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An update on antibiotic susceptibility testing

A summary of the methods currently used and the benefits of understanding intrinsic resistance, which is essential in initial treatment choice

17 July 2018, at 8:34pm

Antibiotic susceptibility testing (AST) is one of the key elements of microbiology, and can have great consequences on the choice of antimicrobial used. After an isolate has been identified, AST should be performed to confirm susceptibility to the chosen empirical antimicrobial agent, and to detect acquired resistance in individual isolates. Once susceptibility patterns have been determined, a clear clinical treatment plan can be developed. 

The most frequently used industry methods are Kirby-Bauer disc diffusion, automated instrument systems, such as the VITEK, or the antimicrobial gradient method using E-tests (Figure 1). All three methods are governed by international and/or European standards which are the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), respectively. CLSI has veterinary guidelines (CLSI Vet), whereas EUCAST currently has a subcommittee, “VETCAST”, which has not yet published veterinary guidelines. 

FIGURE 1 The most frequently used methods of antibiotic sensitivity testing
FIGURE 1 The most frequently used methods of antibiotic sensitivity testing

Kirby-Bauer disc diffusion

One of the most commonly used methods of AST in veterinary laboratories is Kirby-Bauer disc diffusion, which uses small discs impregnated with specific concentrations of antibiotic to determine sensitivity. Pure colonies are taken from a bacterial isolate and mixed with demineralised water to produce a standardised concentration, which is determined by using McFarland Turbidity Standard 0.5. The solution is then spread evenly over a Mueller Hinton agar plate. The antibiotic discs are then placed on the surface of the agar and the plate is incubated for 18 to 24 hours at 37℃. 

The following day, zone diameters are read using either a ruler or callipers. The zone size is then determined as susceptible (also “sensitive”), intermediate or resistant, based on the clinical break point diameters provided by relevant guidelines. Zone sizes differ for each antibiotic and also vary depending on the organism, therefore identification of an isolate prior to AST is essential. While this is a qualitative method, it still provides useful information regarding appropriate use of antimicrobials for the veterinarian.

Automated instrument systems 

Automated instrument systems have greatly improved the standardisation and turnaround time of identification and AST. Most systems utilise reagent cards that contain microlitre quantities of antibiotics and test in a 64-96 well system to implement repetitive turbidimetric monitoring of bacterial growth. Growth is regularly monitored to produce data as quickly as possible. The turbidity of the solution indicates the sensitivity of the isolate based on the minimum inhibitory concentration (MIC). The instrument systems have advanced computer software used to interpret susceptibility patterns for each organism, including “expert systems” for analysing test results for atypical patterns and unusual resistance phenotypes. 

Antimicrobial gradient method 

The antimicrobial gradient method determines AST by establishing an antimicrobial concentration gradient in an agar medium; the point to which the isolate grows determines susceptibility. The isolate is taken from the original growth plate and mixed with saline to a McFarland Turbidity Standard that is determined by the E-test application guide, then spread evenly over a Mueller Hinton agar plate. Plastic E-test strips impregnated with antibiotic on the underside are placed onto the agar surface. The MIC value (as dictated by relevant standards) is determined by the point at which the growth intersects with the strip (at the bottom of the ellipse shaped inhibition area).

Intrinsic resistance 

With all methods of susceptibility testing, each organism has its own reference breakpoints as advised by the EUCAST/CLSI standards. Each organism type must be assigned a target panel of antibiotics with individual reference zone sizes, because zone size diameters for the same antibiotic vary between organisms. With some organisms, reference breakpoints can vary within a genus. Staphylococcus pseudintermedius and S. aureus, for example, have different breakpoints for assessing sensitivity to penicillins and cephalosporins. 

Some organisms also have greater intrinsic resistance, and therefore have a more limited panel of target antibiotics. A prime example of this is Pseudomonas aeruginosa, which has developed intrinsic resistance to multiple first-line antibiotics, so only a limited panel of antibiotics can be tested (Figure 2). Pseudomonas frequently develops resistance to antibiotics during therapy so retesting the isolate is recommended when cases are slow to, or do not, resolve clinically. 

Intrinsic resistance varies among species and within genera, and can greatly aid in decisions regarding initial drug choice. Intrinsic resistance is the innate ability of an organism to resist the action of a specific antimicrobial agent, as a result of its inherent characteristics. For example, enterococci are intrinsically resistant to all drugs in the cephalosporin class. This is because the drug action works

FIGURE 2 Only a limited panel of antibiotics can be tested for Pseudomonas aeruginosa, which has developed intrinsic resistance to multiple first-line antibiotics
FIGURE 2 Only a limited panel of antibiotics can be tested for Pseudomonas aeruginosa, which has developed intrinsic resistance to multiple first-line antibiotics

by binding to a peptidoglycan binding protein, which enterococci do not possess. The EUCAST “Intrinsic Resistance and Exceptional Phenotypes” table can be found on the EUCAST website and contains the intrinsic resistance profile of a multitude of organisms.

Knowledge of intrinsic resistance is essential in initial treatment choice, and can be particularly useful in drug choice for treating topical infections. Gram staining ear cytology smears can greatly narrow down the choice of antibiotics that are available for use (Figure 3). For example, all gram-positive organisms are intrinsically resistant to polymyxin B, and all gram-negative organisms are resistant to fusidic acid. The reader is referred to the EUCAST website for more information on intrinsic resistance patterns. 

Gram-positive bacteria are also intrinsically resistant to aztreonam, temocillin, polymyxin B/colistin and nalidixic acid.

FIGURE 3 Gram staining can greatly narrow down the choice of antibiotics available for use
FIGURE 3 Gram staining can greatly narrow down the choice of antibiotics available for use

Acquired resistance 

Acquired resistance is the ability of an organism to resist the activity of a specific antimicrobial agent to which it was previously, or is expected to be, susceptible (and does not include the antibiotics to which it is intrinsically resistant). Common examples of acquired resistance include methicillin resistant staphylococci (MRS) and extended spectrum beta lactamase producing organisms (ESBLs). MRS are predicted using either molecular methods or inference from sensitivity patterns (penicillin and cefoxitin/oxacillin), combined with detection of beta-lactamase production. 

ESBL-producing organisms develop resistance to all penicillins and cephalosporins. This phenomenon is seen in gram-negative organisms including E. coli, Enterobacter and Klebsiella, and can be detected with disc detection kits. Monitoring trends in acquired resistance can greatly help identify patterns of resistance and incidence locally and internationally. At practice level, it can also aid in detection of any nosocomial (hospital acquired) infection patterns.