Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

Abstract

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP).We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.

Mycobacterium avium ssp . paratuberculosis detection in animals, food, water and other sources or vehicles of human exposure: A scoping review of the existing evidence

“…through irrigation) and seafood. Further research is required to examine how hardy M. paratuberculosis is in its spore-like state and how long it survives in manure storage and composting to better understand the risk of contaminating other potential environmental sources of M. paratuberculosis for humans (Lamont et al, 2012 (Singh et al, 2013;Ahlstrom et al, 2015). The multitude of molecular methods used to date has been a barrier to meaningful synthesis of molecular epidemiological results (Ahlstrom, et al, 2015;Muellner, et al, 2015), therefore as data from whole genome sequencing become more widely used in epidemiological investigations, researchers should seek consensus on target markers to aid in the comparability of results across studies.…”

Modeling the Effect of Direct and Indirect Contamination of On-Farm Bulk Tank Milk with Mycobacterium avium subsp. paratuberculosis

“…Still, MAP isolation in milk sampled at retail stores has been reported (Ellingson et al, 2005;Stephan et al, 2007). It has recently been reported that spore-forming MAP may survive heat treatments of 70°C (Lamont et al, 2012), which could explain the occasional MAP survival after heat treatments. Other possible explanations for the difference between our predictions and the apparent pasteurization inefficiency include the following: (1) inappropriate pasteurization was conducted for the retail store samples; (2) the maximum amount of feces per cow is much higher than the 10 g/cow we used; or (3) the maximum amount of MAP per gram of feces is much higher than that included in our model.…”

Risk Factors for the Presence of Mycobacterium avium spp. paratuberculosis in Human Intestinal Biopsies in Brazil

“…The bacteria inside the clumps would be protected from the heat by those on the outer surface during pasteurization [13,24]. In addition to the formation of clumps, MAP also has the ability to form a spore-like structure that may hinder its elimination and favor bacterial survival in the environment, as observed in aquatic environments, since MAP has been identified in raw milk and untreated water [11,13,26,27].…”

“…Infected patients may develop intestinal problems associated with a genetic propensity, including mutations in the Nod 2, which is gene associated with the processing of modified bacterial products [9,27]. Besides, individuals infected with the agent may contaminate their families through direct contact or fomites of contamination.…”