Microbiological Diagnosis of Animals
Treatment should be aimed at a specific pathogen whenever feasible, and the pathogen should be identified before administration of the antimicrobial is initiated. Care must be taken when predicting the infecting pathogen from historical data because often such data did not discriminate between commensal and pathogen. Furthermore, even when cultures are performed, bacterial culture does not differentiate between infection, colonization, and contamination. Clinically, cytologic evaluation is a fast and relatively inexpensive method to gain some general information about the type of pathogen involved. Examination of a direct smear, scraping, or fine-needle aspirate stained with Wright stain or Gram stain may help to establish the spectra of pathogens involved (eg, gram-positive or gram-negative; rods or cocci) and direct initial antimicrobial treatment. However, microorganisms can grow in stain solutions, leading to artifact or overgrowth on slides. Therefore, stains should be changed frequently, and it is recommended that veterinary practices maintain separate cytological staining setups for blood or effusion samples (clean tub) and for otic, cutaneous, and fecal samples (dirty tub). For more information on cytology sample preparation and evaluation, see Cytology.
Even if the pathogen is correctly identified, the ability to predict its susceptibility pattern is impeded due to increasing antimicrobial resistance and shifting resistance patterns. If the patient has not been previously exposed to antimicrobials, standard expected susceptibility patterns for first-tier drugs (eg, amoxicillin with or without clavulanic acid for E coli, or cephalexin for Staphylococcus pseudintermedius) may be relevant. However, if the pathogen has been exposed to a given antimicrobial (eg, because the current infection is a recurrence, because a different infection was treated with it, or because the pathogen was exposed through another household member), culture and antimicrobial susceptibility testing are recommended to determine the most appropriate treatment.
Numerous state and commercial diagnostic laboratories offer culture and susceptibility testing services. A list of accredited diagnostic laboratories in the US may be accessed through the American Association of Veterinary Laboratory Diagnosticians (AAVLD). The use of an antibiogram, either generated locally for the practice or based on national data, may help identify current susceptibility patterns. Antibiograms report the susceptibility rate of various pathogens to a panel of antimicrobials and list the percentage of tested samples that are susceptible to a particular antimicrobial. Antibiograms vary greatly even between facilities in close proximity, and they reflect current population susceptibility patterns. However, antibiograms do not include minimum inhibitory concentration (MIC) data for pathogens and are best used to direct empiric treatment before culture and susceptibility results are available.
Package insert data or recent literature may also be helpful in drug selection based on population MIC statistics; such data can also be useful for designing dosing regimens. Even in the event of culture submission, empiric antimicrobial treatment may need to be initiated before susceptibility data are received in severely ill patients. If susceptibility data indicate that the isolate is resistant, the empiric treatment should not change if the patient has responded to the chosen drug. Repeated culture in that instance might be important after treatment has been completed. If the patient has not responded to empiric treatment, then the antimicrobial should be changed according to culture and susceptibility results. If the patient fails to respond to antimicrobial treatment directed by culture and susceptibility testing, the data collected before treatment may no longer accurately predict the infecting population because the drug may have changed susceptibility patterns.
Interpretation of Culture and Susceptibility Testing for Animals
Isolation and characterization of the causative pathogen, susceptibility testing, and determination of the MIC provide a sound foundation from which to select the antimicrobial drug and the dosing regimen. However, culture and susceptibility data are only as good as the method by which the sample was collected, handled, and tested. Samples must be collected without contamination, and preferably under aseptic conditions from the site of suspected infection. For information on the collection of culture samples, see Collection and Submission of Laboratory Samples or Clinical Microbiology. Free-catch urine samples, swabs from endotracheal tubes, culture of drain tubes, and swabs from the surface of a contaminated wound are all examples of unacceptable culture samples.
Although swabs are the most common method of collecting specimens, they are often not ideal. They are the preferred method for culture from the nasal passages, pharynx, tonsils, eyes, ears, reproductive tract, skin, and abscesses. Swabs are often contaminated with commensal microbiota during collection, and typically only a small amount of sample can be obtained from swabs. The type of swab used for collection is important: cotton or polyester swabs are preferable; calcium alginate swabs are inappropriate for microbiological assessment. If a swab is to be used for PCR analysis, it cannot be placed in agar or charcoal-based culture medium. It is critical not to dry the samples after collection, and samples should be shipped in a suitable transport medium.
Whenever possible, a tissue or fluid sample is preferred, and handling of the sample is left to the laboratory. For bacteriologic culture of tissue, it is best for each tissue to be collected into separate bags or tubes. For pooled tissue samples, GI tissues should never be pooled with other tissues. For bacteriologic culture of urine, the best recovery of organisms is from samples of at least 1–5 mL of aseptically collected urine. Equally important is proper refrigeration. For example, with a reproduction rate as short as 20 minutes, E coli in an unrefrigerated urine sample can rapidly grow from 101 CFUs (indicating no infection) to > 105 CFUs (indicating infection), potentially masking the true pathogens.
If samples can be transported directly to the laboratory within 24 hours after collection, chilling is appropriate. If the lag time between collection and laboratory delivery is expected to be > 24 hours, then freezing the tissues is preferred. For clostridia and Campylobacter, analysis or freezing must be performed immediately after sample collection to prevent the degradation of toxins. For anaerobic culture, there are several commercial systems for adequately transporting specimens to the laboratory. If commercial systems cannot be used, samples must be collected into a sterile glass tube and the tube completely filled so that no oxygen remains.
For bacteriologic culture of milk, aseptic collection is necessary because contamination of samples is common without strict hygiene measures. The collector should have clean, dry, and preferably gloved hands. Teats must be cleaned, predipped, dried, and antiseptically treated before collection. Then for collection, the teat must be forestripped 2–3 times, and then the samples taken. Milk samples may be refrigerated if culture is performed within 24–48 hours after collection; otherwise, milk samples should be frozen. Bulk tank cultures for milk should be taken from the top of the tank after the tank has been agitated for 10–15 minutes. Samples from the bulk tank should not be taken from the outlet valve. The laboratory must also be selected carefully; it should follow guidelines promulgated by the Clinical Laboratory Standards Institute (CLSI), use veterinary rather than human materials, and be directed by a veterinary clinical microbiologist.
Of the culture and susceptibility procedures routinely used by laboratories, tube dilution (or microdilution) procedures are preferred over agar gel procedures because tube dilution can provide a MIC of the drug toward an isolate of the infecting organism that has been cultured from the patient. The MIC is the minimum concentration of an antimicrobial needed to inhibit visible growth of a single isolate of an organism. The MIC can be used not only to select the drug but also to design the dosing regimen.
Some key points regarding susceptibility testing may facilitate interpretation. The S (susceptible), I (intermediate), or R (resistant) indicator accompanying each MIC is determined by comparison of the MIC of the isolate to MIC break points determined by CLSI. These break points are related to the dose administered, properties of the pharmacokinetics and pharmacodynamics of an antimicrobial, and the bacteria in question.
An organism that is deemed susceptible (S) is inhibited by the serum concentrations of the drug achieved by administration of the recommended dosage.
An organism deemed intermediate (I) is has an extent of inhibition that is approached by the serum concentrations of the drug achieved by administration of the recommended dosing. The intermediate classification implies that the drug may have clinical efficacy in body sites where it accumulates at higher doses, but not in all sites. Sites where drug concentrations may be higher than in the plasma include urine for hydrophilic drugs, local tissues for intra-articular or regional perfusion, and, potentially, abscesses for lipophilic drugs.
An organism deemed resistant (R) is resistant to the concentrations achieved by the drug at the recommended dosage.
Drugs listed as not interpretable (NI) on a culture and susceptibility report require individual interpretation on the basis of the known pharmacokinetics and pharmacodynamics of the drug.
There are some exceptions to the interpretation of culture and susceptibility testing results, generally pertaining to sites where drug concentrations are lower than those achieved in the plasma. These protected sites include the CNS, eyes, prostate, intracellular infections, abscesses for hydrophilic drugs, and poorly vascularized tissues.
Although the actual concentrations tested for all drugs are generally the same, the tested range varies for each drug, as do the break points. The MICs for each drug tested cannot be compared because each antimicrobial has different pharmacokinetics and achieves different plasma or tissue concentrations. For enrofloxacin, for example, tested concentrations generally range from 0.5 to 2 mcg/mL; for amikacin, from 4 to 32 mcg/mL; and for ticarcillin, from 16 to 128 mcg/mL. The current susceptible and resistant CLSI break points, respectively, established for each drug are < 0.5 mcg/mL and > 4 mcg/mL for enrofloxacin, and < 4 mcg/mL and > 64 mcg/mL for amikacin. These break points are adhered to by every laboratory in the US that follows CLSI protocols or guidelines.
The concentrations tested and achieved in a patient vary for each drug, so, for example, a MIC of 0.25 mcg/mL for enrofloxacin should not be interpreted as being better than a MIC of 2 mcg/mL for amikacin. The drug to which the isolate is most susceptible, on the basis of susceptibility data, is the drug with the lowest MIC compared to the maximum plasma concentration (Cmax) achieved in the patient at the recommended dose. In comparisons of multiple drugs to which an isolate is found to be susceptible on culture and susceptibility testing, a better measure of superiority is the number of dilutions away from the break point for each MIC. A drug 3 dilutions away from the break point between susceptible and intermediate is a superior choice to a drug 1 dilution away from the break point. However, it may not necessarily be the best drug to treat the infection, once other host, microbial, and drug factors are taken into consideration. Finally, just because an isolate has been flagged "S" on a susceptibility report does preclude the possibility that the isolate has developed some resistance. Rather, particularly for commonly used drugs, the MIC for a specific isolate may be approaching the CLSI break point and still be deemed susceptible based on the CLSI criteria, despite its having developed some resistance.
Thus, dosages for antimicrobials ideally should err on the side of higher doses (concentration- or time-dependent drugs) or shorter dosing intervals (time-dependent drugs); the more at risk the patient is for treatment to fail, with development of a recurrent, resistant infection, the more important the design of the dosing regimen is. In addition, many synergistic antimicrobial combinations are not tested in culture and susceptibility reports. For example, the combination of a beta-lactam and an aminoglycoside may lower the MIC by 1 dilution. For gentamicin, at a MIC of 4 mcg/mL, E coli is deemed intermediate. However, when gentamicin is combined with penicillin, the MIC decreases to 2 mcg/mL, and the isolate is deemed susceptible.
In addition, data from even appropriately collected samples tested under ideal conditions remain subject to limitations. Testing cannot take into account the impact of distribution to the site of infection, host factors such as inflammation, or microbial factors, including the size of the inoculum. These and other factors may indicate a need to modify the dosing regimen to ensure adequate concentrations at the site of infection. Finally, the route, cost, and safety of the proposed treatment should be taken into account to ensure proper owner compliance and patient safety.
