Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures.

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.

Epidemiology and economics of paratuberculosis.

Johne's disease is the clinical manifestation of Mycobacterium avium subsp. paratuberculosis (MAP) infection and has become widespread since it was first observed in the United States in the early 1900s. MAP is primarily spread through the fecal-oral route, and herds generally become infected by unknowingly purchasing infected animals. The economic losses from the disease are primarily due to decreased milk production, decreased weaning weights in nursing young stock, increased replacement costs, and decreased slaughter value.

A novel cell wall lipopeptide is important for biofilm formation and pathogenicity of Mycobacterium avium subspecies paratuberculosis.

Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's disease. Scanning electron microscopy and colonization levels of the M. ap mutant indicated that the pstA gene significantly contributes to the ability of M. ap to form biofilms. Digital measurements taken during electron microscopy identified a unique morphology for the DeltapstA mutant, which consisted of significantly shorter bacilli than the wild type. Analysis of the lipid profiles of the mycobacterial strains identified a novel lipopeptide that was present in the cell wall extracts of wild-type M. ap, but missing from the DeltapstA mutant. Interestingly, the calf infection model suggested that pstA contributes to intestinal invasion of M. ap. Furthermore, immunoblot analysis of peptides encoded by pstA identified a specific and significant level of immunogenicity. Taken together, our analysis revealed a novel cell wall component that could contribute to biofilm formation and to the virulence and immunogenicity of M. ap. Molecular tools to better control M. ap infections could be developed utilizing the presented findings.

Preliminary characterization of chemically generated Mycobacterium avium subsp. paratuberculosis cell wall deficient forms (spheroplasts).

Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis (MAP) have been isolated from patients with Crohn's disease and sarcoidosis. These MAP CWD may be important in the pathogenesis of these diseases and in Johne's Disease in other animal species. CWD forms are extremely difficult to isolate and generally revert to cell wall competent forms (CWC) when cultured in vitro. Cultured MAP strain 19698 were chemically treated to generate sufficient CWD to compare to CWC organisms by electron microscopy, chemotype profile (matrix solid-phase dispersion and thin layer chromatography), silver-stained SDS-PAGE gels with and without periodic acid treatment and Western blots with antigen recognition by sera from confirmed Johne's positive and Johne's negative cattle. On electron microscopy, CWD organisms were larger and rounder than cell wall competent forms and had lost the majority of their cell walls, being bounded only by a plasma membrane. Chemotype profiles of CWD lacked bands generally associated with cell wall glycolipids. Silver-stained SDS-PAGE gels of CWD demonstrated loss of bands that migrate in the same region as lipoarabinomannan (LAM) and some bands likely representing proteins and weakening of bands that migrate similarly to phosphatidylinositol mannosides (PIM). Western blots of CWD demonstrated bands with loss or attenuation of signal that migrate similarly to LAM and other constituents. In summary, CWD and CWC forms of MAP 19698 had marked differences in morphology, chemotype profile, cell wall constituents, and antigens recognized by Johne's disease positive and negative bovine sera.

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host. RESULTS: Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a steady decline in viability over the next six days. Antibodies against M. paratuberculosis were affinity purified and used in conjunction with transmission electron microscopy to track the development of intracellular bacilli. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. Conversely, when macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed. CONCLUSIONS: We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and viability counts. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years.

Inactivation of Mycobacterium paratuberculosis by pulsed electric fields.

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50 degrees C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log(10) CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50 degrees C for 25 min or at 72 degrees C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log(10) CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.

Presence of bacterial 16S ribosomal RNA gene segments in human intestinal lymph follicles.

BACKGROUND: There is currently no information regarding microbial agents inside the intestinal lymph follicles. METHODS: Biopsy or resected specimens, mostly from macroscopically normal areas, were sectioned with a cryostat. DNA was extracted from microdissected samples, exclusively from the lymph follicle. Amplification of DNA was performed using universal primers designed from conserved regions of bacterial 16S ribosomal RNA (rRNA). Several clones with inserts of around 400 base pairs were subjected to DNA sequence analysis followed by a database homology search. RESULTS: Bacterial 16S rRNA gene segments were detected in the lymph follicle in 2 of 14 (14%) non-inflammatory bowel disease (IBD) cases, 4 of 14 (28%) Crohn disease cases, and in 2 of 5 (40%) ulcerative colitis cases. Nineteen 16S rRNA gene segments were recognized in the eight positive cases. Five segments showed 100% identity to known bacterial 16S rRNAs, namely staphylococcus species, Streptococcus sanguis, and Paracoccus marcusii. However, the other 14 segments showed below 100% identity, indicating either the presence of unknown bacteria or of bacteria without known DNA data. No single identified or unidentified bacterium, characteristic of IBD, including Mycobacterium paratuberculosis and Listeria monocytogenes, was detected. CONCLUSIONS: The present study confirms the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles and paves the way for new investigations into the microbiology of the lymph follicle. Whether or not bacteria inside the lymph follicle is a primary stimulus in IBD has yet to be clarified.

Mycobacterium paratuberculosis. Factors that influence mycobactin dependence.

Mycobacterium paratuberculosis does not produce any detectable mycobactin, an iron-binding compound that is synthesized by most Mycobacterium spp. and necessary for the growth of all mycobacteria. This study examined the influence of various culture conditions on mycobactin dependence in M. paratuberculosis. Using a radiometric growth assay, we found the minimal concentration of mycobactin J necessary for growth of M. paratuberculosis to be 0.006 microM, whereas 1.2 microM (1 microgram/ml) was required for optimal growth. In media without mycobactin at iron concentrations less than or equal to 100 microM, growth of M. paratuberculosis occurred at pH 5.0, but not pH 6.8. Iron concentrations greater than 100 microM did not significantly increase growth at pH 5.0, but at pH 6.8 the growth rate increased with increasing amounts of iron reaching a rate equal to control cultures containing mycobactin. Mycobacterium paratuberculosis appeared to lose mycobactin dependence when subcultured; however, this was subsequently shown to be a result of mycobactin carried over from primary medium. Removal of this contaminating cell-wall-associated mycobactin reestablished mycobactin dependence. We conclude that mycobactin dependence must be carefully determined because it is a key test used in identification of M. paratuberculosis and may be easily influenced by media pH, iron concentration, and mycobactin carryover from primary media.