Use of Susceptibility Testing in Veterinary Medicine Peter D. Constable, BVSc, MS, PhD, DACVIM Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL 61 802 Abstract three categories, susceptible (sensitive), intermediate and resistant, using recommendations from the National There has been increased interest in optimizing Committee for Clinical Laboratory Standards (NCCLS) treatment protocols for antimicrobial agents, with sub­ for testing Veterinary Pathogens.4 The intermediate stantial reliance on susceptibility testing of bacterial term indicates an MIC value that is close to the pathogens isolated from diseased cattle. Antimicrobial breakpoint.5 susceptibility testing of bovine bacterial pathogens has The broth dilution method provides a direct mea­ traditionally used the agar diffusion (Kirby-Bauer) surement of MIC, and determines the ability of the method, which was designed to reflect the antibiotic pathogen to grow in the presence of a known antibiotic concentration in serum and interstitial fluid of human concentration. The broth dilution test is usually per­ patients. The validity of agar diffusion susceptibility formed as a commercially available microdilution test breakpoints derived from humans to the treatment of (Sensititre, Westlake, OH) in a 96 well microtiter plate mastitis, diarrhea and respiratory disease in cattle has that permits the testing of 12 antibiotics in a range of not been established. The use of susceptibility testing eight 2-fold dilutions.5 The microdilution method starts to guide treatment decisions for individual cattle is not by using a sterile loop to remove 3-5 representative colo­ recommended until the breakpoints have been validated nies from a 24-hour bacterial culture plate (use of mul­ as being predictive of treatment outcome. tiple colonies avoids selection of an atypical variant). The bacterial colonies are then suspended in 5 ml of .g (D Introduction 0.9% NaCl and the bacterial suspension standardized l::l ~ by adjusting the turbidity to a 0.5 McFarland standard (") (") 7 (D A number of methods have been used to determine (an index of bacterial concentration approximating 10 V, the susceptibility of bovine pathogens to antimicrobial colony forming units [CFU]/ml). A fixed volume aliquot V, agents: broth dilution, milk dilution (for mastitis patho­ is then transferred to Mueller-Hinton broth, and an gens), agar dilution, determination of mean bactericidal automated inoculation device used to deposit a standard­ concentration, determination of killing kinetics and the ized inoculum into each well of the microtitration tray agar diffusion method (Kirby-Bauer test). The first five containing a geometric progression of dehydrated anti­ methods are quantitative, whereas the agar diffusion microbial agent concentrations. Growth is recorded by method is qualitative.15 Because of issues related to cost monitoring the turbidity of each well, and the first dilu­ and complexity, the broth microdilution method is the tion with non-visible growth considered to be the MIC recommended gold standard method for in vitro suscep­ for that isolate.41 tibility testing, whereas agar diffusion provides a crude, The agar diffusion method is also called the Kirby­ inexpensive and clinically practical method for deter­ Bauer method, and the test procedure has changed little mining in vitro susceptibility. since Bauer, Kirby and others standardized the method Two important concepts (minimum inhibitory con­ in 1966.4•7 Three to 10 representative bacterial colonies centration [MIC], and breakpoints for MIC) need to be are selected from a blood agar plate and suspended in a understood when interpreting the results of suscepti­ fixed volume of sterile 0.9% NaCl to achieve the turbid­ bility testing. The minimum inhibitory concentration is ity of a 0.5 McFarlane standard; the suspension is then the lowest antibiotic concentration (expressed in µg/ml) spread evenly across the surface of an agar plate using that, under defined in vitro conditions, prevents the a sterile cotton swab. Small circular disks of filter pa­ growth of bacteria within a defined period oftime.5 It is per or tablets impregnated with antimicrobial agents generally accepted that MIC values are very repeatable. 6 are placed on the agar plate using flamed forceps or a Statistics such as MIC50 (the median MIC for all iso­ special applicator and gently pressed down to ensure lates) and MIC90 (the MIC value which exceeds or equals contact.7 The agar plate is selected based on the bacte­ the MIC for 90% of the isolates) are frequently used to rial species being tested and incubated at 37°C over­ summarize population data. Breakpoints for MIC are night. During incubation, antibiotics dissolve from the specific MIC values used to assign bacteria to one of filter paper or tablets into the surrounding agar and 11 TH E AABP PROCEEDINGS-VOL. 37 thereby inhibit bacterial growth. The diameter of the eed publication documenting the relationship between zone of inhibition (Figure 1) is measured in mm and is the MIC of bacteria isolated from the site of infection correlated in some manner with the MIC for the bacte­ and clinical outcome in individual cattle administered (Q) ria.4 Interpretative zone diameters differ for each anti­ an antimicrobial agent. Accordingly, the recommended n 0 biotic because of differences in MIC and the diffusion MIC breakpoints appear to be based on in vitro MIC -0 '-< and solubility of the antibiotic in agar. Because agar values, pharmacokinetic/pharmacodynamic data and the "-1 cjq" diffusion is qualitative, the method is inferior to the results of clinical trials indicating efficacy using a stated ;::!"' quantitative broth dilution method. dosage protocol. In other words, we are still using the ....... approach described by Hjerpe in his seminal 1976 pa­ Determination and validation of per on the treatment of bacterial pneumonia in cattle.20 susceptibility breakpoints Many problems exist with the currently used sus­ ceptibility breakpoints for bacteria isolated from cattle. A standardized testing procedure for determining Accurate antimicrobial susceptibility test breakpoints antimicrobial susceptibility has been developed in the should be derived using MIC values for 300 to 600 iso­ United States by the NCCLS. A veterinary subcommit­ lates from representative clinical cases from a large geo­ tee ofNCCLS, called the Veterinary Antimicrobial Sus­ graphic area, 4·6 published pharmacokinetic/ ceptibility Testing (VAST) subcommittee, was formed in pharmacodynamic data for cattle, and clinical and bac­ 1992. The VAST subcommittee published a proposed teriologic cure rates.6 The results of field studies that standard in 1994 and approved standards in 1999 and measure the rate of clinical cure, using clinically rel­ 2002. 4 The VAST committee recommends an official in­ evant end points such as mortality, weight gain, treat­ terpretative MIC breakpoint against specific bacteria ment duration and relapse rate should be reported as a at a stated dosage protocol for a specific disease in a bare minimum. The rate of bacteriologic cure within a species. The MIC breakpoint is determined by consid­ specified time interval, using biologically relevant end­ ering available in vitro susceptibility data, pharmaco­ points such as failure to isolate the same pathogen from kinetic/pharmacodynamic data, and clinical efficacy the affected quarter in cows with mastitis, the feces in data; however, this author is unaware of a single refer- calf diarrhea, or a transtracheal wash in pneumonia, Figure 1. Antimicrobial susceptibility testing using the agar diffusion (Kirby-Bauer) test. The diameter of the zone of inhibition around each antibiotic disk is associated with the MIC value for each antibiotic. Interpretative zone diameters differ for each antibiotic because of differences in the MIC value, diffusion rate and solubility of the antibiotic in agar. Photograph courtesy of Dr. DE Morin. SEPTEMBER, 2004 12 also provides useful data. Clinical and bacteriologic cure trimethoprim/sulfadiazine.4 Susceptibility testing there­ rates may provide a clear breakpoint, or in other situa­ fore usually does not employ the active antibiotic agents tions, this data can be used in conjunction with phar­ present in commercially available antibiotic treatments (Q) macokinetic/pharmacodynamic data to suggest the most for cattle. It is likely that susceptibility test results based n 6 0 appropriate breakpoint. Unfortunately, the ideal ap­ on class representatives rather than the active antibi­ "O otic agent will lead to erroneous results. '< proach to determine accurate susceptibility breakpoints ....."'"I in cattle is hampered by three main difficulties: 1) lim­ {IQ g' ited availability of contemporaneous MIC values from Susceptibility testing in mastitis, calf diarrhea heterogeneous geographic locations, 2) incomplete and pneumonia ► ~ phamacokinetic/pharmacodynamic data, and 3) com­ ....."'"I (') plete absence of published field studies validating the Important considerations for treating bacterial § suggested susceptibility breakpoints. The effect of dis­ diseases in cattle are: 1) administering an antibiotic as Cfl► ease on the pharmacokinetics of antimicrobial agents directed on the label whenever possible, 2) using an Cfl 0 has usually been ignored, but differences in the plasma antimicrobial agent with an appropriate spectrum of (') 3 12 ~- concentration-time profile for oxytetracycline • and activity, 3) selecting an antimicrobial agent