DEVELOPMENT OF A SELECTIVE MEDIUM FOR RHODOCOCCUS EQUI A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Orhan Sahin, D.V.M. ***** The Ohio State University 1997 Master's Examination Committee: Approved by Dr. Joseph J. Kowalski, Adviser Dr. Stephen M. Reed Dr. Kenneth W. Hinchcliff dviser Graduate Program in Veterinary Clinical Sciences Copyright by Orhan Sahin 1997 ABSTRACT Rhodococcus equi is a frequent cause of chronic suppurative bronchopneumonia, lymphadenitis, and enteritis in foals, in most cases during the first four months of life. This study was carried out to develop a selective medium for the organism for its isolation from highly contaminated sources such as soil and feces of grazing herbivores. Twenty-eight clinical isolates of R. equi recovered from lesions of infected foals were tested for susceptibility to antimicrobial agents by disk diffusion and microbroth dilution susceptibility testing methods. In-vitro the most active antibiotics were gentamicin, amikacin, netilmicin, erythromycin and trimethoprim/sulphonamide combination whereas oxacillin, cloxacillin, ceftiofur, ceftazidime, aztreonam, trimethoprim, novobiocin, and nystatin were routinely impotent to tested isolates. Based on these susceptibility patterns of R. equi isolates a selective agar medium was developed. The medium contained aztreonam 100 (μg/ml, novobiocin 25 (μg/ml and nystatin 100 Units/ml with (herein referred to as IST-ANYT) or without (herein called IST-ANY) 0.005 % potassium tellurite. After the medium was proved for its selectivity for R. equi, it was tested with a number of soil and fecal samples for isolation of the organism from a Quarter Horse breeding farm in Columbus, OH. All soil samples (n=10) and fecal specimens from the rectum of healthy mares (n=12) yielded R. equi on either medium after 48 hours of incubation while the organism was not isolates from feces of any yearlings (n=4). These results show the satisfactory capacity of the new selective medium for isolation of R. equi from contaminated sources, and confirm the findings of others that the organism is widespread in equine feces and their soil environment. ii Dedicated to my mother lll ACKNOWLEDGMENTS I express my sincere appreciation to my adviser, Dr. Joseph J. Kowalski, for his guidance, support and encouragement, which made this thesis possible. In another aspect, I wish to thank to my sponsor, Ministry of National Education in Turkey. Thanks go to the other members of my graduate committee, Dr. Stephen M. Reed and Dr. Kenneth W. Hinchcliff for their suggestions and comments. The technical assistance of the persons in Dr. Kowalski's laboratory, especially Al Lawrence is gratefully acknowledged. iv VITA May 1, 1972 ................................. Born-Gaziantep, Turkey 1993 .......................................... D.V.M., Ankara University 1996-present ................................. Graduate Student, The Ohio State University FIELD OF STUDY Major Field: Veterinary Clinical Sciences v TABLE OF CONTENTS Dedication .................................................................................................................. .iii Acknowledgments ...................................................................................................... iv Vita.............................................................................................................................. v List of Tables ............................................................................................................ viii List ofFigures ............................................................................................................ .ix Chapters: 1. Introduction ............................................................................................................. 1 1.1 General characteristics and taxonomic status of Rhodococcus equi .................. 1 1.2 Ecology of Rhodococcus equi ........................................................................ ... 2 1.2.1 Growth characteristics of R. equi in vitro ........................................... .4 1.3 Rhodococcus equi infection in foals ................................................................ .4 1.3 .1 Epidemiology of the infection ................................................................ .4 1.3.2 Clinical manifestations of the disease ...................................................... 6 1.3.3 Pathogenicity of R. equi infection in foals ............................................... 7 1.3.4 Immunity to R. equi infection .................................................................. 8 1.3 .5 Diagnosis of R. equi infection in foals ................................................... 10 1.3.6 Treatment of R. equi infection in foals ................................................. 12 1.3.7 Control and prevention of R. equi infection in foals ............................. 14 1.4 In vitro antimicrobial susceptibility of R. equi strains .................................... 15 1.5 Selective media for R. equi ............................................................................. 16 1. 6 Purpose of the study .................................................................. 18 2. Material and Methods ........................................................................................... 19 2.1 Bacterial isolates ............................................................................................ 19 2.2 Antimicrobials ................................................................................................ 20 vi 2.3 Susceptibility tests .......................................................................................... 21 2.3 .1 Disk diffusion susceptibility test.. ......................................................... 21 2.3.2 Microbroth dilution test. ...................................................................... .22 2.4 Quality control strains .................................................................................. 23 2.5 Incorporation of antibiotics into IST agar .................................................... 23 2.5.1 Aztreonam incorporation .................................................................... 23 2.5.2 Novobiocin incorporation ................................................................... .23 2.5.3 Nystatin incorporation ......................................................................... 23 2.5.4 Tellurite incorporation ...................................................... .24 2.6 Testing the media for selectivity for R. equi ................................................. 24 2.6.1 Inoculation of pure cultures of various organisms ............................... 24 2.6.2 Inoculation of pure R. equi cultures for viable count.. ......................... 24 2. 7 Isolation of R. equi from horse feces ........................................................... .25 2.7.1 Isolation of R.equi after inoculation of horse feces with a laboratory culture of the organism ....................................................................................................... 25 2.7.2 Attempts at isolation of R. equi from horse feces and soil from a Quarter Horse farm in Ohio ..........................................................................26 2.8 Shelf life of the medium .............................................................................. .27 2.9 Statistics ........................................................................................................ 27 3. Results .................................................................................... 28 3 .1 Confirmation of the R. equi isolates evaluated in this study ....................... .28 3 .2 Susceptibility testing .................................................................................... 29 3.3 Antibiotic incorporation .............................................................................. 33 3.4 Selectivity of the medium for R. equi ....................................................... .. 34 3 .4.1 Growth of other organisms ................................................................. 34 3.4.2 Result of viable count.. ....................................................................... 34 3.5 Isolation of R. equi from horse feces .......................................................... .41 3.5.1 Isolation of R. equi in horse feces inoculated with pure culture of the organism ................................................................................................................. 41 3.5.2 Isolation of R. equi from horse feces and soil in an Ohio Quarter Horse farm ............................................................................................. 41 3.6 Shelf life of the medium ............................................................................ .42 4 Discussion ......................................................................................................... 58 Bibliography ......................................................................................................... 66 vii LIST OF TABLES 3 .1 Zone diameter of 19 antibiotics against 28 R. equi strains on Mueller-Hinton agar with 5 % lysed horse blood ..................................... 29 3.2 Minimum inhibitory concentratons of 35 antibiotics aganist 28 R. equi isolates ............................................................................... 31 3.3 In vitro susceptibility of 28 R. equi