342 MICROBIAL DRUG RESISTANCE Volume 11, Number 4, 2005 © Mary Ann Liebert, Inc. Assessment of Accuracy of Disk Diffusion Tests for the Determination of Antimicrobial Susceptibility of Common Bovine Mastitis Pathogens: A Novel Approach EYAL KLEMENT, 1,4 MARCELO CHAFFER, 2,4 GABRIEL LEITNER, 2 ADIN SHWIMMER, 1 SHMUEL FRIEDMAN, 1 ARTHUR SARAN, 2 and NAHUM SHPIGEL 1,3 ABSTRACT A novel approach was used to assess disk diffusion accuracy for determination of antibiotic susceptibility of var- ious bovine mastitis pathogens (Escherichia coli, Staphylococcus aureus, Staphylococcus chromogenes, and Strep- tococcus dysgalactiae). MIC and disk diffusion diameters were compared for 587 bovine mastitis bacterial iso- lates collected in Israel and 3,186 drug–organism combinations. Results were analyzed by ROC curves, Bayesian statistics, and standard descriptive methods. Low correlation was observed between results of disk diffusion and MIC for S. dysgalactiae and all antimicrobial agents, S. aureus and erythromycin and neomycin, and E. coli and gentamicin, neomycin, and polymyxin B. On a few occasions in which correlation was satisfactory, accepted sus- ceptibility breakpoints to some of the antimicrobial agents resulted in high discrepancies with MIC results and new breakpoints were suggested—e.g., 21 mm for S. aureus susceptibility to penicillin G instead of 29 mm rec- ommended by the National Committee for Clinical Laboratory Standards (NCCLS) and 21 mm, resistant, 21–25 mm, intermediate, and 25 mm, susceptible for susceptibility of E. coli to trimethoprim/sulfamethoxa- zole. Thus, this approach enabled determination of the most accurate breakpoints that best fitted the specific prevalence of susceptibility in Israel. Thus, we suggest its adoption by microbiology diagnostic laboratories for the provision of accurate antimicrobial susceptibility results when using the disk diffusion test. INTRODUCTION B OVINE MASTITIS IS A DISEASE with significant economic ram- ifications to the dairy cattle industry, causing annual losses of over two billion dollars in the United States alone. Further- more, it is considered to be the main cause of antimicrobial use in lactating dairy cattle. 8,12,20 The importance of this disease can, perhaps, be best demonstrated by noting that 82% of antibiotic residue violations are mastitis treatment related. 26 These facts emphasize the importance of the appropriate use of antimicro- bial agents for the treatment of bovine mastitis. Because the ad- ministration of antimicrobial agents is highly dependent on the pathogens susceptibility to the agent, 34 the importance of using accurate and reliable susceptibility tests cannot be overstated. The disk diffusion test has long been the most widely ap- plied testing method for antimicrobial susceptibility in veteri- nary medicine due to its ease of use, flexibility, and low cost 34 and is used in many antimicrobial susceptibility reports con- cerning bovine mastitis. 4,7,17–19,24 The results achieved by the use of this method are influenced by many factors, including composition of agar medium, pH, electrolyte concentration in the medium, interaction between antimicrobial agents and agar, density of the inoculum, agar depth, temperature of incubation, and incubation time. 2 Proper interpretation, therefore, requires the application of standardized and quality controlled condi- tions, such as those recommended by the National Committee for Clinical Laboratory Standards (NCCLS). 22 However, even strict compliance with these guidelines can be sometimes in- sufficient of itself to overcome the limitations inherent in the disk diffusion tests. For example, antibiotics that diffuse poorly in agar media, such as polymyxins and vancomycin, lack sat- isfactory correlation between zone inhibition diameters and 1 The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel. 2 The Kimron Veterinary Institute, Beit Dagan 50250, Israel. 3 The Hachakleit Veterinary Services, Caesarea, Israel. 4 Both authors contributed equally to this work.