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

Abstract

OBJECTIVEMycobacterium 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. METHODSUsing 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. RESULTSSignificant 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. CONCLUSIONThis 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.

Thiopurine Drugs Azathioprine and 6-Mercaptopurine Inhibit Mycobacterium paratuberculosis Growth In Vitro

“…At 3 and 5 days postinoculation an aliquot (0.1 ml) was removed for plate count determinations (note that MGIT 960 instrument readings were not taken on these tubes; the tubes merely provided the medium in which the bacterium-drug interaction took place). Bacterial cells were harvested from MGIT tubes by centrifugation, resuspended in PBS, and homogenized to break up bacterial cell clumps by vortexing with glass beads (30). Then, 10-fold serial dilutions (10 0 to 10 Ϫ6 ) were made in PBS, and 100 l of each dilution was plated in quadruplicate on 7H10 agar supplemented 10% OADC (Becton Dickinson, Sparks, MD) and 2 g/ml of mycobactin J (Allied Monitor, Fayette, MO).…”

Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

“…Other mycobacterial species capable of generating smooth-colony morphotypes have been described (1,5,18,19,28). The smooth-colony phenotype of DL2008 is likely to be linked to changes in the hydrophobicity of the cell wall (Fig.…”

Absence of mycobacterium avium subsp. paratuberculosis in Crohnʼs patients

“…[31][32][33][34][35] HEYA slants, considered the "gold standard" for the culture of MAP in Johne's disease, were included as were the broth-based BACTEC 460 and MGIT, both of which supported the recovery of viable MAP in other studies when supplemented with Mycobactin J. 11,35 The addition of an anaerobic culture detection method was based on evidence that suggested that MAP may be capable of anaerobic adaptation. 28,29 Every effort was made to maximize recovery of MAP from buffy coat preparations through careful consideration of which modifications were required for the selected testing methods.…”