Species Identification by Polymerase Chain Reaction of Staphylococcal Isolates from the Skin and Ears of Dogs and Evaluation of Clinical Laboratory Standards Institute Interpretive Criteria for Canine Methcillin-resistant Staphylococcus pseudintermedius Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Graduate School of The Ohio State University By Jennifer Ruth Schissler, DVM Graduate Program in Veterinary Clinical Sciences The Ohio State University 2009 Thesis Committee Dr. Andrew Hillier, Advisor Dr. Lynette Cole Dr. Wondwossen Gebreyes Dr. Paivi Rajala-Schultz Dr. Joshua Daniels Copyright by Jennifer Schissler, DVM 2009 [Type a quote from the document or the summary of an interesting point. You can position the text box anywhere in the document. Use the Text Box Tools tab to change the formatting ofii the pull quote text box.] Abstract The Clinical and Laboratory Standards Institute has published (2008) new interpretive criteria for identification of methicillin resistance in veterinary staphylococci. The sensitivity of the 2008 interpretive criteria compared to previous (2004) criteria was established in thirty canine clinical isolates of mecA gene–positive Staphylococcus pseudintermedius. The minimum inhibitory concentration for oxacillin was determined by broth microdilution. The 2008 breakpoint of > 4µg/ml for methicillin resistance resulted in a sensitivity of 73.3% (22/30). The 2004 breakpoint guideline of ≥ 0.5 µg/ml resulted in a sensitivity of 97% (29/30). For oxacillin disk diffusion, the 2008 interpretive criterion of ≤10 mm for methicillin resistance resulted in a sensitivity of 70% (21/30). Application of the 2004 interpretive criterion of ≤ 17mm resulted in a sensitivity of 100% (30/30). For cefoxitin disk diffusion, the interpretive criterion of ≤ 21mm for methicillin resistance (as used for S. aureus) resulted in a diagnostic sensitivity of 6.7% (2/30). The interpretive criterion of ≤ 24mm (as used for coagulase negative staphylococci) resulted in a diagnostic sensitivity of 43.3% (13/30). The 2008 interpretive criteria produced what we consider to be an unacceptable level of false negative results. This study also established that cefoxitin disk diffusion is an inappropriate screening test for methicillin resistance of canine S. pseudintermedius. ii Pilot experiments to screen novel PCR primers for S. aureus, S. schleiferi, S. intermedius, and S. pseudintermedius were performed using type culture control strains. Target genes included hsp60, sodA, femA, and nuc. Three of 15 primers (20%) failed to produce an amplicon of predicted size. Three (20%) successfully functioned for S. aureus and S. schleiferi. All 4 functional S. intermedius primers demonstrated cross- amplification in S. pseudintermedius type strains. After S. pseudintermedius sequences were available, it was determined that identical to near- identical S .intermedius primer annealing site sequences were present in this species. A primer targeting nuc gene differentiated S. intermedius from S. pseudintermedius type strains. Three sodA primers for S. aureus, S. schleiferi, S. intermedius/pseudintermedius and one nuc primer for S. pseudintermedius were selected for further investigation. A total of 91 isolates of S. pseudintermedius, S. schleiferi, and S. aureus previously identified via select biochemical testing or API ID 32 STAPH identification were used for PCR validation experiments. Identity was confirmed via VITEK2 system. There was 29% (26/91) disagreement between VITEK2 results and results of previous identification methods. Previously identified S. schleiferi provided the greatest disagreement, 82 % (22/28). Supplemental testing was necessary for identification in a majority of isolates. iii The sodA- based primer for S. pseudintermedius provided a sensitivity of 100% and a specificity range of 6.7-22%. The sodA-based primer for S. schleiferi provided a sensitivity of 83% and a specificity range of 56-75%. The sodA-based primer for S. aureus provided a sensitivity of 97% and a specificity of 91%. The nuc- based primer for S. pseudintermedius provided a sensitivity range of 94-100% and specificity range of 22-96%. These primers did not provide sufficient sensitivity and specificity to discriminate between S. pseudintermedius, S. schleiferi, and S. aureus as a sole diagnostic test. iv Dedication This thesis is dedicated first and foremost to my parents Fred and Linda. Thank you for nurturing my intellectual curiousity and supporting me wherever it may take me, even if it is far from home. You have provided great mentorship and friendship. To my brother Eric: I wish you all the best in your future academic endeavors. I am very proud of you- may we celebrate the completion of our theses together. To my brother Bryan: you have my respect for being a good husband and father. It is unfortunate that I couldn’t spend as much time with Freddy and Abby as I would liked the last three years, but I hope they get to know Aunt Jenny and Uncle Aaron soon. To Aaron: I started this journey alone and somehow found the most interesting person I know to spend the rest of it with. Your steadfastness and dedication to us is a blessing. You have been incredibly supportive during the construction of this weighty script. May this thesis be one of our many collective achievements. I hope this tome spends many a dusty year on a bookshelf in our home. To our future children: if you are reading this now, stop here you have already read the most interesting parts. v Acknowledgement Andy Hillier and Lynette Cole, thank you for your guidance, sense of humor and demand for excellence throughout my residency. These three years have been the most challenging and formative thus far on both a personal and professional level. Thank you for this life-changing opportunity. It was my goal to earn your professional trust and respect during my residency. Whever my future may take me, I hope to continue to earn it. Do not be surprised if I continue to seek your input in the future, as your opinions and experiences will remain valuable to me. Wendy Lorch, I admire greatly your academic achievements, your intellect, and your cheerful and helping spirit. Thank you for your friendship in and your guidance. I wish you continued success in the future. Michele Fox, thank you for your friendship. I look forward to our daily conversations and will miss your company. You have been a great model of patience, organization, and proficiency- an ideal technician. I would like to thank the members of my thesis committee: Dr. Gebreyes, Dr. Daniels, Dr. Rajala-Schultz, and Dr. Bannerman for their technical advice and encouragement. vi Vita February 3, 1980………………………………………...Born- Westminster, Colorado 2005……………………………………………........DVM, Colorado State University 2006- Present…………………………….Graduate Teaching and Research Associate, The Ohio State University Publications 1. Schissler JR, Lorch G. Bacterial Dermatitis-Superficial. In: Lavoie J, Hinchcliff KW. Blackwell’s Five –Minute Veterinary Consult: Equine. 2nd Ed. Ames, Wiley-Blackwell. 2008; 122-123. 2. Smirnova NJ, Troyer JL, Schissler J, Terwee J, Poss M, Vandewoude S. Feline lentiviruses demonstrate differences in receptor repertoire and envelope structural elements. Virology 2005 Nov 10; 342(1): 60-76. Fields of Study Major Field: Veterinary Clinical Sciences Studies in Veterinary Dermatology vii Table of Contents Abstract………………………………………………………………………………..ii Dedication……………………………………………………………………………..v Acknowledgement……………………………………………………………………vi Vita……………………………………………………………………………..........vii List of Tables………………………………………………………………………..xvi Chapter 1:Introduction…………………………………………………………….1 Chapter 2: Literature Review……………………………………………………...5 2.1 The Genus Staphylococcus……………………………………………………..5 2.1.1 Taxonomy…………………………………………………….....5 2.1.2 Staphylococci of Veterinary Interest……………………………6 2.1.2.1 S. intermedius and S. pseudintermedius………………...6 2.1.2.2. S. aureus………………………………………………8 2.1.2.3 S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans…………………………………………….10 2.2 Methicillin resistance in veterinary medicine……………………………………10 2.2.1 Methicillin- resistant S. pseudintermedius……………………………………..10 2.2.2 Methicillin- resistant S. aureus…………………………………...14 2.2.3 Methicillin- resistant S. schleiferi……………………………...17 viii 2.3 Identification of Staphylococcus species………………………...19 2.3.1 Phenotypic Identification………………………………19 2.3.1.1 Morphology…………………………………..19 2.3.1.2 Gram stain……………………………………20 2.3.1.3 Coagulase activity……………………………21 2.3.1.4 Catalase activity…………………………….. 21 2.3.1.5 Biochemical tests……………………………21 2.3.1.6.1 Expected results for S. pseudintermedius, S. intermedius, S. schleiferi, S. aureus…………………………………………..22 2.3.1.6.2 Phenotypic testing technologies……………31 2.3.1.6.3 Difficulties and discrepancies in phenotypic technologies …………………………………………32 2.3.2 Genotypic identification……………………………………….34 2.3.2.1 Technologies…………………………………………34 2.3.2.2 PCR for identification of staphylococci……………..35 2.3.2.2.1 Genes of interest for identification………..36 2.3.2.2.2 femA gene…………………………………37 2.3.2.2.3 hsp60 gene…………………………………38 ix 2.3.2.2.4 sodA gene………………………………..38 2.3.2.2.5 nuc gene…………………………………39 2.4 Detection of Methicillin resistance in staphylococci ……………………..40 2.4.1 Phenotypic identification………………………………………40 2.4.1.1 Oxacillin salt agar……………………………………41 2.4.1.2 Oxacillin minimum inhibitory concentration by broth microdilution…………………………………………………42 2.4.1.3 Oxacillin disk diffusion………………………………43 2.4.1.4