Antimicrobial susceptibility testing

Introduction

Microbiology laboratories have an important role in tackling antimicrobial resistance (AMR), largely through routinely performed antimicrobial susceptibility testing (AST). AST is used to detect resistance in clinically important bacteria and provide information about which antibiotic therapeutic options will be effective against a given bacterial pathogen. Standardised AST data are also used to track the emergence and spread of antibiotic resistance in human and animal populations, and are an integral part of a One Health approach to AMR surveillance.

This module focuses on phenotypic methods used for AST. These rely on observable characteristics of a bacterial strain, such as whether it can grow at particular concentrations of the antibiotic being tested. Phenotypic methods for AST are based on measuring the minimum inhibitory concentration (MIC) of an antibiotic agent, and the availability of a clinical breakpoint. These terms are explained in the following sections.

It is also possible to use genotypic methods to find out whether an antibiotic will work against a pathogen. Genotypic methods detect the presence of resistance genes or mutations conferring resistance in a bacterial isolate. To get the full resistance profile for an organism, however, would require multiple different commercial test kits or whole genome sequencing; at the moment, phenotypic methods remains more widely used in clinical laboratories. Genotypic methods are covered in the Testing for methods of resistance module.

After completing this module, you will be able to:

• describe the principles of AST
• understand the concepts of phenotypic methods for AST
• understand the process of disk diffusion testing and the relevant quality measures needed
• understand the principles of other phenotypic methods, including automated methods
• understand that the method used may depend on the organism being tested
• understand the concept of MICs and breakpoints
• recognise relevant international guidelines, and know where to find them and when to use them
• recognise that additional testing may be needed to identify/confirm resistance mechanisms
• recognise the importance of procedures designed to ensure the quality of AST testing in your workplace.

1 The theory underpinning AST

You should be familiar with the Introducing antimicrobial resistance module before starting this section.

AST determines which antibiotics a specific microorganism is susceptible to. This information is used to predict which antibiotic therapies are likely be effective.

Susceptible microorganisms are inhibited or killed by the antibiotic, and it is inferred that infections caused by these organisms can be treated with the antibiotic. Conversely, resistant microorganisms are not inhibited by the recommended antibiotic dose, so an infection caused by a resistant microorganism is unlikely to be cured by the antibiotic.

1.1 Minimum inhibitory concentrations

The minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic agent that inhibits the visible growth of a micro-organism. MICs are determined by incubating the micro-organism with the antibiotic agent at a range of different concentrations.

The classic test is broth dilution: this is done by doing doubling dilutions of the antibiotic agents to give a range of concentrations, each one twice as concentrated as the one before. Each tube in the dilution series is then inoculated with a fixed amount of bacterial inoculum. The tubes are incubated, and after 18–24 hours the dilution series is examined for bacterial growth which appears as turbidity, or cloudiness (Figure 1). A variation is to use a microdilution method: again, the doubling dilutions are used and bacterial inoculum added, but this time the test is performed using much smaller volumes, usually in a microtitre plate. The turbidity can be read visually, or using a special plate reader. Broth dilution or microdilution methods are considered the Gold Standard for AST; however, broth dilution is rarely used in routine diagnostic laboratories. The microdilution method is often used in automated systems, which you will learn about later in the module.

Figure 1 Determining the MIC using a manual broth dilution method. Tubes containing increasing concentrations of antibiotic (from right to left in the figure) and a fixed amount of inoculum are examined for turbidity following incubation.

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A photo of seven test tubes with increasing concentrations of antibiotic: 64 µg/ml, 32 µg/ml, 16 µg/ml, 8 µg/ml, 4 µg/ml, 2 µg/ml and 1 µg/ml.

Figure 1 Determining the MIC using a manual broth dilution method. Tubes containing increasing concentrations of antibiotic (from right ...

It is important to note that the MIC of an antibiotic is the concentration at which this antibiotic is bacteriostatic – it stops bacterial growth. However, the test does not show whether there are any viable bacteria still in the suspension. The concentration of antibiotic that kills bacteria is known as the minimum bactericidal concentration (MBC). The MBC will often be a higher concentration of antibiotic than the MIC. You would need to subculture the clear tubes to see if any live bacteria were still present in order to find the MBC.

MICs are used to evaluate the antibiotic efficacy of compounds: the lower the MIC, the less antibiotic is required to inhibit the growth of an organism, so the more susceptible it is. However, the value of a MIC is influenced by the method used to determine it, so MICs are only meaningful if they are compared to other values that were obtained using the same standardised method.

MICs are not very useful clinically on their own to predict treatment success or failure. It is therefore necessary to set breakpoints using the MIC. If the MIC of an isolate is below the breakpoint, the organism is considered susceptible. Breakpoints are discussed in the following section.

1.2 Interpretive criteria

A breakpoint is the MIC that is considered to be the cut-off between susceptible and resistant isolates. It can be an epidemiological cut-off (ECOFF) value or a clinical breakpoint; in both cases, values above the breakpoint signify resistance, while values below the breakpoint signify susceptibility.

1.2.1 Epidemiological cut-off (ECOFF)

The ECOFF is defined as the highest MIC value of isolates that are not known to have resistance and are therefore considered representative of wild type bacterial isolates. Bacteria are defined as ‘wild type’ in the absence of a detectable acquired or mutational resistance mechanism to the antibiotic being considered. ECOFFs reflect the ability to detect resistance mechanisms that may, or may not, be clinically significant. As noted above, ECOFFs are distinct from clinical breakpoints. Clinical breakpoints distinguish between isolates where antibiotic therapy is likely to succeed or fail and take into account the antibiotic dose which a patient can safely receive. You will look at these in the next section.

The starting point for defining the ECOFF for a given pathogen-antibiotic combination is an MIC distribution. This gives the MIC values for a number of bacterial isolates that have been extensively characterised and investigated for possible resistance mechanisms. Typically, the wild type MIC distribution spans between three and five two-fold dilution steps, with the ECOFF marking the upper end of the distribution plot (Figures 2 and 3).

Figure 2 The MIC distribution of amoxicillin for Staphylococcus aureus (EUCAST, 2016).

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A graph of MIC distribution, with a wild type population at 0.12–1 mg/L and a resistant population at 32–256 mg/L.

Figure 2 The MIC distribution of amoxicillin for Staphylococcus aureus (EUCAST, 2016).

In Figure 2, bacteria with detectable resistance mechanisms are clearly distinguishable from the wild type populations. This is because most resistance in this instance is due to a beta-lactamase enzyme made by bacteria. The gene for the enzyme can be acquired by S. aureus, so some isolates may have the gene while some do not. The beta-lactamase enzyme breaks down the amoxicillin, so the antibiotic does not work against the resistant strains. This results in the susceptible bacteria (those that do not produce the enzyme) and resistant bacteria (those that do produce the enzyme) having MIC distributions that are clearly separate.

However, for other pathogen-antibiotic combinations the MIC distribution of bacteria with acquired resistance mechanisms may be closer to, or in some cases overlap with, the wild type MIC distribution (Figure 3).

Figure 3 The MIC distribution of benzylpenicillin for Streptococcus pneumoniae. Wild type strains are represented in blue (EUCAST, 2016).

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A graph of MIC distribution, with a wild type population at 0.004–0.06 mg/L and a resistant population at 0.12–4 mg/L.

Figure 3 The MIC distribution of benzylpenicillin for Streptococcus pneumoniae. Wild type strains are represented in blue (EUCAST, 2016).

In human and animal health, ECOFF values are mainly used in surveillance rather than to guide therapy. If a clinical breakpoint is available, it should always be used for decisions on treatment rather than an ECOFF value.

Clinical breakpoints are available for most domesticated animal species such as dogs, cats, horses, cattle and pigs. Human clinical breakpoints are often used when veterinary-specific breakpoints are not available. ECOFF values are occasionally used in animal health for some host species where there are few or no clinical breakpoints, for example in aquaculture.

1.2.2 Clinical breakpoints

AST provides prescribers with information that can be used to determine which antibiotic therapy is appropriate. Clinical breakpoints are used for this.

The clinical breakpoint is based on the MIC and the safely achievable tissue concentrations expected when a patient or animal is given a standard antibiotic dose. It is a cut-off value that defines isolates as susceptible or resistant in clinical practice given a standard treatment regimen. Remember that if the MIC of an isolate is less than or equal to the clinical breakpoint for a given antibiotic, then the bacteria are considered susceptible (S) to treatment with that antibiotic, following a standard dosing regimen. Isolates with MICs higher than this breakpoint are considered as having resistance (R) to treatment with the antibiotic in question.

For some pathogen-antibiotic combinations, there is an ‘intermediate’ zone between definite susceptibility and definite resistance. Isolates that have an MIC in this ‘intermediate zone’ are susceptible to increased antibiotic exposure (I) (Kahlmeter and the EUCAST Steering Committee, 2019). The benzylpenicillin MIC distribution for S. pneumoniae that we looked at earlier is a good example of this type of pathogen-antibiotic combination (Figure 4). The antibiotic can still potentially be used, depending on the site of the infection, but it must be at a higher dose. It may for example be effective in sites where high concentration can easily be achieved, such as in the lung (pneumonia), but not in sites such as the central nervous system (meningitis) due to lower levels crossing the blood-brain barrier. This should be made clear in the laboratory report.

Figure 4 Interpreting MICs in the ‘intermediate’ zone (Kahlmeter and the EUCAST Steering Committee, 2019).

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A graph of MIC distribution, with a susceptible (normal exposure) population at 0.004–0.06 mg/L, a susceptible (increased exposure) population at 0.12–2 mg/L and a resistant population at 4 mg/L.

Figure 4 Interpreting MICs in the ‘intermediate’ zone (Kahlmeter and the EUCAST Steering Committee, 2019).

1.2.3 How breakpoints are set

Clinical breakpoints are set collaboratively by a committee of people with a range of backgrounds, including clinical microbiology and infectious diseases.

Once the committee agrees on an antibiotic breakpoint concentration, this tentative breakpoint is published for consultation and refined before the final breakpoint concentration is agreed. The published guidelines and breakpoints are reviewed regularly, and it is important to always refer to the most up-to-date guidelines available.

Animal and human breakpoints are not necessarily the same, as animals may metabolise some antibiotics differently and so may achieve different tissue concentrations.

2 International guidelines for AST

2.1 Why the guidelines are important

Most clinical and veterinary microbiology laboratories adhere to international quality standards set by an organisation such as the International Standards Organisation (ISO) to standardise and inform laboratory best practice. In addition, to ensure uniformity, consistency, repeatability and reliability between different laboratories, standard operating procedures (SOPs) for AST should integrate international guidelines developed by either the US-based Clinical and Laboratory Standards Institute (CLSI) (CLSI, n.d.), or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2019). CLSI and EUCAST separately produce guidelines, standards and associated criteria that are used to perform and interpret AST results. You will look at some of the laboratory methods that underpin these standards later in this module.

2.2 Which set of standards should you use?

CLSI and EUCAST use similar methodologies and criteria to set the reference standards for AST, but there are some differences between the two (Cusack et al., 2019), such as the type of agar used for certain bacteria. Both are recognised by the World Health Organization’s Global Antimicrobial Resistance Surveillance System (GLASS) and the World Organisation for Animal Health (OIE). However, EUCAST guidelines are developed as part of an open and public consultation, are freely available, and are more widely used outside of North America (WHO, 2020).

CLSI and EUCAST guidelines are used to produce antimicrobial susceptibility data from clinical samples that can be analysed at both a local and a national level. The interpretation of these results may then be used to inform animal and public health policy recommendations, and also as part of a One Health approach to AMR surveillance. One Health is a concept that recognises the interdependency of animal health and human health, and the ecosystems in which they evolve.

CLSI and EUCAST guidelines are used to guide treatment decisions by veterinarians and clinicians.

The same antibiotic may be used to treat bacterial disease in animals and humans, but separate reference standards should be used to determine treatments in veterinary and human medicine since animals and people may metabolise the same drug differently.

‘Human’ reference standards are published by both systems but only CLSI (CLSI VET) currently provides guidelines and standards specifically for use in veterinary medicine and research. EUCAST VET guidelines and standards are still in development (EUCAST, 2020a). When results are used to inform veterinarians’ clinical decisions, it is important to use veterinary guidelines. When the results of tests for isolates of animal origin are to be compared with human health results in a One Health approach to AMR surveillance, the same human reference standards should be used for all tests so that valid comparisons can be made.

3 Performing phenotypic AST: the disk diffusion test

The majority of microbiology laboratories use disk diffusion testing for AST. This section focuses on performing these laboratory tests. It is assumed that you are reasonably familiar with good laboratory practice and good microbiological practice; however, if you are unfamiliar with basic microbiology techniques, you may find it helpful to look at the Quality assurance and AMR surveillance module from this course.

The most commonly used disk diffusion method is the Kirby-Bauer method, which uses disks impregnated with antibiotics to test their effectiveness on a specific bacterial isolate.

You may also be familiar with the older Stokes method, where the isolate and a control organism are compared on the same plate. The Kirby-Bauer method is recommended here because it produces more accurate and reproducible data, which is essential for surveillance and comparison.

3.1 Performing the Kirby-Bauer method

A bacterial culture is diluted to a defined standard. It is then spread uniformly across a culture plate and filter paper disks impregnated with a specified concentration of the antibiotics are added. Once the disk is applied to the surface of the plate, the antibiotic diffuses into the surrounding media, creating a gradient of antibiotic with the highest concentration nearest the disk.

Following incubation, if the pathogen is susceptible to an antibiotic agent, there will be an area around the disk where bacteria are unable to grow due to the concentration of antibiotic. However, the further the antibiotic diffuses away from the disk, the lower its concentration in the agar is, until it has dropped below the MIC for the given bacterial-antibiotic combination. In this part of the agar it will no longer be able to inhibit the growth of the susceptible bacteria.

This results in a clear zone, where the concentration of antibiotic is high enough to prevent the growth of susceptible bacteria. This clear area is known as a zone of inhibition (Figure 5). Its size is determined by the MIC of the bacteria: a low MIC for more susceptible organisms gives a bigger zone. However, the size of the zone is also influenced by the size and weight of the antibiotic molecules. Small, light molecules diffuse further through the agar, so you cannot assume that a large zone automatically means the isolate is susceptible. You must always measure and compare the zone size with the guidelines for that particular antibiotic.

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Figure 5 The disk diffusion method.

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(Left) An inoculated plate with six different antibiotic disks prior to incubation. (Right) The same plate after incubation at the temperature and duration defined in the SOP (generally 18 hours at 35ºC).

Figure 5 The disk diffusion method.

Once the size of the zone of inhibition has been measured it must be compared to a database of zone diameter breakpoints to determine whether the bacteria are susceptible or resistant to the antibiotic in question. (You will look at how this is done in Section 3.3.)

EUCAST and the CLSI publish guidance for how to perform disk diffusion tests, and the following sections are based on this guidance. Note that you should have SOPs in your workplace derived from the guidelines. The SOPs should include written step-by-step instructions, which everyone doing the tests has to follow. Note also that McFarland standards, while expensive to buy, are straightforward to prepare. Guidance on preparing McFarland standards is given in Table 2 of the EUCAST Disk Diffusion Test Methodology (version 9.0) (EUCAST, 2020a).

The list below shows the key stages of the disk diffusion test. The first three stages include a video from EUCAST demonstrating this stage.

If you can, you should watch Videos 1–3 now. If however you are unable to access these videos, slides 1–21 of a slidecast of EUCAST’s disk diffusion method cover these first three stages (EUCAST, 2020a).

1. Prepare and standardise the inoculum:

2. Inoculate the plate:

3. Apply antibiotic disks and incubate:

4. Measure the zones of inhibition.
5. Interpret the results.

You will look at stages 4 and 5 later in this module. Note that how each step is performed may depend on the pathogen-antibiotic combination being tested. For example, the type of culture plate and the incubation time may vary for different pathogens. Your workplace’s SOP should describe in detail how to perform each stage for all the pathogen-antibiotic combinations that you are working with.

3.2 Setting up the test

Now try the following activity.

3.3 Measuring and interpreting the test

After incubation, the zone sizes are measured and interpreted. The size of the zone of inhibition depends on the MIC of the antibiotic for the strain being tested, and the size of the antibiotic molecules.

It is also important to note that the zone size does not give the exact MIC: if it is important to know this, then a dilution method or Etest should be used (see Section 4).

Just like the previous steps of the disk diffusion test, EUCAST and the CLSI recommend international standards for measuring and interpreting disk diffusion test results. The measured zone size should be compared against the zone sizes given by whichever method has been used to set up the tests to decide whether the isolate is susceptible or resistant (see Figure 6). Remember that in veterinary laboratories, for diagnosis purposes, CLSI VET guidelines should ideally be used to decide whether or not to use an antibiotic to treat a sick animal. Caution is advised if human guidelines are used for isolates of animal origin (for example, if CLSI VET guidelines are unavailable, or there is no breakpoint given for the animal species being treated).

Figure 6 Zones of inhibition for different antibiotics after incubation of the plate.

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EUCAST/CLSI guidelines are used to interpret the zones of inhibition for each antibiotic tested. The diameter of the inhibition zone is compared with the breakpoint zone size. If the diameter of the zone of inhibition is equal to or greater than the size that indicates ‘susceptible’ in the guidance document, the isolate is considered susceptible (S).

Figure 6 Zones of inhibition for different antibiotics after incubation of the plate.

Video 4 describes stages 4 and 5 of the recommended standards for reading disk diffusion tests:

If you are unable to watch Video 4, slides 22 onwards of EUCAST’s disk diffusion method slidecast cover the same material (EUCAST, 2020a).

Do not worry if you are not familiar with the terms ‘swarming’ and ‘haemolysis’ that are mentioned in the video and slidecast – different pathogens may behave somewhat differently in the disk diffusion test, but for this course you are only concerned with the basic principles of measuring zones of inhibition.

As with the previous steps of the disk diffusion test, the exact way that zones of inhibition are measured can depend on the pathogen-antibiotic combination. Your workplace’s SOP should describe in detail how to measure the zone of inhibition for the pathogen-antibiotic combinations that you are working with. If it does not, then refer to the EUCAST guidelines.

Once the diameter of the zone of inhibition has been measured, the results need to be interpreted to determine whether the microorganism is susceptible or resistant to the antibiotic. This is done using zone diameter breakpoint tables.

As for the MIC breakpoints you looked at in Section 1.1, if a zone diameter measurement falls between the susceptible (S) and resistant (R) zone diameter breakpoint given in the breakpoint table, then the result is interpreted as susceptible to increased exposure to the antibiotic (I).

You can practise using breakpoint tables in Activity 4. Select the clinical or veterinary option to access appropriate data.

4 Other methods

4.1 Advantages and disadvantages of different types of test in practice

Gradient diffusion testing

Gradient diffusion testing is used when it is important to know the precise MIC for a given pathogen-antibiotic combination. Usually, commercial MIC gradient test strips are used. These have a high concentration of antimicrobial at one end of the strip, and the concentration gradually decreases along the length of the strip. Markings on the strip indicate the antimicrobial concentration at each point.

Advantages of gradient diffusion testing are that it can be used:

• for certain organism–antimicrobial combinations where disk diffusion testing is hard to interpret for technical reasons, for example glycopeptides in Staphylococcus
• for some organisms, like certain fungi, where disk diffusion testing is unreliable
• in laboratories without access to an automated system for MIC determination – the procedure is very similar to disk diffusion testing, and a laboratory that is able to perform disk diffusion testing should be able to perform gradient diffusion testing relatively easily.

Figure 7 Gradient diffusion Etest.

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The plate is inoculated, the strip is applied, and then the plate is incubated as for disk diffusion. The MIC value can then be read off the strip at the end of the zone of inhibition.

Figure 7 Gradient diffusion Etest.

Automated systems or semi-automated systems

Automated systems or semi-automated systems that use dilution methods are used by large microbiology laboratories with high throughput, including many clinical laboratories in higher-income countries. These systems are usually relatively costly to buy and run.

Advantages of such automated systems are that they generate precise MICs, can test large panels of antibiotics and are less labour-intensive than other methods. Further advantages include capability for interpretive testing of resistance mechanisms (see the Testing for mechanisms of resistance module) and speciation of microorganisms.

Figure 8 BD Phoenix Automated System.

4.2 How the test organism determines the method used

Disk diffusion is good for organisms such as Staphylococci and Enterobacterales, which grow well under standard conditions and in a 24-hour time frame.

5 Quality procedures in AST

As well as providing results to guide antimicrobial prescribing, microbiology laboratories act as ‘data collectors’ in local AMR surveillance processes and have a critical role in ensuring that the data is accurate, reliable, valid and comparable. Whilst AST should reliably report the susceptibility of a bacterial pathogen to a given antibiotic, the results of these tests can be affected by many factors. To minimise variability, laboratories use various quality procedures that identify and correct or reduce errors. Laboratory quality procedures, when implemented appropriately and effectively, increase the confidence of everyone who uses AMR data (such as clinicians, veterinarians, patients, farmers and policy-makers) in the value of the test results.

You will learn about quality procedures in the Quality assurance and AMR surveillance module, but for now we focus on factors that can affect the results of AST and those measures that are taken to ensure data quality.

5.1 Factors affecting disk diffusion tests

As you have learned, the size of the zone of inhibition in a disk diffusion test depends both on how effective the antibiotic is at preventing bacterial growth and how fast it can move through the agar (molecular weight and size of the molecule). However, several other factors can affect the outcome of these tests and lead to incorrect results.

If the inhibition zone is bigger than it should be, the test organism will appear more susceptible to the antibiotic than it really is. Conversely, a too small zone of inhibition suggests the test organism is more resistant to the antibiotic than it really is. You will learn more about how different factors can lead to errors in the test results in Activity 7.

5.2 Standard operating procedures (SOPs)

Following well-written SOPs can minimise the likelihood of variables affecting the outcome of a test by including step-by-step instructions and quality procedures. You will learn about SOPs in the Quality assurance and AMR surveillance module.

5.3 Use of control strains

One important quality procedure for AST is the use of control strains.

Control strains are bacterial isolates that have been widely used and have a known, well characterised antibiotic susceptibility profile. They are usually obtained from a reputable source, such as the American Type Culture Collection (ATCC). Both susceptible and resistant control strains are usually used, and the results are compared to standard tables that ensure that they are within acceptable ranges.

Using control strains means that laboratory staff can ensure that:

• the medium performs satisfactorily
• the strength of the antibiotic used in the disk diffusion test is correct
• the incubation conditions are appropriate, for example the correct temperature is maintained
• tests are performed correctly.

6 Antimicrobial susceptibility testing and your workplace

You should now have a better understanding of AST, the factors that affect it and the importance of quality procedures to ensure the test results are accurate and reliable. Before you end this module complete the final activity, which will help you to think about how this knowledge applies to your workplace.

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. 

• End-of-module 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.

8 Summary

In this module you have learned about AST and other phenotypic methods of testing for antibiotic resistance. You have seen the importance of quality procedures to ensure that the results of these tests are uniform, consistent, repeatable and reliable. You have also been introduced to the EUCAST and CLSI internationally recommended standards that allow data from different laboratories to be compared.

You should now be able to:

• describe the principles of AST
• understand the concepts of phenotypic methods for AST
• understand the process of disk diffusion testing and the relevant quality measures needed
• understand the principles of other phenotypic methods, including automated methods
• understand that the method used may depend on the organism being tested
• understand the concept of MICs and breakpoints
• recognise relevant international guidelines, and know where to find them and when to use them
• recognise that additional testing may be needed to identify/confirm resistance mechanisms
• recognise the importance of procedures designed to ensure the quality of AST testing 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.

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

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: © Arindam Ghosh/iStock/Getty Images Plus.

Figures 2 and 3: EUCAST (2016) Splitting the wild type and resistant populations, 12 May 2016.

Figure 4: adapted from SIR – new definitions 2019, © eucast.org.

Figures 5–8: © Liz Sheridan.

Resources

EUCAST Disk Diffusion Test Methodology: this document has been produced in part under ECDC service contracts and made available at no cost by EUCAST at no cost to the user and can be accessed on the EUCAST website www.eucast.org. EUCAST recommendations are frequently updated and the latest versions are available at www.eucast.org.

Standard Operating Procedures: Bacteriology – Veterinary Sector: Indian Council of Medical Research.

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.