Department of Medicine & Epidemiology, University of California-Davis, Davis, CA, USA
In recent years, there has been a frightening rise in the prevalence of multidrug-resistant bacterial infections in dogs and cats. Because of this, whenever possible, veterinarians should make attempts to confirm a suspected bacterial infection by requesting microscopic evaluation of direct smears, culture and susceptibility by a laboratory before the choice is made to administer an antimicrobial drug. Cytology can permit the rapid preliminary diagnosis of infection. Whether organisms seen are cocci or rods helps guide the clinician to select an appropriate empiric therapy, if necessary, while awaiting the results of culture and susceptibility testing. Important terms used to describe resistant bacterial infections are as follows:
Beta lactam: Antimicrobial drug that includes a beta-lactam ring (all penicillins, cephalosporins and carbapenems such as meropenem). These bind to penicillin binding proteins (PBPs) (bacterial enzymes that catalyze bacterial cell wall formation) and cause bacterial lysis.
Beta lactamases: Bacterial enzymes that destroy the beta lactam ring (associated with resistance to beta-lactams). These include a variety of penicillinases. Beta lactamase inhibitors are drugs that inhibit these enzymes and include clavulanic acid and sulbactam.
ESBLs: Extended spectrum beta lactamases. These are bacterial enzymes that destroy critical beta-lactam drugs needed for treatment of resistant bacterial infections in humans (by definition, third generation cephalosporins such as cefuroxime, cefotaxime, ceftazidime). They are generally expressed by gram-negative enteric bacteria such as E. coli and Klebsiella.
MRS: Methicillin-resistant staphylococcus. These organisms express an altered penicillin binding protein (PBP2a) that does not bind beta-lactam drugs. Therefore, they are resistant to penicillins, cephalosporins and carbapenems.
MOR: Multidrug resistance. By definition, this is resistance to 3 or more classes of antimicrobial drugs (e.g., cephalosporins, fluoroquinolones, and aminoglycosides).
Methods of Susceptibility Testing
Clinical microbiology laboratories will perform susceptibility testing for most aerobic bacteria, with the exception of streptococci. Streptococci from dogs and cats are almost always susceptible to penicillins. Most laboratories also do not routinely perform susceptibility testing on anaerobes, which also mostly have predictable susceptibilities, although resistance in anaerobes is increasing and some anaerobes, such as Bacteroides fragilis, have a high prevalence of β-lactamase enzyme production.
Susceptibility testing can be performed using dilution methods or diffusion methods. The minimum inhibitory concentration (MIC) is the lowest concentration of antimicrobial drug that inhibits visible growth of an organism over a defined incubation period, most commonly 18 to 24 hours, and is determined using dilution methods, which involve exposing the organism to 2-fold dilutions of an antimicrobial drug. The concentration range used varies with the drug and the organism being tested. Standard protocols are published by the Clinical and Laboratory Standards Institute (CLSI) that specify medium composition and pH, inoculum size (determined on the basis of turbidity measurements), inoculation procedures, agar depth and incubation conditions, as well as quality control requirements. Because failure to comply with these protocols can lead to erroneous results, veterinarians should always attempt to use laboratories that follow CLSI or EUCAST protocols.
The most widely used dilution method is broth microdilution, whereby 2-fold dilutions of antimicrobials are made in a broth media in a microtiter plate. Pre-prepared frozen or freeze-dried plates are available commercially for inoculation (e.g., Sensititre™ plates, TREK Diagnostic Systems). The results can be determined using visual examination of the plates for the inhibition of bacterial growth, or by the use of semi automated or automated instrumentation. The MIC for each antimicrobial drug tested against the organism is reported to the clinician on the susceptibility panel. It is the lowest concentration of antibiotic (usually in mg/ml) that inhibits growth of the organism in vitro, and the lower the MIC, the more potent the antimicrobial is at inhibiting bacterial growth.
Diffusion methods include gradient diffusion (also known as Etest®) and disk diffusion. The Etest involves use of a plastic strip coated with an antimicrobial gradient on one side and an MIC interpretive scale on the other side. An agar plate is inoculated with the organism of interest so that subsequent growth of the organism will form a “lawn”, rather than individual colonies. The strips are applied to the surface of the plate, with the lowest concentration towards the center. The antimicrobial drug diffuses into the medium, which results in an elliptical zone of growth inhibition around the strip. The MIC is read at the point of intersection of the ellipse with the MIC scale on the strip. Although the strips are expensive, Etests have the advantage of being adaptable to use with fastidious organisms and anaerobes if susceptibility testing of these organisms is deemed necessary. Disk diffusion involves application of commercially available drug-impregnated filter paper disks to the surface of an agar plate that has been inoculated to confluence with the organism of interest, and is also known as Kirby-Bauer antibiotic testing. Commercially available, mechanical disk-dispensing devices can be used to apply several disks simultaneously to the surface of the agar. The drug diffuses radially through the agar, the concentration of the drug decreasing logarithmically as the distance from the disk increases. This results in a circular zone of growth inhibition around the disk, the diameter of which is inversely proportional to the MIC. The zone diameters are interpreted on the basis of guidelines published by CLSI and the organisms are reported as susceptible, intermediate or resistant.
Breakpoints and Definition of Susceptible vs. Resistant Organisms
Once susceptibility testing has been performed, organisms are classified on the susceptibility panel report as “susceptible” (S), “resistant” (R), and, in some cases, of “intermediate” (I) susceptibility. This refers to a predicted in vivo situation, rather than in vitro susceptibility. The growth of “susceptible” isolates should be inhibited by concentrations of antimicrobial agent that are usually achievable in blood and tissues using normal dosage regimens. “Intermediate” isolates have MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than those for susceptible isolates. This category implies clinical efficacy in body sites where the drugs are normally concentrated (e.g., enrofloxacin and amoxicillin in urine) or when a higher-than-normal dose of a drug can be used, and acts as a buffer zone in order to prevent technical factors from causing major discrepancies in interpretations. With the exception of UTIs, ‘intermediate’ should be interpreted as ‘resistant’. “Resistant” isolates should continue to grow in the face of the usually achievable concentrations of the drug in blood and tissues.
In order to determine if an in vivo response is likely, the laboratory refers to breakpoints, or clinical cut-off MICs (or, for disk diffusion testing, cut-off zone diameters), which are established, published, and revised regularly by committees associated with standards agencies such as the CLSI. If the MIC determined in the microbiology laboratory is lower than the published breakpoint, then the organism is defined as susceptible. The breakpoint is not reported to the clinician. Breakpoints are established on the basis of multiple factors, which include 1) a knowledge of MIC distributions and resistance mechanisms for each organism-drug combination, 2) clinical response rates in humans and animal models, 3) how the drug is distributed and metabolized in the body (pharmacokinetics), and 4) whether the drug is concentration-dependent or time-dependent as it relates to antibacterial effect (pharmacodynamics). Zone diameter breakpoints for disk diffusion testing are determined by correlation with MIC values. For simplicity, breakpoints are established for bloodstream infections, and are based on a specific dosage regime for the antimicrobial drug tested, which are selected by the standards agency involved. Because some antimicrobials are concentrated extensively in urine, some veterinary laboratories may report urine MIC panels, which provide breakpoints for lower urinary tract infections, which are higher than corresponding serum MIC breakpoints. These have been controversial because the possibility of concurrent pyelonephritis cannot always be ruled out. Breakpoints are often re-evaluated when new mechanisms of resistance appear in bacteria or when new data are generated that improve understanding of the pharmacokinetics and pharmacodynamics of an antimicrobial drug.
The veterinary clinician should always remember that the list of drugs reported in the susceptibility panel is simply just a list of drugs tested. They are not suggestions from the laboratory for patient care. The clinician should always ask the following questions, when faced with a susceptibility panel result:
1. Is this organism that was cultured likely to be the cause of disease? (i.e., Should I treat this organism?)
2. Are any of the drugs shown as “susceptible” the appropriate drugs for treatment of the bacterial species cultured?
3. Assuming the drugs are active against the bacterial species isolated, are the drugs the right drugs for the patient in question?
4. Will they achieve adequate concentrations at the site of infection?
5. What route of administration is necessary and can the antimicrobials be administered by the route that is most appropriate for my patient?
6. Could adverse drug reactions occur in this patient with these antimicrobials?
7. Could drug interactions occur in this patient with these antimicrobials?
8. Is the antimicrobial drug currently being administered the most appropriate for the infection I am trying to treat?