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Growth, Congo Red Agar Colony Morphotypes and Antibiotic Susceptibility Testing of Mycobacterium avium subspecies paratuberculosis

Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Maryland

Chiew G. Ko, MSc

Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Maryland

James D. Dick, PhD, MS

Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Maryland

Paul B. Jones, PhD

Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina

Jay L.E. Ellingson, PhD

Marshfield Clinic Laboratories – Food Safety Services, Marshfield, Wisconsin

REPRINT REQUESTS: Jay L.E. Ellingson, Ph.D., Marshfield Clinic Laboratories, Food Safety Service, Marshfield Clinic, 1000 North Oak Avenue, Marshfield, WI 54449, Email: ellingson.jay{at}marshfieldclinic.org

OBJECTIVE

Mycobacterium avium subspecies (subsp.) paratuberculosis (MAP) is the causative agent of Johne’s disease in ruminants and has been associated with Crohn’s disease in humans. We sought to test growth rates and susceptibilities of various strains of MAP in two available growth media.

DESIGN

Paired comparison design.

METHODS

Using the BACTEC macrobroth radiometric growth system and Congo Red-staining agar media, we determined inherent differences in growth characteristics of three bovine and two human strains of MAP and compared susceptibility results obtained in each growth system.

RESULTS

Significant differences were observed in growth rate as well as mycobactin J dependence between strains and between a laboratory-adapted isolate of the same strain in the macrobroth system. Similarly, colonial morphology and Congo Red staining on agar media were observed. Two strains, one human and one bovine, demonstrated a 100% rough transparent colony with white coloration on Congo Red agar, while one bovine isolate exclusively grew as a smooth opaque colony with red coloration on Congo Red agar. The remaining strains exhibited mixtures of these two colonial morphotypes on agar media. Comparative susceptibility results between the BACTEC radiometric macrobroth method and the agar proportionality method showed good correlation for most antibiotics/inhibitors tested. However, erratic or poor growth in the macrobroth system prevented minimal inhibitory concentration determinations for two bovine strains by this method.

CONCLUSION

This study demonstrates the variability in the colonial morphology of MAP on Congo Red agar as well as the correlation of antibiotic susceptibility results between the BACTEC macro broth method and the agar proportionality method. This study also emphasizes the need for the development of improved, standardized culture and susceptibility test methods for MAP.

Key Words: Mycobacterium paratuberculosis • Antibiotic susceptibility • Paratuberculosis • Crohn disease • Microbiological techniques • Inflammatory bowel disease

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