org/ 10.3109/ 00313021003631338 (Accessed 19 February, 2021)
OpenStax (2021) Microbiology [Online]. Available from: https://openstax.org/ details/ books/ microbiology (Accessed 23 February, 2021).
Pitout, J.D.D. and DeVinney, R. (2017) Escherichia coli ST131: a multidrug-resistant clone primed for global domination [Online]. Available from: https://www.ncbi.nlm.nih.gov/ pmc/ articles/ PMC5333602/ (Accessed 19 February, 2021).
Prof Koch’s guide to perfect blood cultures (2010) Vimeo video added by Lindsey Knight [Online]. Available from: https://vimeo.com/ 7978606 (Accessed 19 February, 2021).
Public Health England (PHE) (2015a) UK Standards for Microbiology Investigations ID 24: identification of Salmonella species [Online]. Available from: https://www.gov.uk/ government/ publications/ smi-id-24-identification-of-salmonella-species (Accessed 19 February, 2021).
Public Health England (PHE) (2015b) UK Standards for Microbiology Investigations ID 20: identification of Shigella species [Online]. Available from: https://www.gov.uk/ government/ publications/ smi-id-20-identification-of-shigella-species (Accessed 19 February, 2021).
Public Health England (PHE) (2018) UK Standards for Microbiology Investigations B11 (6.5): Investigation of swabs from skin and suerficial soft tissue infections [Online]. Available from: https://assets.publishing.service.gov.uk/ government/ uploads/ system/ uploads/ attachment_data/ file/ 766634/ B_11i6.5.pdf (Accessed 22 February, 2021).
Public Health England (PHE) (n.d.) Collection: Standards for microbiology investigations (UK SMI) [Online]. Available from https://www.gov.uk/ government/ collections/ standards-for-microbiology-investigations-smi (Accessed 19 February, 2021)
Sarowska, J., Futoma-Koloch, B., Jama-Kmiecik, A., Frej-Madrzak, M., Ksiazczyk, M., Bugla-Ploskonska, G. and Choroszy-Krol, I. (2019) Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports [Online]. DOI: 10.1186/ s13099-019-0290-0 (Accessed 22 February, 2021)
Setting up an API20E (2011) YouTube video, added by Dr Kimmitt [Online]. Available from: https://www.youtube.com/ watch?v=PXIis18qN9k (Accessed 19 February, 2021).
Theory of MALDI-TOF Mass Spectrometry (2016) YouTube video, added by ARCC Chem [Online]. Available from: https://www.youtube.com/ watch?v=8R1Oyqx5KfE(Accessed 24 February, 2021).
Vijayakumar, S., Biswas, I. and Veeraraghavan, B. (2019) Accurate identification of clinically important Acinetobacter spp.: an update [Online]. DOI: https://doi.org/ 10.2144/ fsoa-2018-0127 (Accessed 19 February, 2021).
World Health Organization (WHO) (2015) Global Antimicrobial Resistance Surveillance System: Manual for early implementation [Online]. Available from: https://apps.who.int/ iris/ bitstream/ handle/ 10665/ 188783/ 9789241549400_eng.pdf;sequence=1 (Accessed 19 February, 2021).

Acknowledgements

This free course was collaboratively written by Liz Sheridan and Sarah Palmer, and reviewed by Priya Khanna, Melanie Bannister-Tyrrell, Skye Badger, Claire Gordon, Natalie Moyen and Hilary MacQueen.

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:

Images

Module image: Yulia Koltyrina/123RF.

Figures 1, 2 and 4: L. Sheridan.

Figures 3, 5, 6, 8 and 9: E. Tinnion and L. Sheridan.

Activity 11 (top to bottom): S. aureus: National Institutes of Health/Stocktrek Images/Getty Images; S. pneumoniae: Eye of Science/Science Photo Library; A. baumannii: Janice Haney Carr (public domain image); E. coli: David McCarthy/Science Photo Library; K. pneumoniae: Cultura Creative RF/Alamy Stock Photo; N. gonorrhoeae: National Institute of Allergy and Infectious Diseases, National Institutes of Health, ‘Neisseria gonorrhoeae bacteria’, https://www.flickr.com/ photos/ nihgov/ 24503565430 – this file is licensed under a Creative Commons Attribution-NonCommercial 2.0 Generic (CC BY-NC 2.0) licence, https://creativecommons.org/ licenses/ by-nc/ 2.0/; S. typhimurium: Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin, Germany in ‘A novel data-mining approach systematically links genes to traits’ (2005) PLOS Biology 3(5): e166, https://doi.org/ 10.1371/ journal.pbio.0030166 – this file is licensed under a Creative Commons Attribution 2.5 Generic (CC BY 2.5) licence, https://creativecommons.org/ licenses/ by/ 2.5/; Shigella: Stephanie Rossow/Science Photo Library/Getty Images.

Figure 7: adapted from Ng, L.S.Y., Thean Yen Tan, T.Y. and Yeow, S.C.S. (2010) ‘A cost-effective method for the presumptive identification of Enterobacteriaceae for diagnostic microbiology laboratories’ [online] DOI: https://doi.org/ 10.3109/ 00313021003631338

Tables

Table 3: based on Global Antimicrobial Resistance Surveillance System: Manual for Early Implementation, World Health Organization, surveillance methods, p. 5, copyright 2015, https://apps.who.int/ iris/ bitstream/ handle/ 10665/ 188783/ 9789241549400_eng.pdf .

Text

Activity 3: adapted from Public Health England (2018) ‘UK Standards for Microbiology Investigations: investigation of swabs from skin and superficial soft tissue infections’ available online at: https://www.gov.uk/ government/ publications/ smi-b-11-investigation-of-skin-superficial-and-non-surgical-wound-swabs – reproduced under the terms of the Open Government Licence v3.0, https://www.nationalarchives.gov.uk/ doc/ open-government-licence/ version/ 3/.

Video

Video 1: The Open University.

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