Experimental pieces of evidence for Mycobacterium ulcerans dormancy

Abstract

ABSTRACTBackgroundWhether Mycobacterium ulcerans, the etiological agent of the neglected Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, is not established.MethodologySix M. ulcerans strains were exposed to a progressive depletion in oxygen for two months, using a previously described Wayne model of dormancy; and further examined by microscopy using DDD staining, microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls.Principal Findings/ConclusionsM. ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile Red-stained intracellular lipid droplets after DDD staining and a 14-20-day regrowth when exposed to ambient air, diagnosing dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, needs to be investigated in the natural ecological niches where M. ulcerans thrive and in Buruli ulcer lesions.Author summaryMycobacterium ulcerans is an environmental opportunistic pathogen of mammals and humans, causing a subcutaneous necrotizing infection named Buruli ulcer. Molecular detection of M. ulcerans DNA revealed different ecological niches where M. ulcerans may thrive, but the molecular biology approach does not catch the physiological state of M. ulcerans in these different ecological niches. Thus, the reservoir and the mode of transmission of M. ulcerans remain elusive. Here, we investigated experimental dormancy of M. ulcerans by using a previously described Wayne model of dormancy coupled with microscopy using DDD staining, microcalorimetry and subculture. Our findings demonstrate for the first time that some M. ulcerans strains exhibit a physiological state of dormancy; potentially limiting isolation and culture of M. ulcerans from environmental niches.