Joseph E. Rubin and Peter Damborg The veterinary diagnostic microbiology laboratory plays a key role in the practice of evidence‐based antimicrobial therapy by providing culture and susceptibility information to practitioners. Before the introduction of antimicrobials, we were largely powerless to treat invasive infections. The antimicrobial age began with the familiar story of the discovery of penicillin in 1928 by Alexander Fleming. By the early 1940s, that Penicillium notatum extract was successfully used against infections caused by organisms ranging from Staphylococcus aureus to Neisseria gonorrhoeae (Aronson, 1992; Bryskier, 2005). Unfortunately, the evolutionary power of bacteria resulted in the rapid emergence of antimicrobial resistance. Susceptibility testing is now vital for effective therapeutic decision making. Although veterinary laboratories utilize many of the same basic microbiological techniques as human diagnostic labs, they face some unique challenges. These challenges include the difficulty in cultivation of fastidious veterinary‐specific organisms, availability of species customized antimicrobial panels for susceptibility testing, and lack of validated veterinary breakpoints. In the clinical setting, the goal of antimicrobial susceptibility testing (AST) is to help clinicians choose the optimal antimicrobial therapy. The decision to undertake culture and susceptibility testing depends on the site of infection, state of the patient, prior history of infections and antimicrobial use, co‐morbidities and underlying disease, and the predictability of the susceptibility patterns of the most likely pathogen(s). For example, susceptibility testing is generally not indicated in horses with “strangles,” as Streptococcus equi is uniformly susceptible to the drug of choice, namely penicillin (Erol et al., 2012). Conversely, culture and susceptibility testing should always be done in cases of recurrent urinary tract infections in dogs. Early methods used to assess the susceptibility of organisms to antimicrobials were developed by individual labs and lacked standardization; the first effort to standardize susceptibility testing was published in 1971 (Ericsson et al., 1971). National standards organizations responsible for guidelines for conducting and interpreting antimicrobial susceptibility tests were subsequently created. In the United States, the Clinical and Laboratory Standards Institute (CLSI) formed in the late 1960s as the National Committee for Clinical Laboratory Standards (NCCLS) and was tasked with developing a standard for disk diffusion antimicrobial susceptibility testing (Barry, 2007). While standardization of methods yields more comparable data between labs, heterogeneity in interpretive criteria persists. In 1997, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was formed to harmonize both testing methods and interpretive criteria throughout Europe. In North America, the CLSI methodologies are used for both human and veterinary diagnostics. For the most common nonfastidious veterinary bacterial pathogens (e.g. Enterobacterales and staphylococci), susceptibility testing is straightforward using a common method. For testing fastidious and anaerobic organisms, specific standards have been developed but are not covered in this chapter. The available standardized antimicrobial susceptibility tests yield quantitative data (minimum inhibitory concentration [MIC] or inhibition zone diameter) that can be interpreted in different ways as described later in this chapter. Testing methods can be divided into two distinct categories: diffusion and dilution based. Two types of diffusion tests are available. The size of the inhibitory zone is a function of the rate of drug diffusion, thickness of the media, concentration of drug in the disk, and the susceptibility of the organism, making method standardization necessary for interpretive criteria to be applied (Figures 2.1, 2.2). Figure 2.1 Disk diffusion: The results of the disk diffusion test can be influenced by he depth of the medium (A and B, increase in zone of inhibition; C, decrease in zone of inhibition) or the quality of the inoculum (D, false increase in zone of inhibition; E, false decrease in zone of inhibition; F, mixed culture, false decrease in zone of inhibition). Disk diffusion testing is done using 4 mm thick Mueller–Hinton agar plates and antimicrobial impregnated filter paper disks (CLSI, 2018a,b). Room‐temperature plates are inoculated with a lawn of bacteria drawn from a McFarland 0.5 (approximately 108 CFU/ ml) suspension using a sterile swab. Plates are allowed to dry for up to 15 minutes before the disk is applied and are then incubated for 16–20 hours at 35°C (+/– 2°C) at room atmosphere. After incubation, the diameter of the inhibitory zone is measured (Figure 2.2A). Owing to differences in antimicrobial diffusion rate, amount of drug included in disks, and pharmacodynamic interactions, the size of the inhibitory zone corresponding to resistance breakpoints is unique to each drug/organism combination. The relative clinical appropriateness of different antimicrobials can therefore not be determined by simply comparing inhibitory zone diameters. Gradient tests (e.g., Etest®) are conducted in the same way as disk tests. These strips contain a gradient of antimicrobial from low to high concentrations corresponding to printed MIC values on the strip. Following incubation, the apex of the teardrop zone of inhibition indicates the MIC of the organism (Figure 2.2B). Figure 2.2 Antimicrobial susceptibility testing methods. Diffusion‐based tests are technically simple to perform and versatile, allowing customization of test panels to bacterial and patient species and type of infection. Dilutional susceptibility testing can be done using either broth or agar media and yields MIC data. By convention, MICs are measured on a doubling dilution series (… 0.12 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml …). An antimicrobial free control medium must always be included as a positive control for bacterial growth. The lowest concentration without bacterial growth defines the MIC, except for the sulfonamides and trimethoprim, where an 80% reduction in growth compared to the control constitutes inhibition. For agar media dilution, Mueller–Hinton agar plates are prepared incorporating doubling dilutions of antimicrobial. Antimicrobial stock solutions at 10 times the test concentration are prepared using the solvents and diluents recommended by the CLSI (CLSI, 2018b). To prepare media, antimicrobial stock solution is added in a 1:9 ratio to molten Mueller–Hinton agar no hotter than 50 °C, and poured into sterile petri dishes. Separate plates are prepared for each antimicrobial concentration test. Plates must not be stored for more than seven days prior to use and for some drugs (e.g., imipenem), they must be prepared fresh on the day of use. Room‐temperature plates are inoculated with approximately ~104 CFU using either a multi‐spot replicator or manually by pipette. To prevent discrete samples from mixing, plates are left on the bench top for up to 30 minutes for the bacterial spots to be absorbed prior to incubation. Plates are incubated in room air at 35 °C for 16–20 hours and examined for growth (Figure 2.2C). This technique is very labor intensive, hence its use is mainly limited to research. For broth dilution, Mueller–Hinton broths containing doubling dilutions of antimicrobial are prepared. As in agar dilution, antimicrobial stock solutions at 10 times the final concentration are prepared and added to test medium in a 1:9 ratio. Each antimicrobial concentration is dispensed into separate vials and inoculated with bacteria to yield a final concentration of 5 × 105 CFU/ml. A McFarland 0.5 inoculum is typically made in either sterile water or saline and then aliquoted into the Mueller–Hinton broth to yield the final concentration. Growth is indicated by turbidity or a cell pellet, and the MIC is defined by the lowest concentration where growth is not seen. Commercially prepared microdilution plates (Figure 2.2D) allow a large number of bacterial isolates to be tested efficiently without the need to prepare antimicrobial dilutions or large volumes of media in house. In some systems, microdilution plates can be inoculated and read automatically, thereby reducing hands‐on time. Some automated systems (e.g., Vitek® 2) work by regularly comparing the growth of test and antimicrobial‐free control cultures to provide a growth index from which susceptibility is predicted. In this way, results can be produced faster than the 16–20 hours typically required for incubation of MIC assays. Unfortunately, these commercially prepared plates are more expensive compared to the supplies required for disk diffusion, and the flexibility to change antimicrobial panels is limited (Figure 2.2E). Antimicrobial susceptibility testing results can be interpreted in different ways. The epidemiological cut‐off (ECOFF) for an antimicrobial agent is defined as the highest MIC for organisms without phenotypically detectable acquired resistance.1 Isolates with MICs above the ECOFF have therefore acquired resistance mechanisms that differentiate them from wild‐type organisms of the same species. ECOFFs are established by evaluating the MIC distributions of large isolate collections. While ECOFFs are invaluable research tools, e.g., for surveillance of emerging resistance, they do not take into account achievable drug concentrations in target tissue and should not be used as a first tool to guide therapy of patients. In fact, as shown in Figure 2.3, ECOFFs may differ from clinical breakpoints (CBPs), which are used for categorical interpretation of antimicrobial susceptibility test results for clinical use: susceptible = high probability of success following treatment, intermediate = treatment possible when the drug concentrates at the target site or when the dosage can be increased, and resistant = low probability of success following treatment. In 2024, a new category called susceptible dose dependent (SDD) was added for some drug–organism combinations by the breakpoint‐setting association CLSI. SDD implies that susceptibility of an isolate depends on the dosage regimen that is used in the patient. One reason for adding the SDD category was that to the clinician, “intermediate” too often means “resistant” because they do not appreciate the full definition of “intermediate.” Nevertheless, the SDD definition can appear confusing, as it partially overlaps with the intermediate category. For further elaboration, readers are encouraged to consult material provided by the CLSI (CLSI, 2024). CBPs are established by taking into account ECOFFs as well as pharmacokinetic data and, when available, clinical outcome data following treatment. CBPs are specific to animal species, dosing regimen (dose, route of administration, and frequency), disease, and target pathogen. Internationally recognized veterinary‐specific CBPs are currently only published by the CLSI (CLSI, 2024), although the veterinary subcommittee of EUCAST (VetCAST) also has veterinary CBPs in the pipeline. Figure 2.3 In this MIC distribution graph, the observed gentamicin MICs for 80 001 E. coli isolates are displayed. MICs are listed on the x‐axis in μg/ml and the number of isolates inhibited at each concentration is listed on the y‐axis. The EUCAST epidemiological cut‐off (ECOFF) differentiating wild‐type organisms from those with an acquired resistance mechanism is compared with the CLSI clinical resistance breakpoint (CLSI, 2024). In this example, E. coli may possess an acquired gentamicin resistance mechanism without crossing the threshold into clinical resistance. Unfortunately, a general lack of validated veterinary‐specific CBPs is a serious limitation for veterinarians trying to practice evidence‐based medicine. For example, there are no validated CBPs for any pathogens causing enteric disease, for important (but difficult to culture) pathogens like Mycoplasma, or for pathogens in certain animal species including fish and exotic/wildlife species (e.g., rodents, lagomorphs, reptiles, amphibians, and birds). Current veterinary‐specific CBPs from the CLSI are listed in Table 2.1, but as breakpoints are continuously being developed, readers are advised to always seek the most updated guideline documents. Extrapolation of nonapproved breakpoints should be done with extreme caution; in some instances CBPs from other infection sites, bacterial species, animal species or humans can be adapted. One should consider consulting with a clinical microbiologist or clinical pharmacologist when deviating from animal‐, infection‐, and dosage‐specific CBPs. In practice, the application of antimicrobial susceptibility test results for clinical use is often reduced to susceptible = good treatment choice and resistant = bad treatment choice, rather than a thorough analysis of the susceptibility profile. Interpretive reading is a more biological approach that incorporates knowledge of intrinsic drug resistance, exceptional resistance phenotypes, indicator drugs, and consideration of antimicrobial selection pressure (Livermore et al., 2001). Interpretive reading is used to detect specific resistance phenotypes such as methicillin resistance or the production of extended‐spectrum beta‐lactamases (ESBLs). Some of these tests are organism specific, and use across species or genera may not yield reliable results. For example, the CLSI recommends that either cefoxitin or oxacillin resistance may be used as an indicator of mecA‐mediated methicillin resistance in S. aureus, while only oxacillin resistance reliably predicts mecA in S. pseudintermedius (CLSI, 2023, 2024; Papich, 2010). In Enterobacterales, the susceptibility profile to a combination of third‐generation cephalosporins with and without clavulanic acid, cefoxitin, and cloxacillin is useful for phenotypically differentiating ESBLs from AmpC type beta‐lactamases (CLSI, 2023, 2024). Table 2.1 Drugs with veterinary‐specific CLSI resistance breakpoints according to the VET01S‐Ed7 document (CLSI, 2024). Abbreviations used for bacteria are shown in the footnote.
2
Antimicrobial Susceptibility Testing Methods and Interpretation of Results
Antimicrobial Susceptibility Testing Methods
Diffusion‐based Methods
Dilution‐based Methods
Interpretation of Susceptibility Testing Results
Drug
Animal species
Dog
Cat
Cattle
Horse
Pig
Amikacin
Ent, PA, Staph spp., Streptococcus spp.
Ent, PA, SA, S. equi subsp. equi and zooepidemicus
Amoxicillin‐clavulanate
Ent, Staph spp. (SST), EC, Enterococcus spp., K. pneumoniae, Proteus mirabilis/vulgaris (ur)
Ent, PM, Staph spp., Strep spp. (SST), EC, Enterococcus spp., K. pneumoniae, PM, P. mirabilis/vulgaris, Staph spp., Strep spp. (ur)
Ampicillin
Ent, Enterococcus spp. SP, Strep spp. (SST), EC, Enterococcus spp., Proteus mirabilis (ur)
Ent, PM, Staph spp., Strep spp. (SST), EC, Enterococcus spp., PM, P. mirabilis, Staph spp., Strep spp. (ur)
Ent (metritis), HS/MH/PM (resp)
SA (resp, SST), S. equi subsp. equi and zooepidemicus (resp), Ent
APP, BB, PM, S. suis (resp)
Cefazolin
Ent (SST), EC, K. pneumoniae, P. mirabilis (ur), SA, SP (resp, SST, ur), beta‐hem Strep spp. (gen, resp, SST, ur), PM (resp, SST)
Ent, beta‐hem Strep spp. (gen, resp)
Cefoperazone
EC, Staph spp., S. agalactiae, S. dysgalactiae, S. uberis (mastitis)
Cefovecin
EC, P. mirabilis (ur), SP, beta‐hem Strep spp. (SST)
EC (ur), PM (SST)
Cefpodoxime
EC, P. mirabilis (ur, wound/abscess), SA, SP, S. canis, PM (wound/abscess)
Ceftazidime
Ent, PA (SST)
Ceftiofur
EC, SA, S. agalactiae, S. dysgalactiae, S. uberis (mastitis), HS/MH/PM (resp)
S. equi subsp. zooepidemicus (resp)
APP, PM, S. Choleraesuis, S. suis (resp)
Cephalexin
EC, K. pneumoniae, P. mirabilis (ur), EC, SA, SP, beta‐hem Strep spp. (SST)
Cephalothin
SA, SP, beta‐hem Strep spp. (SST)
Chloramphenicol
Ent, Enterococcus
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