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BMC Microbiology Nov 2012Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne's disease or paratuberculosis and is included within the Mycobacterium avium...
BACKGROUND
Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne's disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as 'Sheep' or 'S-type' and 'Cattle' or 'C-type'. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis.
RESULTS
The presence of LSP(A)4 and absence of LSP(A)20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III.
CONCLUSION
This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.
Topics: Animals; Cattle; DNA Transposable Elements; DNA, Bacterial; Electrophoresis, Gel, Pulsed-Field; Genetic Variation; Genotype; Minisatellite Repeats; Molecular Typing; Mycobacterium avium subsp. paratuberculosis; Polymerase Chain Reaction; Sheep
PubMed: 23164429
DOI: 10.1186/1471-2180-12-264 -
Frontiers in Cellular and Infection... 2017Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium subsp. (). We previously demonstrated that isolates from sheep...
Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium subsp. (). We previously demonstrated that isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1--inositol mannosides of the cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence 's envelope fluidity ultimately affecting pathogenicity. To test this hypothesis, we investigated the responses of two isolates from cattle (K10 isolate) and sheep (2349/06-1) to the bovine and ovine macrophage environment by measuring the FAs content of extracellular and intracellular bacteria. For this purpose, macrophages cell lines of bovine (BOMAC) and ovine (MOCL-4) origin were infected with the two isolates of for 4 days at 37°C. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. Using this approach, we demonstrated that the FAs composition of extracellular and 7H9-grown bacteria was highly conserved within each isolate, and statistically different from that of intracellular bacteria. Analysis of FAs composition from extracellular bacteria enabled the distinction of the two strains based on the presence of the tuberculostearic acid (18:0 10Me) exclusively in the K10 strain of . In addition, significant differences in the content of Palmitic acid and cis-7 Palmitoleic acid between both isolates harvested from the extracellular environment were observed. Once the infection established itself in BOMAC and MOCL-4 cells, the FAs profiles of both isolates appeared conserved. Our results suggest that the FAs composition of might influence its recognition by macrophages and influence the survival of the bacillus within host macrophages.
Topics: Animals; Cattle; Cell Line; Cell Wall; Chromatography, Gas; Fatty Acids; Host-Pathogen Interactions; Macrophages; Microbial Viability; Mycobacterium avium subsp. paratuberculosis; Sheep
PubMed: 28377904
DOI: 10.3389/fcimb.2017.00089 -
Viruses Dec 2023Johne's disease (JD), a chronic infectious enteritis of ruminants, causes major economic losses in the dairy industry globally. This enteritis is caused by subsp....
Johne's disease (JD), a chronic infectious enteritis of ruminants, causes major economic losses in the dairy industry globally. This enteritis is caused by subsp. (MAP). Currently there is no cure for JD and test-based culling has proved ineffective at preventing the spread. To isolate new mycobacteriophages (mbps) that can potentially be used to control JD transmission and infection on dairy farms, we optimized an isolation protocol by fecal spiking and the testing of different isolation solution compositions. Using this protocol, we successfully enhanced the yield of mbps from spiked fecal samples, elevating it from less than 1% to 59%. With this method, we isolated 14 mbps from 475 environmental samples collected from MAP-positive dairy farms, after in-sample enrichment with MAP and the fast-growing . The sample sources included soil, manure pits, lactation barns, feces, milk, and drain water. After fingerprinting these mbps by restriction enzyme profiling, we concluded that 12 were distinct and novel. Further characterization of their host range revealed that eight were capable of lysing multiple MAP strains. We also studied the cross-resistance, lysogeny, the effect of pH and their antimycobacterial properties in milk replacer. Each novel mbp showed limited cross-resistance and prophage immunity and showed no reduction in the titer in a range of pHs after 4 h. The novel phages were also able to reduce the mycobacterial counts to zero after 8 h in milk replacer. In conclusion, these novel mbps could be considered to be used in the control strategies of JD on farms.
Topics: Female; Animals; Mycobacterium avium subsp. paratuberculosis; Mycobacteriophages; Bacteriophages; Farms; Enteritis
PubMed: 38257721
DOI: 10.3390/v16010020 -
Frontiers in Immunology 2023The induction of an effective immune response is critical for the success of mRNA-based therapeutics. Here, we developed a nanoadjuvant system compromised of Quil-A and...
The induction of an effective immune response is critical for the success of mRNA-based therapeutics. Here, we developed a nanoadjuvant system compromised of Quil-A and DOTAP (dioleoyl 3 trimethylammonium propane), hence named QTAP, for the efficient delivery of mRNA vaccine constructs into cells. Electron microscopy indicated that the complexation of mRNA with QTAP forms nanoparticles with an average size of 75 nm and which have ~90% encapsulation efficiency. The incorporation of pseudouridine-modified mRNA resulted in higher transfection efficiency and protein translation with low cytotoxicity than unmodified mRNA. When QTAP-mRNA or QTAP alone transfected macrophages, pro-inflammatory pathways (e.g., NLRP3, NF-kb, and MyD88) were upregulated, an indication of macrophage activation. In C57Bl/6 mice, QTAP nanovaccines encoding Ag85B and Hsp70 transcripts (QTAP-85B+H70) were able to elicit robust IgG antibody and IFN- ɣ, TNF-α, IL-2, and IL-17 cytokines responses. Following aerosol challenge with a clinical isolate of significant reduction of mycobacterial counts was observed in lungs and spleens of only immunized animals at both 4- and 8-weeks post-challenge. As expected, reduced levels of were associated with diminished histological lesions and robust cell-mediated immunity. Interestingly, polyfunctional T-cells expressing IFN- ɣ, IL-2, and TNF- α were detected at 8 but not 4 weeks post-challenge. Overall, our analysis indicated that QTAP is a highly efficient transfection agent and could improve the immunogenicity of mRNA vaccines against pulmonary , an infection of significant public health importance, especially to the elderly and to those who are immune compromised.
Topics: Animals; Mice; Mycobacterium avium; Mycobacterium tuberculosis; Interleukin-2; RNA; RNA, Messenger
PubMed: 37359562
DOI: 10.3389/fimmu.2023.1188754 -
Infection and Immunity Apr 2019Members of the complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide...
Members of the complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted strains. The study revealed the rapid genetic evolution of in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates.
Topics: Adaptation, Biological; Aged; Aged, 80 and over; Animals; Bacterial Proteins; Cells, Cultured; Cytokines; Evolution, Molecular; Female; Genetic Variation; Humans; Macrophages; Male; Mice; Mice, Inbred C57BL; Middle Aged; Mycobacterium avium; Mycobacterium avium-intracellulare Infection; Phylogeny; Polymorphism, Single Nucleotide
PubMed: 30642899
DOI: 10.1128/IAI.00323-18 -
PloS One 2012Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking...
Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.
Topics: Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gas Chromatography-Mass Spectrometry; Humans; Lipopolysaccharides; Macrophages; Mycobacterium avium
PubMed: 23028991
DOI: 10.1371/journal.pone.0045411 -
Clinical Microbiology and Infection :... Mar 2019To determine MIC distributions for Mycobacterium chimaera, Mycobacterium intracellulare, Mycobacterium colombiense and Mycobacterium avium, and to derive tentative... (Comparative Study)
Comparative Study
OBJECTIVES
To determine MIC distributions for Mycobacterium chimaera, Mycobacterium intracellulare, Mycobacterium colombiense and Mycobacterium avium, and to derive tentative epidemiological cut-off (ECOFF) values.
METHODS
A total of 683 bacterial isolates (M. chimaera, n = 203; M. intracellulare, n = 77; M. colombiense, n = 68; M. avium, n = 335) from 627 patients were tested by broth microdilution according to CLSI protocol M24-A2 on Sensititre RAPMYCOI plates. MICs were interpreted based on CLSI breakpoints for clarithromycin, and tentative breakpoints for amikacin, moxifloxacin and linezolid. Tentative ECOFFs were determined by visual approximation and the ECOFFinder algorithm.
RESULTS
Modal MIC, MIC and MIC values were within ± one dilution step from the respective aggregated data set for 47/48 (97.9%), 48/48 (100%) and 48/48 (100%) species-drug combinations. Clarithromycin wild-type populations were mostly classified as susceptible (MIC 4-8 mg/L; S ≤8 mg/L). Rifabutin MICs were lower than those of rifampicin. Tentative moxifloxacin, linezolid and amikacin breakpoints split wild-type populations. No ECOFFs could be set for rifampicin, ethambutol, ciprofloxacin, isoniazid, trimethoprim/sulfamethoxazole and doxycycline because of truncation of MIC distributions. Agreement between the visually determined and the modelled 97.5% ECOFFs was 90.9%. All 99.0% ECOFFs were one titre step higher than by visual approximation.
CONCLUSIONS
Drug susceptibility patterns of M. chimaera are comparable to those of closely related species. Except for clarithromycin, breakpoints for Mycobacterium avium-intracellulare complex should be re-evaluated. Statistical determination of the 99.0% ECOFF may be superior to visual approximation.
Topics: Anti-Bacterial Agents; Drug Resistance, Bacterial; Humans; Microbial Sensitivity Tests; Mycobacterium avium; Mycobacterium avium Complex; Mycobacterium avium-intracellulare Infection
PubMed: 29906595
DOI: 10.1016/j.cmi.2018.06.010 -
Journal of Applied Microbiology Sep 2013Measure adherence and biofilm formation by cells of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus on common household plumbing materials...
AIMS
Measure adherence and biofilm formation by cells of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus on common household plumbing materials namely stainless steel, glass, zinc-galvanized steel, copper and polyvinyl chloride (PVC).
METHODS AND RESULTS
Coupons in a CDC biofilm reactor were exposed to cell suspensions containing 10(5) NTM colony forming units (CFU) per ml and adherence measured for 6 h. Biofilm formation (increased numbers of adherent CFU) was measured weekly to 21 days in the absence of substantial numbers of suspended mycobacterial cells. Adherence was rapid and substantial with 2000-15 000 CFU cm(-2) adhering within 1-6 h at room temperature. Biofilm numbers reached as high as 10(7) CFU cm(-2) . Biofilm-grown cells of Myco. avium were more adherent compared with suspension-grown cells.
CONCLUSIONS
Mycobacterium avium, Myco. intracellulare and Myco. abscessus readily adhered and formed biofilms on all types of plumbing materials. Factors influencing adherence and biofilm formation were species, plumbing material and prior growth.
Topics: Bacterial Adhesion; Biofilms; Household Articles; Humans; Mycobacterium avium; Mycobacterium avium Complex; Nontuberculous Mycobacteria; Sanitary Engineering; United States
PubMed: 23742161
DOI: 10.1111/jam.12272 -
PloS One 2012Mycobacterium avium is the principal etiologic agent of non-tuberculous lymphadenitis in children. It is also a known pathogen for birds and other animals. Genetic...
BACKGROUND
Mycobacterium avium is the principal etiologic agent of non-tuberculous lymphadenitis in children. It is also a known pathogen for birds and other animals. Genetic typing of M. avium isolates has led to a proposal to expand the set of subspecies to include M. avium subsp. hominissuis. Isolates associated with disease in humans belong to this subspecies.
METHODOLOGY/PRINCIPAL FINDINGS
Peripheral blood mononuclear cells from six healthy blood donors were stimulated in vitro with ten isolates of M. avium avium and 11 isolates of M. avium hominissuis followed by multiplex bead array quantification of cytokines in supernatants. M. avium hominissuis isolates induced significantly more IL-10 and significantly less IL-12p70, TNF, IFN-γ and IL-17 when compared to M. avium avium isolates. All strains induced high levels of IL-17, but had very low levels of IL-12p70.
CONCLUSION/SIGNIFICANCE
The strong association between M. avium subsp. hominissuis and disease in humans and the clear differences in the human immune response to M. avium subsp. hominissuis compared to M. avium subsp. avium isolates, as demonstrated in this study, suggest that genetic differences between M. avium isolates play an important role in the pathogenicity in humans.
Topics: Humans; Interferon-gamma; Interleukins; Leukocytes, Mononuclear; Mycobacterium avium; Tuberculosis; Tumor Necrosis Factor-alpha
PubMed: 22506018
DOI: 10.1371/journal.pone.0034391 -
Euro Surveillance : Bulletin Europeen... 2016Mycobacterium avium represents a health concern for both humans and pigs. The characterisation of its subspecies is an important step improving the understanding of the...
Mycobacterium avium represents a health concern for both humans and pigs. The characterisation of its subspecies is an important step improving the understanding of the epidemiology and the control of this pathogen. Ninety-two human M. avium strains were selected for a retrospective study. Subspecies determination by rpoB sequencing and IS1245/IS901 analysis showed that 98.9% of Belgian human M. avium strains belong to the subspecies hominissuis (MAH). Some of these MAH strains present particular IS1245/IS901 profiles (absence of IS1245 and false IS901 detection provoked by the presence of ISMav6). In addition, 54 MAH strains isolated from submandibular lymph nodes of Belgian pigs with lymphadenitis were included in this study. Genotyping of human and porcine isolates was performed using multispacer sequence typing (MST). In total, 49 different MST types were identified among pig (n = 11) and human (n = 43) MA isolates, with only five shared by both hosts. Among these MST types, 34 were newly identified. Our findings demonstrate the extensive genetic diversity among MAH isolates. Some genotypes were more prevalent in human or pigs but no correlation was observed between MST type and place of residence or the farm of origin for human and porcine isolates respectively, suggesting an environmental source of infection.
Topics: Animals; Belgium; Genetic Variation; Genotype; Humans; Minisatellite Repeats; Mycobacterium avium; Phylogeny; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Retrospective Studies; Sequence Analysis, DNA; Swine; Swine Diseases; Tuberculosis
PubMed: 26835872
DOI: 10.2807/1560-7917.ES.2016.21.3.30111