Bacterial culture and antimicrobial susceptibility testing — basic information that every veterinary practitioner should know

Bacterial infections are common in veterinary medicine, with increasing prevalence of oxacillin (methicillin)-resistant staphylococci identified in 24 to 50% of isolates (1,2). Effective antibiotic stewardship is crucial to preserve antibiotic efficacy for both humans and companion animals, as part of the One Health initiative (3). Veterinarians frequently encounter bacterial skin infections, and updated guidelines for pyoderma are now available [International Society for Companion Animal Infectious Disease (ISCAID)] (4). The objective of this column is to highlight the importance of skin cultures and provide tools for interpreting them.

Veterinarians provide samples and clinical history, including details of current and previous antibiotic treatments. Microbiologists and laboratory personnel are responsible for growing bacteria, identifying them, and performing antimicrobial susceptibility testing (AST). Veterinary microbiologists are Board-certified specialists; collaboration between professionals is essential for optimizing test results and developing an effective treatment plan.

OBTAINING SAMPLES

The first step is to know where and how to sample. Moist lesions, such as pustules or undercrusts, typically harbor more bacteria than dry lesions (4). However, if no fresh lesions are present, clinicians can moisten the swab with culture media to improve retrieval. Cytology can provide valuable preliminary information. For instance, if cocci are observed, cocci should be cultured. Alternatively, if rods are present, the clinician must consider whether these bacteria are the true pathogen. Extra care is needed when sampling perimucosal areas and feet, as these regions can harbor normal flora or environmental bacteria. For example, strains of Pseudomonas aeruginosa may be present in tap water, and wet feet or lips may carry Pseudomonas more as a contaminant than a pathogen (5). Therefore, it is advised that gloves be used when swabbing, to avoid contaminating the swab with your own skin bacteria.

WHEN SHOULD A CULTURE BE DONE?

Although not always necessary, cultures are never contraindicated. Due to the expense of tests in veterinary medicine, practitioners often face challenging decisions. As a guideline, cultures should be considered under the following circumstances (4,6):

1. The patient has received antibiotics in the past year, especially beta-lactams or quinolones.

2. The patient has been hospitalized.

3. There is treatment failure with current antibiotics (< 50% improvement within 2 wk, or new lesions have appeared during treatment).

4. Rod-shaped bacteria are observed in cytology.

5. The patient has a history of bacterial resistance.

If a client declines doing a culture, advise them that a culture should be done if the first-line treatment fails. Culture results and AST guide treatment and document bacterial resistance, with implications for pet owners. If a pet is on antibiotics but bacteria are still present in cytology, a culture may be warranted. However, false negatives can occur. Ideally, antibiotic treatment should be discontinued 48 h before sample collection (7). If stopping treatment is not feasible for the patient’s well-being, inform the laboratory, as enrichment or different growth media may be used.

WHAT INFORMATION SHOULD BE INCLUDED ON THE LABORATORY REQUEST?

The laboratory request should include species, breed, and the body site from which the sample was taken. The latter is crucial, as normal flora may vary among sites. This information also helps the laboratory determine which antibiotics to test. For instance, topical antibiotics may be tested on ear samples but not on mucosal samples.

WHAT BACTERIA SHOULD BE EXPECTED ON A SKIN CULTURE REPORT?

On the skin, > 90% of cultured bacteria will be Staphylococcus spp. (4). In cases of superficial pyoderma, any bacteria other than Staphylococcus should be considered suspect and correlated with cytology.

Deep pyoderma cultures can yield a variety of bacteria, e.g., Staphylococcus, Streptococcus, Enterococcus, etc. Cultures are essential for deep pyodermas, as they require prolonged antibiotic treatment. Tissue samples are preferable for such cultures. Feet furunculosis may involve enteric bacteria such as E. coli and Enterococcus spp., which have diverse resistance patterns, making empirical treatment risky.

Mucocutaneous pyoderma affects the junction between the epidermis and dermis. Topical treatments are often impractical for these sites; systemic treatment is usually required. Culturing is strongly recommended before choosing a treatment. Caution must be taken to interpret results, as normal flora bacteria can be isolated. Although there are no specific guidelines for this condition, longer courses of systemic antibiotics (3 wk to start, then re-evaluate) are often necessary (4).

WHAT IS ENRICHMENT AND WHEN IS IT USED?

Enrichment steps for samples likely containing commensal flora are not recommended, as bacteria recovered from enrichment broth may represent normal flora rather than pathogenic organisms. In veterinary dermatology, enrichment is generally reserved for deep pyoderma samples, tympanic bulla samples if diluted, and for patients currently receiving antibiotics. Otherwise, enrichment can lead to unreliable results. To promote antimicrobial stewardship, veterinary microbiologists typically do not report normal commensal flora (8).

WHAT IS CONSIDERED WHEN CULTURING A SKIN SAMPLE?

Standard culture media support the growth of common bacteria (e.g., Staphylococcus, Streptococcus, E. coli, Enterococcus, Pseudomonas, and Proteus spp.). Samples should be submitted in an appropriate transport medium (e.g., Stuart or Amies medium) and transportation delays minimized. Enriched non-selective media, such as Columbia blood agar, is often used for routine culture, whereas for suspected Gram-negative bacteria, selective media such as MacConkey agar may also be used. More fastidious bacteria, e.g., Actinomyces spp., Nocardia spp. and mycobacteria, require longer incubation periods and can be involved in subcutaneous abscesses. These bacteria are not visible with Romanowsky stains (e.g., Diff-Quick). Clinicians should note any suspicion of these bacteria on the submission form so that appropriate media and incubation periods can be chosen.

HOW ARE BACTERIA IDENTIFIED?

Most laboratories now use Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight (MALDI-TOF) technology for precise bacterial identification via mass spectrometry (9). Bacteria identification results can be obtained rapidly, allowing antibiotic selection based on antibiotic spectrum of activity against the bacteria identified. This is particularly useful in urgent care situations. For most cases of superficial pyoderma, clinicians can wait for AST results, which are typically available 48 h after bacterial identification and 36 to 72 h after sample submission or receipt by the laboratory. The actual timing can vary, depending on factors such as bacterial load and species.

WHAT IS ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST) AND HOW IS IT DETERMINED?

Antibiotic susceptibility is determined in vitro using standardized methods recognized by the Clinical and Laboratory Standards Institute (CLSI) (10). Common methods include minimal inhibitory concentration (MIC) determination by broth microdilution and zone diameter measurement using the disc diffusion method (Kirby-Bauer). The CLSI VET09 (11) defines bacterial susceptibility as follows: A bacterial isolate is considered susceptible if its minimum inhibitory concentration (MIC) is at or below the breakpoint, or if the zone diameter of inhibition is at or above the susceptible breakpoint. This means that the antimicrobial agent, when administered at the recommended dosage for the infection site, will likely achieve concentrations sufficient to inhibit bacterial growth, leading to probable clinical efficacy.

WHICH ANTIBIOTICS ARE TESTED?

Laboratories consider several factors when deciding which antibiotics to test and report. Commercial devices may be used to obtain MICs with standardized plates or cards. The panel of antibiotics tested is usually standard for each animal species and bacterial type. Laboratories using the Kirby-Bauer method have more flexibility in choosing antibiotics.

Once antibiotic susceptibility of a bacterial isolate has been assessed, the laboratory should follow selective or cascade reporting practices, as recommended by the ACVIM (8). This approach involves reporting only specific antibiotic results based on the case and on principles of good antimicrobial stewardship. Results for additional antibiotics will only be reported if first-line treatments are resistant or if special patient considerations apply (e.g., allergic reactions, renal or hepatic diseases). The body site will also influence reporting; for instance, some antibiotics can only be applied topically and will be reported only when a topical treatment is feasible. In addition, selective reporting may prioritize first-generation or narrow-spectrum antibiotics if they are effective, such as first-generation cephalosporins, whereas third-generation antibiotics may only be reported if first-line drugs are likely to be ineffective due to resistance (11).

WHAT ARE ANTIMICROBIAL BREAKPOINTS AND HOW ARE THEY DETERMINED?

Clinical breakpoints are established considering the bacteria, animal species, infection site, and the pharmacokinetics and pharmacodynamics (PK/PD) of the antimicrobial. For example, a breakpoint might be available for MIC and zone diameter for Staphylococcus spp. isolated from a dog’s skin or soft tissue infection treated with clindamycin at 5.6 mg/kg BW, PO, q12h. Breakpoints help to relate in vitro results to clinical outcomes. If specific breakpoints are unavailable, the laboratory may extrapolate from human breakpoints, which can affect validity. Breakpoints are sometimes available for only 1 method (e.g., MIC), which can limit interpretation if another method (e.g., Kirby-Bauer) is used.

WHAT IS THE MINIMAL INHIBITORY CONCENTRATION (MIC) AND HOW IS IT USED?

The MIC represents the minimal concentration needed to inhibit bacterial growth in vitro. It is especially useful when compared to clinical breakpoints to predict treatment effectiveness. If a clinical breakpoint is unavailable, the MIC can help if the antibiotic concentration achievable at the infection site is known, considering dosage and PK/PD. If the concentration at the infection site exceeds the MIC, treatment is likely to be effective. The MIC values are useful when breakpoints exist but cannot be compared across antibiotics for the same bacteria. For example, a susceptible Staphylococcus from a dog might have a breakpoint of ≤ 0.5 μg/mL for clindamycin and ≤ 2 μg/mL for cephalexin. Despite the higher MIC for cephalexin, both antibiotics are susceptible and should be equally effective at recommended dosages (12).

WHY IS AST NOT PROVIDED FOR ALL BACTERIA REPORTED?

Not all bacteria have established clinical breakpoints for susceptibility testing. Laboratories may not perform AST if no breakpoint exists for a specific bacterium. In addition, some methods, such as Kirby-Bauer, only test rapidly growing bacteria (24 h). For example, Corynebacterium spp. and Actinomyces are slow growing bacteria and lack veterinary-specific breakpoints. When reliable AST cannot be conducted or interpreted, a literature review may help determine which antibiotics to use. Diagnostic laboratories strive to provide valid and repeatable results, whereas research contexts may allow more flexibility.

IS BACTERIAL CULTURE AND AST RECOMMENDED FOR EXTERNAL OTITIS?

Bacterial cultures of the ear should always be interpreted in conjunction with cytology, as the bacterial culture and cytology correlate only 60% of the time (13). Sometimes, opportunistic pathogens can also be common contaminants. For example, Proteus spp., a member of the Enterobacterales Order, can overgrow on culture media, but it might be present only in small amounts as a fecal contaminant rather than being the actual pathogen. Correlating culture results with cytology is crucial for identifying the true culprit. As AST is less relevant for topical antibiotics, which often exceed MICs, but remains useful for documenting bacterial resistance in a patient. For suspected otitis media, culture and AST are crucial for selecting an appropriate systemic antibiotic. Antimicrobial susceptibility testing and clinical breakpoints are developed for systemic administration, not local or topical treatments, as concentrations achieved with topical treatments can be significantly higher than plasma concentrations (11).

WHAT IS INTRINSIC RESISTANCE AND WHY IS IT RELEVANT?

Intrinsic resistance refers to the inherent ability of certain bacteria to resist the effects of specific antibiotics, due to characteristics that are encoded in their genome. For practitioners, knowing about intrinsic resistance is crucial for several reasons:

1. Avoiding treatment errors: Intrinsic resistance helps in avoiding ineffective treatments for infections caused by intrinsically resistant bacteria.

2. Interpreting AST results: Intrinsic resistance aids in the correct interpretation of AST results, distinguishing between intrinsic and acquired resistance.

3. Empirical treatment: Intrinsic resistance informs the selection of empirical treatment while awaiting AST results.

Intrinsic resistances of bacteria most frequently encountered in veterinary dermatology are in Table 1.

TABLE 1.

Intrinsic resistances of bacteria most frequently encountered in veterinary dermatology.

Bacteria	Intrinsic resistance (CLSI Vet09) (12)
Staphylococcus spp.	Polymyxin B
Streptococcus spp.	None. Beta-hemolytic streptococci are predictably susceptible to penicillins and therefore may not need to be tested
Pseudomonas aeruginosa	Penicillin, Ampicillin/Amoxicillin, Amoxicillin-clavulanate, Cephalexin, Cefpodoxime, Cefovecin, Clindamycin, Erythromycine, Lincosamide, Tetracyclines, Chloramphenicol, Trimethoprim, TMS, Rifampin, Imipenem, Fusidic acid, Glycopeptides (Vancomycin)
Enterococcus spp.	Fusidic acid, Trimethoprim, Polymyxins Although not classified as intrinsic resistance, aminoglycosides (except for high level resistance), cephalosporins, clindamycin and TMS may appear active in vitro but are not effective clinically and should not be reported as susceptible
Proteus mirabilis	Tetracycline, Polymyxin B, Clindamycin, Fusidic acid, Glycopeptides (Vancomycin), Macrolides, Rifampin
Proteus vulgaris	Ampicillin/Amoxicillin, Cephalexin, Tetracycline, Polymyxin B, Clindamycin, Fusidic acid, Glycopeptides (Vancomycin), Macrolides, Rifampin
Pasteurella multocida	None
Actinomyces	None

Unlike acquired resistance, which develops through mutations or acquisition of resistance genes, intrinsic resistance is a natural feature of the bacterium (14) and can result from:

1. Lack of affinity: The antibiotic has no or very low affinity for the bacterial target.

2. Impaired drug entry: The antibiotic cannot effectively penetrate the bacterial cell.

3. Efflux Pumps: The bacterium has efflux pumps that actively expel the antibiotic.

4. Degrading enzymes: The bacterium produces enzymes that degrade the antibiotic.

In conclusion, the choice of sampling site, communication with the laboratory, the expertise of veterinary microbiologists, and judicious interpretation of results all help to select an appropriate treatment for the patient. Minimum inhibitory concentrations cannot be compared to one another, and laboratories that report them do not always provide the information needed to use them clinically. Clinicians are advised to read current guidelines for treatment recommendations. Knowledge of intrinsic resistances and common pathogens helps clinicians to correctly interpret culture and AST results.