Testing for mechanisms of resistance

Introduction

In this module you will learn about phenotypic and genotypic laboratory techniques that are used to test for common mechanisms of antimicrobial resistance (AMR). World Health Organization (WHO) priority pathogens for surveillance in human health, and pathogens subject to surveillance and monitoring as described by the World Organization for Animal Health (OIE) and the Food and Agriculture Organization for the United Nations (FAO) are considered in some detail. You were introduced to these pathogens in the Isolating and identifying bacteria module.

You will start by looking at resistance patterns of global concern in human and animal health, and the importance of knowing the underlying resistance mechanism involved. You will further learn how data derived from screening for specific resistance mechanisms and confirmatory testing, differs from antimicrobial susceptibility testing (AST) data, and the role it plays in AMR surveillance. At a more practical level you will study the principles and applications of relevant laboratory techniques using wellcharacterised examples, such as carbapenemase-producers, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producers. Examples of resistance mechanisms directly relevant to your work and quality control (QC) measures related to these techniques will also be raised.

Basic knowledge of concepts such as the emergence and spread of ‘resistance genes’ will be 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 these free online courses: Understanding antibiotic resistance and Inheritance of characters

By the end of this module you should be able to:

• give examples of the resistance patterns/resistant organisms causing global concern
• give examples of the resistance patterns/resistant organisms encountered in your work
• understand the difference between screening and confirmatory testing
• describe some of the phenotypic methods commonly used for screening and confirmation of resistance mechanisms
• understand how genotypic methods can be used, and the advantages/disadvantages of genotypic versus phenotypic methods
• understand how more data from detailed testing for resistance mechanisms/genes contributes to AMR surveillance
• apply your knowledge of these laboratory tests to interpret data relevant to your work
• know the importance of procedures designed to ensure the quality of these laboratory tests in your workplace.

1 Resistance problems of global concern

1.1 Priority human and animal pathogens

Bacteria isolated from clinical sources and animals may have acquired resistance to one or more antimicrobials. A minority of these are both virulent and have the capacity to spread widely and cause problems on a global scale.

Of the eight WHO-listed human pathogens which are the focus of the Global Antimicrobial Resistance Surveillance System (GLASS) some are exclusive to humans, some can be zoonotic, and some affect both humans and animal species.

In contrast, different bacteria are the main focus of active AMR surveillance in healthy animals, of which some are commensals and some zoonotic pathogens (EFSA, n.d.; FAO, n.d.). In some countries, such as France, Denmark and the USA, bacterial pathogens from certain animal species are included in routine AMR surveillance, for example, livestock-associated MRSA in pigs and Salmonella in cattle (CDC, 2019; DVFA, n.d.). AMR data on these bacterial pathogens are generated from routine laboratory investigations when animals display clinical disease and from controls at abattoirs on healthy animals.

For background information see the EFSA Panel on Biological Hazards (2009) and WHO (2018).

1.2 WHO priority human pathogens for antimicrobial R&D

The WHO (2017a) has also developed a different list of specific organism-resistance combinations which have the most urgent need for research and development (R&D) into new treatment approaches. Nine of the twelve organisms listed are multi-drug resistant (MDR)Gram-negative pathogens, highlighting the growing threat of this group to human health. Organisms are grouped by level of priority: critical, high and medium.

Priority 1: CRITICAL

• Acinetobacter baumannii – carbapenem-resistant
• Pseudomonas aeruginosa – carbapenem-resistant
• Enterobacterales – carbapenem-resistant, ESBL-producing.

Priority 2: HIGH

• Enterococcus faecium – vancomycin-resistant (VRE)
• Staphylococcus aureus, – methicillin-resistant (MRSA), vancomycin-intermediate and vancomycin-resistant (VISA, VRSA)
• Helicobacter pylori – clarithromycin-resistant
• Campylobacter species ­– fluoroquinolone-resistant
• Salmonella species – fluoroquinolone-resistant
• Neisseria gonorrhoeae – cephalosporin-resistant, fluoroquinolone-resistant

Priority 3: MEDIUM

• Streptococcus pneumoniae – penicillin-non-susceptible
• Haemophilus influenzae – ampicillin-resistant
• Shigella species – fluoroquinolone-resistant

Figure 1 Scanning electron micrograph of Campylobacter jejuni

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The Campylobacter species is listed by WHO as a high level of priority among the twelve organisms that are multi-drug resistant pathogens, highlighting the growing threat of this group to human health.

Figure 1 Scanning electron micrograph of Campylobacter jejuni

1.3 Resistance patterns in your workplace

Most of the GLASS and WHO priority R&D pathogens, including those of One Health importance, can be cultured easily; some have specific growth requirements (see the Isolating and identifying bacteria module). Some of the resistance patterns can be identified by routine AST; others, for instance ESBL production or VRE, may need additional testing to identify and confirm their presence. You will find out more about these methods later in the module.

2 Antimicrobial resistance

2.1 Resistance mechanisms

The resistance mechanisms that bacteria have evolved to counter the action of antimicrobials are sophisticated. In the Introducing antimicrobial resistance module you learned about the main mechanisms of antimicrobial resistance. See if you can remember them in Activity 5.

Activity 5 Antimicrobial resistance mechanisms

Timing: Allow 10 minutes

Part 1

Figure 2 shows an overview of the main mechanisms of antimicrobial resistance. Use the drop-down list to match the mechanism to the appropriate part of the diagram.

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Discussion

Figure 2 Main mechanisms of bacterial resistance

1. Preventing antimicrobial entry
2. Increasing efflux
3. Modifying the antimicrobial
4. Destroying the antimicrobial
5. Modifying the target
6. Protecting the target
7. Amplifying the target.

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Unlike hydrolysing enzymes which in general provide high levels of resistance, some mechanisms only produce low-level resistance on their own. However, different mechanisms can combine to increase the level of resistance provided. For example, porin loss combined with an over-expression of efflux pumps can lead to carbapenem resistance which is hard to distinguish on AST from the hydrolysing activity of a carbapenemase, and which has the same effect clinically in reducing the efficacy of treatment.

The β-lactam-hydrolysing enzymes are interesting in that they can sometimes be blocked by using a second substance along with the antimicrobial. This enzyme blocker is called an inhibitor, for example clavulanic acid, which can be administered alongside an antimicrobial, and acts to neutralise the hydrolysing enzyme and protect the antimicrobial during treatment (Drawz and Bonomo, 2010). An example of a commonly used antimicrobial inhibitor combination is co-amoxiclav, which contains amoxicillin and clavulanic acid.

2.2 Spread of antimicrobial resistance in bacteria

Some species of bacteria have intrinsic resistance – an innate ability to resist the action of an antimicrobial which can lead to treatment failure. Resistance can also be acquired. It can develop by mutation, with the newly resistant bacterial clone expanding and spreading under evolutionary pressure from the presence of the antibiotic. However, the main healthcare concern is acquired resistance through horizontal transfer of resistance genes mainly carried by mobile genetic elements, in particular plasmids.

Resistance can be shuffled between chromosomal and plasmid DNA on small mobile genetic elements, facilitating recombination and promoting the rapid evolution of new resistant forms. Resistance genes can remain on the plasmid or be integrated into the chromosome; chromosomal mutations can also find their way onto plasmids. This, coupled with horizontal gene transfer which can so easily lead to MDR strains, is the main cause of antimicrobial resistance spreading through Gram-negative bacterial populations – including between different species. Transmissible resistance mediated by plasmids and other mobile genetic elements (such as transposons or insertion sequences), is therefore of huge global concern in both human and animal health.

The current greatest AMR threat to modern medicine is from plasmids with a carbapenemase gene plus resistance to multiple antimicrobials. In any of the three organisms in the WHO ‘critical’ R&D category, carbapenem-resistance leads to infections that can only be treated with combinations of drugs or toxic ‘third line’ antimicrobials, for example, colistin. These plasmids can lead to untreatable infections, hence the high priority placed on these pathogen-AMR combinations.

3 Pathogen-AMR combinations

In this section we will explore in more detail some of the specific pathogen resistance patterns in human and animal health.

3.1 Gram-negative pathogens

Gram-negative pathogens are a significant cause of morbidity both in community-acquired infection such as enteric fever and urinary tract infection (UTI) and in healthcare-associated infections (HCAI). It is notable that all the organisms in the WHO priority R&D ‘critical’ category are all Gram-negative: P. aeruginosa,A. baumannii and Enterobacterales.

In animal health, Gram-negatives are found as gut commensals and as pathogens. They are an important source of resistance genes getting into the human food chain, especially as it is hard to totally prevent contamination of meat with faecal material at slaughter.

Figure 3 Salmonella and E. coli are a common cause of health problems in piglets, leading to antibiotic use.

Production of β-lactamases and carbapenemases are the biggest problem with infections caused by Gram-negative organisms owing to the relative lack of antimicrobials available outside these classes to treat them. Quinolone resistance is also a problem.

3.1.1 β-lactam resistance and ESBLs

The resistance genes for β-lactamases are located either on the bacterial chromosome or on plasmids, leading to both intrinsic and acquired resistance. The genes have mutated and evolved so that each one represents a ‘family’ of related genes. Two separate systems are used to classify β-lactamases: a structural scheme based on primary structure (Ambler, 1980; Hall and Barlow, 2005); and a functional scheme based on chemical reactivity and DNA sequence (Bush and Jacoby, 2005). The Ambler classification is based on classes A-D (Table 1), whereas the Bush‒Jacoby has numbered groups. This may seem complex but fortunately only a few subdivisions of the main classes of β-lactamases are widespread enough to mention specifically here in the context of surveillance.

ESBL-producers among Enterobacterales are significant as they cannot be treated with the usual ‘workhorse’ antimicrobials ‒ amoxicillin and cephalosporins. They also tend to be MDR as several resistance genes may be carried on the same plasmid. This means that often no oral options are available because the majority of second- and third-line drugs to which the organisms are still susceptible can only be given intravenously. The presence of ESBL is also a driver of carbapenemase resistance as the choice of drug to treat them is usually a carbapenem.

ESBL-producers, mainly E. coli and Klebsiella, are also widely distributed in poultry, including those which have had little direct exposure to antimicrobials themselves. They are readily transmitted to humans via food consumption, for example by handling uncooked meat or consuming vegetables fertilised with animal manure; they can also be transferred in the other direction from humans to livestock (Subramanya et al., 2020; Uttapoln et al., 2019).

Figure 4 Market day, Bahir Dar, Ethiopia

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ESBL-producers, mainly E. coli and Klebsiella, are widely distributed in poultry which is frequently on sale in markets and readily transmitted to humans, for example by handling uncooked meat or consuming vegetables fertilised with animal manure. The bacteria can also be transferred from humans to livestock.

Figure 4 Market day, Bahir Dar, Ethiopia

Cephalosporin hydrolysing enzymes of clinical and veterinary importance are shown in Table 2.

Table 2 Cephalosporin hydrolysing enzymes important in human and animal health

3.1.2 Carbapenem resistance and carbapenemases

The greatest current concern for human health is from carbapenem resistance. For example, Acinetobacter, Klebsiella and Pseudomonas aeruginosa are important causes of outbreaks in tertiary and critical care. MDR- and pan-resistant strains are a problem in many critical care units. They are frequently resistant not just to carbapenems but also to all other available antimicrobials. Once they have colonised the environment in a unit, they are very hard to get rid of.

Carbapenem-resistant Enterobacterales (CPE), predominantly Klebsiella and E. coli, have been found in wild animals, livestock and pets, with evidence of direct transfer to humans (Kock et al., 2018).

The main classes of carbapenemases of clinical and veterinary importance are shown in Table 3.

Table 3 Carbapenemases important in human and animal health (abbreviations: KPC – Klebsiella pneumoniae carbapenemase; VIM – Verona integron-encoded metallo-beta-lactamase; IMP – Imipenemase metallo-beta-lactamase; NDM – New Delhi metallo-β-lactamase; OXA-48 – Oxacillin-48 carbapenemase)

3.1.3 Fluoroquinolone resistance

Fluoroquinolone antimicrobials are available orally including for severe infections. They can be used for intracellular organisms – in particular, S. typhi against which many other antimicrobials are not effective as they are unable to enter the mammalian cell – and are a very useful class of antimicrobials. However, fluoroquinolone resistance among Gram-negative organisms is widespread and two pathogen-AMR combinations are listed in the WHO ‘high’ category for R&D priority: fluoroquinolone-resistant Salmonellae and Campylobacter species. Fluoroquinolone-resistant Shigella species are of ‘medium’ R&D priority. Resistance is mostly via modification of the antimicrobial target, as a result of one or more chromosomal mutations.

Figure 5 Healthy free-range pigs in Svaneti, Georgia

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Resistance to antimicrobials is less likely to occur in free-range animals like pigs and poultry than those reared in intensive and overcrowded environments where use of antibiotics is common.

Figure 5 Healthy free-range pigs in Svaneti, Georgia

Both fluoroquinolone-resistant Campylobacter and Salmonella infections are major zoonoses and there are few antimicrobial treatment options. The infections can easily be acquired from food of animal origin if food hygiene is not strictly applied. In contrast, fluoroquinolone-resistant Shigella infections are not acquired from food animals and in general, only affect humans and other primates. Reliance is on good hygiene and sanitation to prevent spread, so they are widespread in contexts where this is difficult to achieve.

3.2 Gram-positive pathogens

In human health, two Gram-positive pathogen-AMR combinations are in the WHO ‘high’ R&D category: S. aureus (MRSA, VISA and VRSA) and VRE: vancomycin-resistant E. faecium. MRSA is also a growing problem in animal health.

3.2.1 β-lactam resistant S. aureus

Resistant strains of S. aureus pose a big threat to both human and animal health worldwide, with MRSA S. aureus resistant to β-lactam antimicrobials of particular importance.

MRSA is easily transmitted between patients and is a very important HCAI pathogen globally.

MRSA is also found increasingly in livestock (Livestock-associated MRSA, LA-MRSA) and companion animals and transmits readily between humans and other species (Butaye et al., 2016; Chen and Wu, 2020).

Figure 6 A boy feeds a Vietnamese Pot-bellied pig on a farm in Thailand. © Anant Kasetsinsombut

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Resistant strains of S. aureus pose a big threat to both human and animal health worldwide, with MRSA S. aureus resistant to β-lactam antimicrobials of particular importance. MRSA is easily transmitted between hospital patients and is also found increasingly in livestock, so care must be paid to how these are reared.

Figure 6 A boy feeds a Vietnamese Pot-bellied pig on a farm in Thailand. © Anant Kasetsinsombut

Resistance occurs by two main mechanisms. The most common form is due to the presence of the mecA gene on a mobile genetic element carried by all MRSA strains. MecA encodes an altered penicillin-binding protein (PBP) which confers resistance to all penicillin and inhibitor combinations and almost all cephalosporins, other than novel ‘anti-MRSA’ ones such as Ceftobiprole. There is another similar gene, mecC.

This type of resistance is different from staphylococcal β-lactamases. These enzymes only cause resistance to penicillins (pencillin, ampicillin and amoxicillin) and the organism remains susceptible to isoxazolyl-penicillins (flucloxacillin and cloxacillin), and first generation cephalosporins. As such, these basic β-lactamase producers do not pose a public health problem at the moment as they remain relatively easy to treat.

3.2.2 Vancomycin-resistant S.aureus

Vancomycin is important against S. aureus as it can be used for MRSA strains or when β-lactams cannot be used. VISA refers to organisms with intermediate resistance while hVISA refers to subpopulations of organisms with intermediate resistance in a single culture. VRSA organisms are fully resistant to vancomycin. VISA, hVISA and VRSA therefore refer to S.aureus strains with varying degrees of resistance to vancomycin and using a number of resistance mechanisms. They have been found in increasing numbers in recent years in isolates from both humans and livestock and are a potential future threat (Al-Amery et al., 2019; Cong et al., 2020; Shariati et al., 2020).

Of greatest concern is the presence of the VanA genes, originally found in vancomycin-resistant Enterococci (VRE) (see Section 3.2.3), but other mechanisms can also lead to antimicrobial treatment failure and so are still important.

3.2.3 Vancomycin-resistant Enterococci

Enterococci are gastro-intestinal commensals of humans and other animals. They are moderately virulent, mainly in the context of healthcare i.e. they are largely a HCAI pathogen, although they can cause urinary tract infection in healthy individuals. They cause infections related to prosthetic materials such as intravascular devices, mechanical heart valves and joint implants, that can be extremely difficult to treat.

VRE strains of Enterococcus, most commonly Enterococcus faecium, cause outbreaks among cancer and critical care patients with infections that are hard to treat and require expensive antimicrobials.

E. faecium is intrinsically resistant to β-lactams, including amoxicillin which is active against other Enterococci such as E. faecalis, which means that the loss of vancomycin sensitivity is important. Other species exist that are intrinsically resistant to vancomycin e.g. E. gallinarum and E. casseliflavus, but these only cause sporadic infections in humans (CDC, 2010).

There are a number of resistant phenotypes and genes involved in VRE, the main ones of clinical concern being those with the vanA phenotype. This is mediated by the vanA operon, a two-component system which leads to high level resistance to both vancomycin and teicoplanin (the most commonly used glycopeptide antimicrobials). The vanA operon is transmitted via a plasmid so can move between strains and species. It is found chiefly in E. faecium and occasionally in E. faecalis. Other resistant phenotypes, for example vanB, are of less clinical or veterinary importance (Cetinkaya et al., 2000).

Figure 7 Dairy-source foods derived from cows and goats are a potential source of zoonoses.

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Both fluoroquinolone-resistant Campylobacter and Salmonella infections are major zoonoses and there are few antimicrobial treatment options. The infections can easily be acquired from food of animal origin if food hygiene is not strictly applied.

Figure 7 Dairy-source foods derived from cows and goats are a potential source of zoonoses.

Enterococci of animal origin are important from the One Health perspective as a source of resistance genes entering the human food chain.

3.3 Miscellaneous pathogens of interest

3.3.1 Respiratory and central nervous system pathogens

Figure 8 Scanning electron micrograph of S. pneumoniae

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High-level penicillin resistance in S. pneumoniae (Figure 8) can lead to treatment failure in respiratory tract infections. Even low-level resistance is a problem for infections of the central nervous system, for example meningitis.

Figure 8 Scanning electron micrograph of S. pneumoniae

High-level penicillin resistance in S. pneumoniae can lead to treatment failure in respiratory tract infections. Even low-level resistance is a problem for infections of the central nervous system, for example meningitis. This is because only small concentrations of drug can be achieved in the brain because of the presence of the blood-brain barrier (Linares et al., 2010).

Penicillin resistance in S. pneumoniae is acquired differently to the resistance mechanisms described so far. It is generally the result of cumulative mutations and resistance acquisition leading to resistant strains, rather than by a single transmissible genetic element. An association has often been observed between rates of antimicrobial use and prevalence of resistance in this organism (Albrich et al., 2004; WHO, 2001).

Haemophilus influenzae is a cause of vaccine-preventable invasive disease (HiB) in children, mainly causing respiratory tract infection or meningitis. Despite the ongoing success of the global vaccine programme, resistance to amoxicillin, in both HiB and non-typeable H. influenzae (NTHi) strains, remains a problem as amoxicillin would normally be the treatment of choice (Su et al., 2020; Van Eldere et al., 2014).

3.3.2 Sexually transmitted pathogens

MDR isolates of N. gonorrhoeae are increasing, with fluoroquinolone resistance a particular problem as these infections may not have any treatment options.

Resistance to third generation cephalosporins (ceftriaxone) is also starting to lead to strains which are almost impossible to treat, even with intramuscular injection of antimicrobials rather than oral ones (Costa-Lourenço et al., 2017).

4 Identifying resistance mechanisms

4.1 Why knowing the mechanism is important

4.2 The approach used to determine the resistance mechanism

Once AST has confirmed that a bacterial isolate is resistant to antimicrobial(s), two approaches can be used, either separately or in combination, to identify the resistance mechanism involved ‒ phenotypic methods and genotypic methods.

Phenotypic tests focus on characteristics of the bacteria (i.e. phenotype) that can be readily observed or tested for in the laboratory. They use the results of these observations/tests to infer the resistance mechanism (and thus the gene) involved. In contrast, genotypic tests identify the gene responsible for the resistance directly.

You will learn more about these two approaches in the following sections.

Figure 9 Tests to identify resistance mechanisms can be performed in a clinical microbiology laboratory.

5 Phenotypic tests

Phenotypic tests are carried out more frequently than genotypic tests as they are easier to perform and require less sophisticated/specialised equipment. They include the following, discussed in more detail below:

• use of indicator antimicrobials
• testing for inducible resistance
• synergy tests
• use of interpretative panels
• antigen detection tests
• colorimetric tests.

5.1 Use of indicator antimicrobials

Indicator antimicrobials can be used alongside normal AST. The principle of this test is to use the minimum inhibitory concentration (MIC) to a specific indicator antimicrobial to which resistance implies the mechanism you are looking for. Organisms with an MIC above the ECOFF or clinical breakpoint (measured by inhibition zone size) may have the mechanism of concern.

In these instances, once you know that the organism is resistant to the indicator, you can then extrapolate that it will be resistant to every antimicrobial in that class. However, this only works in a few specific cases. For example, cefoxitin resistance in S. aureus implies mecA is present, so the Staphylococcus must be an MRSA (see Section 3.2.1).

Certain indicator antimicrobials can also be used to detect non-wild type strains, without knowing the precise resistance mechanism. Resistance to these antimicrobials means a resistance mechanism of some sort is present. For example, a nalidixic acid, norfloxacin or pefloxacin MIC higher than the ECOFF or clinical breakpoint implies a fluoroquinolone resistance mechanism in organisms including Salmonella, Streptococcus and H. influenzae – the specific antibiotic used will depend on the species being tested. Similarly, oxacillin is used to look for penicillin resistance mechanisms in S. pneumoniae.

5.2 Testing for inducible resistance

Some resistance mechanisms are expressed only when induced by the presence of another antimicrobial. These strains can become resistant during antibiotic treatment, so it is important to know if they are present. For certain pathogen-antimicrobial combinations, it is therefore possible to test for the resistance mechanism using a form of AST called the 'D' test.

The ‘D’ test can be used to detect inducible clindamycin (DA) resistance in S. aureus. In Figure 10, erythromycin (E) has diffused into the agar and interfered with the effect of the adjacent antibiotic, DA. The effect of E has been to induce the expression of DA in S.aureus organisms present in the agar and allow the bacteria to grow closer to the DA-impregnated disk. This is shown by flattening of the DA inhibition zone on the side next to the E disk.

Figure 10 The ‘D’ test for inducible clindamycin (DA) resistance in S .aureus.

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Flattening of the DA inhibition zone on the side next to the E disk (indicated) is the result of induced resistance to DA by erythromycin (E).

Figure 10 The ‘D’ test for inducible clindamycin (DA) resistance in S .aureus.

5.3 Synergy tests

Synergy refers to the phenomenon where adding a second agent enhances the activity of the first one. The underlying principle of synergy testing is that the MIC is reduced, or the inhibition zone size increased if the added substance has a synergistic effect – even though the effect might not be large enough to be clinically useful for treatment. This can be done, for example, by modified AST such as commercial combination disk testing (CDT) kits double disk synergy testing (DDT), or using an automated system such as Vitek II^TM, Sensititre^TM or Phoenix^TM.

See Section 9.1.2 for examples of synergy testing.

5.4 Use of interpretative panels

Interpretative panels are tests against multiple antimicrobials set up in parallel to look for particular patterns in resistance and sensitivity. For example, AST using a panel of different cephalosporins can be used to imply a particular type of SOP for the test isolate.

Interpretative panels can be done as individual tests, but in practice they are mostly done using an automated system such as Vitek II^TM, Sensititre^TM or Phoenix^TM.

Figure 11 Interpretative panel test from Phoenix^TM

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Image A: The Phoenix automated system showing the test panels in place. Image B: An individual test panel. The wells on the test panel contain substrates which change colour due to bacterial action. The ones on the left are for biochemical reactions used to identify the species. The ones on the right determine the MIC to a range of antimicrobials. There are four different concentrations of antimicrobial for each and growth appears as a change from blue to pink. The machine detects this and analyses the resulting pattern to work out what the likely resistance mechanism is – for example β-lactamase, carbapenemase et cetera, as well as the MIC.

Figure 11 Interpretative panel test from Phoenix^TM

5.5 Antigen detection tests

Antigen detection tests are mainly used to identify antibiotic hydrolysing enzymes, such as ESBLs and carbapenemases. The enzymes are detected by lateral flow tests to detect a specific enzyme.

The example test in Figure 12 is negative but has passed the control. A positive test would have a line by ‘T’ as well. Lateral flow tests are easy to perform though sometimes hard to interpret if the test line appears faintly.

Figure 12 Lateral flow test

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An example of a lateral flow test. The test substance is introduced into the well ‘S’ then moves up the filter paper by capillary action where it meets the test antibodies ‘T’ then the control ‘C’.

Figure 12 Lateral flow test

5.6 Colorimetric tests

Colorimetric tests work on the principle that a substrate changes colour when broken down by an enzyme. This principle is used in chromogenic media (e.g. the commercially available CHROMagar^TM agars developed for detection of carbapenemase producers, Acinetobacter, MRSA etc.). As well as having substrates for the enzymes, some of them are also selective, that is they inhibit the growth of susceptible organisms and species which are not of interest and only support those to be isolated. Some also change colour for a particular species so can be used to identify the species as well as the resistance mechanism.

Another example is nitrocefin – a chromogenic cephalosporin – which can be used to screen for β-lactamases (Video 1). Nitrocefin is broken down by all known β-lactamases leading to a colour change from colourless to red and so works as an indicator antimicrobial. Although it can be used to test whether a β-lactamase is present it will not discriminate between types of β-lactamase. It is routinely used to look for β-lactamase in N. gonorrhoeae and H. influenzae.

Download this video clip.Video player: Video 1

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Video 1 Nitrocefin test for β-lactamases

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This is a commercially produced test. The sticks contain the colourless β-lactam, nitrocefin. The end of the stick is dipped in the culture and incubated in moist conditions for five minutes. If any β-lactamase is present it will break down the nitrocefin, producing a red colour.

Video 1 Nitrocefin test for β-lactamases

6 Genotypic tests

Genotypic tests are widely recognised as the best way to unambiguously prove that a specific resistance gene is present. They are also the only way of characterising suspected new resistance mechanisms.

Many genotypic tests are only done in research and reference laboratories as they all involve molecular biology techniques. However, a few are available as rapid diagnostic kits for commercial polymerase chain reaction (PCR) platforms. The underlying principle is that new and previously known genes can be sequenced and based on this, PCR tests can be developed to look for a specific gene.

6.1 PCR tests

PCR is carried out using:

• commercial or in-house primers
• microarrays
• commercial ‘plug-in’ kits.

Benefits of commercial PCR kits include accuracy and speed. Days can be saved on turnaround times for results in the clinical setting, allowing appropriate antimicrobial treatment or infection control intervention to start earlier.

However, the negatives include:

• high cost per test
• expensive and high-maintenance equipment required
• specimens must still be cultured to look for resistance to other antimicrobials ‒ for example, if the isolate is confirmed to be MRSA, it will be β-lactam-resistant but there is no information about possible resistance to macrolides, tetracyclines et cetera.

6.2 Genome sequencing techniques

Whole genome sequencing (WGS) can also be used to look at the bacterial genome and its associated plasmids. WGS is primarily a research tool. It might be used for instance to study a newly identified mechanism of resistance or to study the different genes found in a particular setting. It could be used to understand how the resistant organisms had spread in an institution by constructing a phylogenetic tree of the bacteria in a particular species and seeing how this related to the different plasmids and resistance genes. It is also possible to sequence just the resistance genes, which is a little faster and less complex.

However, for now sequencing is still very expensive and time consuming. It also needs bioinformatics expertise to interpret the results, so even a reference laboratory is unlikely to be doing this routinely at present.

7 Genotypic versus phenotypic methods

8 Screening for resistance mechanisms

8.1 Theory behind screening and confirmatory testing

Many of the phenotypic and genotypic tests used to identify the resistance mechanism are time-consuming and expensive. Therefore, to reduce the total number of tests performed it is customary to screen for potential resistance mechanisms first and then confirm the mechanism on those isolates that screen positive.

The initial screening tests are done to identify organisms which may have a particular mechanism. This could involve either:

• a.routine screening of samples for asymptomatic carriage of resistance by AST, for example using samples from healthy food animals, or from asymptomatic hospital patients (for infection control purposes)
• b.testing a human or other animal clinical isolate for resistance.

Confirmatory tests then prove whether the organism has the resistance mechanism or not (EUCAST, 2017).

Confirmation is a necessary step to avoid over-reporting of the organisms which do show resistance but do not have the mechanism of concern.

Figure 13 AST in a clinical microbiology laboratory. Screening samples saves time and reduces workload.

8.2 Screening for resistance mechanism in practice

The choice of screening method depends on what sort of samples you are looking at, for example selective media are used for faecal samples from healthy animals, or as part of routine AST for clinical samples.

For more information about how specific screening tests work in combination with confirmatory tests in practice, see Section 9.

9 Confirming resistance mechanisms in practice

Once screening has enabled the identification of isolates which may have the resistance mechanism you are investigating, various methods can be used to confirm whether that mechanism is present or not (EUCAST, 2017).

The following sections illustrate how confirmatory tests work in practice for ESBLs, carbapenemases and in Gram-positive organisms.

9.1 How to confirm the presence of an ESBL

9.1.1 Screening

It is important that the correct antimicrobials are used to screen for ESBLs in routine AST as only some cephalosporins are useful. If the wrong antimicrobial is used the ESBL may be missed. A combination of cefotaxime and ceftriaxone, or cefpodoxime alone will allow all types of ESBL to be detected.

9.1.2 Synergy testing

To confirm that an ESBL is the cause of the resistance the usual approach is to test for synergy with clavulanic acid for ceftazidime, cefotaxime and cefepime, by one of several methods including:

• commercially available combination disk test (CDT) (Figure 14),
• double disk synergy test (DDST) (Figure 15)
• automated systems such as VITEK II^TM or Phoenix^TM .

Figure 14 Combination disk testing using commercial Rosco disks: A) Rosco disk inoculator; B) Rosco test plate.

Key:

• CTX+C = cefotaxime + clavulanic acid
• CTXCX = cefotaxime + cloxacillin
• CTXCC = cefotaxime + clavulanic acid + cloxacillin
• CAZ+C = ceftazidime + clavulanic acid
• CAZCX = ceftazidime + cloxacillin
• CAZCC = ceftazidime + clavulanic acid + cloxacillin.

Activity 10 Interpreting a DDST

Timing: Allow 5 minutes

How would you interpret the results of the double disk synergy test in Figure 15? Does the test organism have an ESBL?

Figure 15 Double disk synergy test set up to compare the zone for cefotaxime alone with the zone for cefotaxime adjacent to the disk with the beta lactamase inhibitor (clavulanic acid).

Answer

The test organism has an ESBL.

Discussion

The cefotaxime is inactivated by the ESBL (no zone) on its own but works ‒ preventing growth and leaving a clear zone – in the part of the agar where the clavulanic acid has diffused out from its disk. The clavulanic acid is protecting the cefotaxime from the ESBL. (Note the clavulanic acid disk contains co-amoxiclav (AMC) so there is also amoxicillin present. This combination has given a small zone around the disk, but not enough for this antibiotic to be useful in clinical practice.)

9.1.3 Alternatives to synergy testing

Alternatives to synergy testing include:

• colorimetric testing (biochemical)
• lateral flow test (immunological) to detect the ESBL such as NG-Test CTX-M MULTI^TM immunochromatographic assay

• MALDI-TOF (spectrometric)
• commercial PCR tests (genotypic).

9.2 Carbapenemase confirmation and identification

Meropenem is the best indicator antimicrobial to screen for carbapenemases in Gram-negatives. Resistance is implied if the MIC is raised or the inhibition zone size reduced.

It is then necessary to determine whether any resistance detected is due to a carbapenemase and not to a combination of resistance mechanisms. For example:

• ESBL or AmpC enzymes combined with decreased permeability as the result of alteration or down-regulation of porins, can also lead to carbapenem resistance
• OXA-48-like enzymes are another challenge. Organisms producing these can look susceptible to cephalosporins so it is necessary to rely on the meropenem as an indicator here. Most other carbapenemases hydrolyse all β-lactams; the OXA-48-like enzymes are unusual and easy to miss because of this unexpected sensitivity.

9.2.1 Carbapenemases in Enterobacterales

Confirmation of carbapenemase in Enterobacterales can be done by several methods in basic laboratories:

• Biochemical (colorimetric) tests, for example the CarbaNP^TM test which detects the hydrolysis of carbapenem, thus confirming the presence of a carbapenemase enzyme
• Immunochromatographic tests to detect specific carbapenemases. Lateral flow tests are now becoming more readily available and are practical to use in most laboratories (Figure 16)
• For laboratories with a suitable commercial, rapid PCR platform, PCR tests for example, Cepheid, Xpert® and Carba-R, are easy to use and can identify several carbapenmases.

Figure 16 Lateral flow test kit for carbapenemases. Note this kit can be used for several different enzymes

Other methods are possible in reference and research laboratories:

• combination disk testing – this follows the same principles as for ESBL and can potentially identify specific carbapenemases. This is more likely to be used in a reference laboratory.
• CIM (Carbapenem Inactivation Method) test and detection of carbapenem hydrolysis via MALDI-TOF are also possible, but at present do not add anything extra in the routine laboratory context.
• genotypic methods.

9.2.1 Carbapenemases in non-Enterobacterales

In non-Enterobacterales such as Acinetobacter, carbapenem resistance is due to a variety of carbapenemases with a range of porins and drug efflux pumps (Hsu et al., 2017). In Pseudomonas resistance is frequently due only to increased efflux or porin loss without a carbapenemase being present. However, carbapenemases are found increasingly in Pseudomonas and as it is hard to discriminate the mechanism in practice; the organism may need to be sent to the reference laboratory if it is important to be sure of the mechanism.

9.2.3 Carbapenemases in practice

9.3 Confirming mechanisms in Gram-positive organisms

9.3.1 MRSA strains of S. aureus

Common screening methods for MRSA from swabs include colorimetric tests using chromogenic agar or mannitol salt agar. However, other tests such as the disk diffusion type tests or automated systems can be used, for example for clinical samples from invasive infections (PHE, 2020). Cefoxitin is now the recommended antimicrobial to use to confirm MRSA as it is less affected by conditions than oxacillin or methicillin and also detects mecC strains. Therefore, cefoxitin resistance confirms the MRSA phenotype.

9.3.2 Vancomycin-resistant S. aureus

Vancomycin resistance in S. aureus (VRSA) is an uncommon but increasing problem. It is worth learning how to test for it accurately.

Note: if you are aiming to treat a human patient or if the investigation is part of surveillance in animals with a One Health approach use EUCAST; if the aim is to treat an animal then vetEUCAST/vetCLSI guidelines (CLSI, 2020) should be used. EUCAST tables should tell you the breakpoint is 2 for Staphylococcus.

If the MIC of your Staphylococcus is vanA mechanism of resistance (e.g. modified cell wall as a result of exposure to the antimicrobial during treatment or via glycopeptide animal growth promoters used in farming). However, it is reported as susceptible for clinical and surveillance purposes.

If the MIC is >2 (resistant), then vanA is a possibility. In this case, the isolate should be sent to the reference laboratory for further testing which usually involves molecular detection of thevanA genes.

9.3.3 Vancomycin-resistant Enterococcus

When dealing with strains of Enterococcus resistant to vancomycin (VRE) the important thing is to distinguish between species that are intrinsically resistant to glycopeptide antimicrobials such as vancomycin and have no public health significance, and those which harbour acquired resistance genes.

For acquired resistance genes there are two types:

• the vanA phenotype, which are both vancomycin- and teicoplanin resistant
• the vanB phenotype which are just vancomycin-resistant.

E. faecium is the most common species to have acquired resistance and the pattern of vancomycin and teicoplanin resistance in this organism can be used to infer the presence of vanA or vanB. Gradient diffusion test strips to measure the MIC are a reliable and straightforward method to use to confirm vanA and vanB(Figure 17).

Figure 17  E-test for VRE ‒ E. faecium with a) teicoplanin and b) vancomycin. The breakpoint for teicoplanin and vancomycin are 2 mg/L and 4 mg/L respectively.

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To interpret the E-test you read the figure where the zone of inhibition hits the test strip. There is no zone at all for the vancomycin and for teicoplanin there is only a small zone ‒ it is many dilutions higher than the breakpoint.

Figure 17  E-test for VRE ‒ E. faecium with a) teicoplanin and b) vancomycin. The breakpoint for teicoplanin and vancomycin are 2 mg/L ...

Note that disk diffusion testing is relatively unreliable for many other antimicrobials in Enterococci. It is essential to follow the guidelines very closely. Most antimicrobials do not have breakpoints for these species defined by EUCAST so it is only recommended to test the antimicrobials that do, otherwise you will end up with results that cannot be interpreted.

This example illustrates how important it is to identify the Enterococcal species. However, it is not easy to tell the difference between species on biochemical tests alone. E. faecium and E. faecalis tend to be non-motile while the other species are motile and most isolates of E. casseliflavus/E. flavescens have a distinct yellow pigment, which can be observed by collecting growth from an agar plate on a swab. However, unless the laboratory has access to MALDI-TOF it may be necessary to send isolates to the reference laboratory.

9.3.4 Penicillin-resistant Streptococcus pneumoniae

Penicillin resistance of S. pneumoniae is commonly screened for with an oxacillin disk in a simple disk diffusion test. This detects non-wild type organisms if the zone size is reduced implying an increased MIC. If necessary, the MIC is then confirmed by a gradient diffusion test (Figure 18). Normally this would only be done for organisms causing an invasive infection. In general, it is seldom necessary to know the specific mechanism.

Figure 18 E-test for a S. pneumoniae isolate

10 How knowing the resistance mechanism contributes to AMR surveillance

10.1 The current picture

Globally, data on AMR and mechanisms are far from complete. For example, there are good data from Europe on isolates from humans and the food chain as a result of a longstanding surveillance scheme run by ECDC and EFSA, but even within these data there is variation between countries. If each centre or country only reports a few isolates this can bias the data. For countries without established regional surveillance schemes the existing data are very patchy. This makes it hard to understand the extent and distribution of resistance.

Two types of data are available:

• rates of mechanisms of resistance for specific species of organisms, for example ESBLs, CPRs, VREs (Figures 19 and 20)
• spatial distribution of particular types of resistance mechanism, for example the NDM carbapenemases or CTX-M ESBLs (Figure 21).

These data are still however far from complete.

Figure 19 Percentage of isolates in 2019 with resistance to third generation cephalosporins in K. pneumoniae (Data from ECDC, 2019)

Figure 20 Spatial distribution of combined resistance to ciprofloxacin and erythromycin in Campylobacter coli isolates from a) broilers and b) fattening pigs (EFSA and ECDC, 2021).

Figure 21 World-wide distribution of carbapenemases: (top) NDM-producers in Enterobacteriaceae and P. aeruginosa; (bottom) OXA-48-like producers in Enterobacteriaceae; (abbreviations) NDM = New Delhi metallo-β-lactamase; OXA-48 = oxacillinase-48 (Bonomo et al., 2018).

10.2 Importance of resistance mechanism data to AMR surveillance

Information about the resistance mechanism associated with clinical and veterinary isolates is useful at local, national and international levels.

Locally, for example within a hospital, knowing the mechanism can be helpful in order to track the spread of a particular gene and/or organism. It may also help to identify whether cases are genuinely linked. This can help the infection control team define whether it is a genuine outbreak or just sporadic cases. Additional testing would be needed to confirm whether strains are related, but it is more likely if they have the same resistance mechanism and early actions can be put in place to limit transmission. If it is an outbreak, understanding how the organism has spread makes it easier to work out the best measures to control the outbreak. For example, is it spreading from person to person or via water contamination?

On a national or international scale, data about resistance mechanisms help to identify priorities for additional surveillance and control, and for R&D. The data can enable early intervention to contain resistance mechanisms of global concern. If mechanisms can be prevented from becoming widespread, this reduces risk to patients from infections which are hard to treat and saves money in healthcare. This is even more important in low-resource settings where antimicrobial choices are limited because of financial constraints.

Maximise

Figure 22 Possible transmission routes for NDM-type carbapenem-resistant E. coli (NDM-EC) among humans, animals and environmental sources in the backyard farm: a) the production mode in backyard farms in rural China; b)possible NDM-EC transmission routes.

The genotypic data can also be used to help understand how transmission from food animals to the human food chain occurs (an example is shown in Figure 22, where the investigators studied the resistance genes in samples from around the farm environment). This in turn can show how this transmission can be prevented. With good quality data it is much easier to implement mechanisms to prevent transmission. This may involve complex interventions such as political, social, and financial measures which may be costly or have negative impacts on food producers in the short term so might be resisted. Data are essential to give the political drive to intervene.

Antimicrobial resistance mechanism data can also help to understand and control international transmission chains. An example could be medical tourism, where people travel to other countries either for cheaper treatment or for treatment which is not available where they live. They risk bringing back and spreading resistant strains to their home country on their return, especially if they have further hospital treatment. This is an important route of spread of AMR and is also a political issue. Again, having good data is vital in order to control spread by this route.

Finally, linking AMR resistance mechanism data to antimicrobial use is very useful to inform strategies for antimicrobial stewardship (see the Antimicrobial stewardship modules).

11 Importance of quality control

In previous modules you learned how microbiology laboratories act as ‘data collectors’ and have an important role in ensuring that test results are accurate, reliable, valid and comparable. You further learned about different quality procedures that laboratories put in place to identify, correct and/or reduce errors. Such measures are essential if confidence in AMR surveillance data is to be maintained.

You will learn more about quality control measures in the Quality assurance and AMR surveillance module.

12 Testing for resistance mechanisms in your workplace

You have now learned about some of the more clinically important mechanisms of antimicrobial resistance and how to test for them.

As long as your laboratory is identifying the species and mechanisms accurately, you can be confident that you are providing reliable data for surveillance. You will then be making an important contribution to fighting antimicrobial resistance both locally and around the world.

13 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.

14 Summary

In this module you have learned about phenotypic and genotypic laboratory techniques used to test for common mechanisms of resistance in important pathogens. You have looked at resistance patterns of global concern, and you have seen how knowledge of the underlying resistance mechanism feeds into AMR surveillance. At a practical level, you have explored screening and confirmatory tests used for pathogen-resistance combinations relevant to your workplace.

You should now be able to:

• give examples of the resistance patterns/resistant organisms causing global concern
• give examples of the resistance patterns/resistant organisms encountered in your work
• understand the difference between screening and confirmatory testing
• describe some of the phenotypic methods commonly used for screening and confirmation of resistance mechanism
• understand how genotypic methods can be used, and the advantages/disadvantages of genotypic versus phenotypic methods
• understand how more data from detailed testing for resistance mechanisms/genes contributes to AMR surveillance
• apply your knowledge of these laboratory tests to interpret data relevant to your work
• know the importance of procedures designed to ensure the quality of these laboratory tests in your workplace.

References

Acknowledgements

This free course was collaboratively written by Sarah Palmer and Liz Sheridan, and was reviewed by Priya Khanna, 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: © funtap/123RF

Figure 1: EYE OF SCIENCE/SCIENCE PHOTO LIBRARY

Figure 2: The Open University

Figure 3: Liz Sheridan

Figures 4 and 5: Liz Sheridan

Figure 6: © Anant Kasetsinsombut/123 Royalty Free

Figure 7: Liz Sheridan

Figure 8: EYE OF SCIENCE/SCIENCE PHOTO LIBRARY

Figures 9, 10, 11 and 12: Liz Sheridan

Figures 13, 14, 15, 16, 17 and 18: Liz Sheridan

Figure 19: © ECDC. Dataset provided by ECDC based on data provided by WHO and Ministries of Health from the affected countries

Figure 20: EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control), 2021. The European Union Summary Report on Antimicrobial Resistance in zoonotic and indicator bacteria from humans, animals and food in 2018/2019. EFSA Journal 2021;19(4):6490, 179 pp. https://doi.org/10.2903/j.efsa.2021.6490. This file is licensed under the Creative Commons Attribution-NoDerivatives Licence http://creativecommons.org/licenses/by-nd/4.0/

Figure 21: Bonomo, R.A., Burd, E.M., Conly, J., Limbago, B.M., Poirel, L., Segre, J.A. and Westblade, L.F. (2018) 'Carbapenemase-Producing Organisms: A Global Scourge', Clinical Infectious Diseases, Vol. 66, Issue 8, 15 April 2018, Pages 1290–1297. Oxford University Press.

Figure 22: Li, J. et al. (2019) Inter-host Transmission of Carbapenemase-Producing Escherichia coli among Humans and Backyard Animals, Environmental Health Perspectives, Vol. 127, No. 10, https://doi.org/10.1289/EHP5251

Videos

Video 1: Liz Sheridan

Tables

Table 1: With kind permission of Prof Peter Hawkey, personal communication, University of Birmingham

Table 5: European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2021) ‘Clinical breakpoints – bacteria’, EUCAST.

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