Mycobacterium Avium Subsp. Hominissuis Research Articles

Effects of nutritional and ambient oxygen condition on biofilm formation in Mycobacterium avium subsp. hominissuis via altered glycolipid expression

Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of nontuberculous mycobacteriosis, the representative case of environment-related infectious diseases the incidence of which is increasing in industrialized countries. MAH is found in biofilm in drinking water distribution system and residential environments. We investigated the effect of gaseous and nutritional conditions, and the role of glycopeptidolipids (GPLs) on biofilm-like pellicle formation in MAH. Pellicle formation was observed under 5% oxygen in Middlebrook 7H9 broth containing 0.2% glycerol and 10% albumin-dextrose-catalase enrichment but not under normoxia or in nutrient-poor media. An analysis of 17 environmental isolates revealed that hypoxia (5% oxygen) preferentially enhanced pellicle formation both in plastic plates and in glass tubes, compared with hypercapnia (5% carbon dioxide). Wild-type strains (WT) developed much thicker pellicles than GPL-deficient rough mutants (RM). WT bacterial cells distributed randomly and individually in contrast to that RM cells positioned linearly in a definite order. Exogenous supplementation of GPLs thickened the pellicles of RM, resulting in a similar morphological pattern to WT. These data suggest a significant implication of eutrophication and hypoxia in biofilm-like pellicle formation, and a functional role of GPLs on development of pellicles in MAH.

Molecular typing and genetic characterization of Mycobacterium avium subsp. hominissuis isolates from humans and swine in Japan.

Mycobacterium avium subsp. hominissuis (MAH) causes disease in both humans and swine; however, the genetic variations in MAH isolates are unclear. The aim of this study was to elucidate the genetic variations in MAH isolates from humans and swine in Japan. We analysed the 16S-23S rDNA internal transcribed spacer (ITS) sequence and variable number of tandem repeats (VNTRs) using the Mycobacterium avium tandem repeat loci, prevalence of ISMav6 and clarithromycin resistance for MAH isolates from patients with pulmonary MAC (pMAC) disease (n=69), and HIV-seropositive and blood culture-positive (HIV-MAC) patients (n=28) and swine (n=23). In the minimum spanning tree based on VNTR analysis, swine MAC isolates belonged to a cluster distinguishable from that of human pMAC isolates. Isolates from HIV-MAC were scattered throughout both clusters. The three major distinct sequevars, Mav-A, Mav-B and Mav-F, were determined according to 16S-23S rDNA ITS sequence analysis in addition to three new sequevars, Mav-Q, Mav-R and Mav-S. Mav-A and Mav-F comprised the majority of human pMAC strains; in contrast, Mav-B predominated in swine isolates. Distribution of ITS sequevars in the minimum spanning tree based on VNTR analysis showed similar clusters of isolates from different origins, i.e. human pMAC, HIV-MAC and swine. These results, together with ISMav6 possession and clarithromycin resistance, revealed the genetic diversity of MAH strains recovered from humans and swine. Molecular epidemiology and genetic characterization in the present study showed the distinctive genetic evolutionary lineage of MAH strains isolated from human pMAC diseases and swine.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A–H (sized 6.2–73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Experimental Infection of Goats with Mycobacterium avium subsp. hominissuis: a Model for Comparative Tuberculosis Research

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic pathogen that causes infections in man and animals. In this study, 18 goat kids were inoculated orally with a high dose of MAH. One group of goats (n=9) developed severe clinical disease for up to 2-3 months post inoculation (mpi). At necropsy examination, there were ulcerative and granulomatous lesions in gut-associated lymphoid tissue and granulomas with extensive necrosis in the lymph nodes (LNs) of the cranial mesenteric lymphocentre (CMLNs). Culture revealed growth of MAH in all lesions with systemic spread. A second group of goats were healthy at the end of the trial (13 mpi); however, all had extensive granulomas in the CMLNs, but no extra-intestinal spread of bacteria. Moderate faecal shedding occurred in all goats up to 2 mpi. Microscopical characterization of the granulomas revealed solid non-necrotic, necrotic, calcified and fibrocalcified granulomas with resemblance to those seen in human and bovine tuberculosis. The two different courses of disease, with highly heterogenic lesions, systemic spread in goats with severe clinical disease and the development of granulomas of all stages in the surviving goats, makes the experimental infection of goats with MAH a valuable model for tuberculosis research. This model might allow new insights into host-pathogen interaction and anti-mycobacterial compound testing.

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis.

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4(+), CD8(+), γδ, B lymphocytes and plasma, CD25(+), CD68(+), MHC-II(+), Ki67(+) and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4(+) T cells, but only few epitheloid macrophages were observed in severely sick goats at 2-3mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.

Evidence for genes associated with the ability of Mycobacterium avium subsp. hominissuis to escape apoptotic macrophages.

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacteria that infects immunocompromised humans. MAH cases are increasing in incidence, making it crucial to gain knowledge of the pathogenic mechanisms associated with the bacterium. MAH infects macrophages and after several days the infection triggers the phagocyte apoptosis. Many of the intracellular MAH escape the cell undergoing apoptosis leading to infection of neighboring macrophages. We screened a transposon bank of MAH mutants in U937 mononuclear phagocytes for the inability to escape macrophages undergoing apoptosis. Mutations in genes; MAV_2235, MAV_2120, MAV_2410, and MAV_4563 resulted in the inability of the bacteria to exit macrophages upon apoptosis. Complementation of the mutations corrected the phenotype either completely or partially. Testing for the ability of the mutants to survive in macrophages compared to the wild-type bacterium revealed that the mutant clones were not attenuated up to 4 days of infection. Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice. Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms. The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process. The study reveals a pathway of escape from macrophages, not shared with Mycobacterium tuberculosis.

Microaggregate-associated protein involved in invasion of epithelial cells by Mycobacterium avium subsp. hominissuis

The environmental opportunistic pathogen Mycobacterium avium subsp hominissuis (MAH), a member of the nontuberculous mycobacteria (NTM) cluster, causes respiratory as well as disseminated disease in patients such as those with chronic respiratory illnesses or AIDS. Currently, there is no effective method to prevent NTM respiratory infections. The formation of mycobacterial microaggregates comprises of phenotypic changes that lead to efficient adherence and invasion of the respiratory mucosa in vitro and in vivo. Microaggregate adhesion to the respiratory epithelium is mediated in part through the mycobacterial protein, MAV_3013 (MBP-1). Through DNA microarray analysis, the small hypothetical gene MAV_0831 (Microaggregate Invasion Protein-1, MIP-1) was identified as being upregulated during microaggregate formation. When MIP-1 was overexpressed in poorly-invasive Mycobacterium smegmatis, it provided the bacterium the ability to bind and enter epithelial cells. In addition, incubating microaggregates with recombinant MIP-1 protein enhanced the ability of microaggregates to invade HEp-2 cells, and exposure to anti-MIP-1 immune serum reduced the invasion of the host epithelium. Through protein-protein interaction assays, MIP-1 was found to bind to the host protein filamin A, a cytoskeletal actin-binding protein integral to the modulation of host cell shape and migration. As visualized by immunofluorescence, filamin A was able to co-localize with microaggregates and to a lesser extent planktonic bacteria. Invasion of HEp-2 cells by microaggregates and planktonic bacteria was also inhibited by the addition of anti-filamin A antibody suggesting that filamin A plays an important role during infection. In addition, at earlier time points binding and invasion assay results suggest that MBP-1 participates significantly during the first interactions with the host cell while MIP-1 becomes important once the bacteria adhere to the host epithelium. In summary, we have unveiled one more step associated with MAH crossing the respiratory mucosa.

Mycobacterium avium MAV_2941 mimics phosphoinositol-3-kinase to interfere with macrophage phagosome maturation

Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV_2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV_2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV_2941 action and demonstrated that MAV_2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV_2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV_2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV_2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV_2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome.

Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.

Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85kb in the four other strains. The genes present in the islands differed among strains and included phage- and plasmid-derived genes, integrase genes, hypothetical genes, and virulence-associated genes like mmpL or mce genes.

Relatedness of Mycobacterium avium subspecies hominissuis clinical isolates of human and porcine origins assessed by MLVA

Mycobacterium avium subsp. hominissuis (MAH) is an important opportunistic pathogen, infecting humans and animals, notably pigs. Several methods have been used to characterize MAH strains. RFLP and PFGE typing techniques have been used as standard methods but are technically demanding. In contrast, the analysis of VNTR loci is a simpler, affordable and highly reliable PCR-based technique, allowing a numerical and reproductive digitalization of typing data. In this study, the analysis of Mycobacterium avium tandem repeats (MATRs) loci was adapted to evaluate the genetic diversity of epidemiological unrelated MAH clinical strains of human (n=28) and porcine (n=69) origins, collected from diverse geographical regions across mainland Portugal. These MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country. Some of the human strains shared identical VNTR profiles with porcine isolates. Our study shows that the VNTR genotyping using selected MATR loci is a useful analysis technique for assessing the genetic diversity of MAH isolates from Portugal. This typing method could be successfully applied in other countries toward the implementation of a worldwide open-access database of MATR-VNTR profiles of MAH isolates, allowing a better assessment of the global epidemiology traits of this important pathogenic species.

Intra-subspecies sequence variability of the MACPPE12 gene in Mycobacterium avium subsp. hominissuis

The PE (Pro-Glu) and PPE (Pro-Pro-Glu) multigene families are unique to mycobacteria, and are highly expanded in the pathogenic members of this genus. We determined the intra-subspecies genetic variability of the MACPPE12 gene, which is a specific PPE gene in Mycobacterium avium subsp. hominissuis (MAH), using 334 MAH isolates obtained from different isolation sources (222 human isolates, 145 Japanese and 77 Korean; 37 bathroom isolates; and 75 pig isolates). In total, 31 single-nucleotide polymorphisms (SNPs), which consisted of 16 synonymous SNPs and 15 nonsynonymous SNPs, were determined through comparison with the MACPPE12 gene sequence of MAH strain 104 as a reference. As the result, the 334 MAH isolates were classified into 19 and 13 different sequevars at the nucleic acid level (NA types) and amino acid level (AA types), respectively. Among the 13 AA types, only one type, the AA02 type, presented various NA types (7 different types) with synonymous SNPs, whereas all other AA types had a one-to-one correspondence with the NA types. This finding suggests that AA02 is a longer discernible lineage than the other AA types. Therefore, AA02 was classified as an ancestral type of the MACPPE12 gene, whereas the other AA types were classified as modern types. The ubiquitous presence of AA02 in all of the isolation sources and all different sequevars classified by the hsp65 genotype further supports this classification. In contrast to the ancestral type, the modern types showed remarkable differences in distribution between human isolates and pig isolates, and between Japanese isolates and Korean isolates. Divergence of the MACPPE12 gene may thus be a good indicator to characterize MAH strains in certain areas and/or hosts.

High phylogenetic proximity of isolates of Mycobacterium avium subsp. hominissuis over a two decades-period

The genetic diversity of 47 human isolates of Mycobacterium avium subsp. hominissuis (MAH) was determined by MIRU-VNTR genotyping. Sixteen unique VNTR patterns and eight clusters including a total 39 isolates were detected; six clusters included strains isolated during at least a 15-year period. Minimum spanning tree analysis showed that 14 VNTR patterns, occurring either as clustered or unique isolates, differed from the nearest one for one allelic variation and that the remaining two patterns differed for two allelic variations. The high phylogenetic proximity of the isolates, even over a long time period, indicates that the MAH genotype is highly homogeneous and conserved.

Survival of three Mycobacterium avium subsp. hominissuis isolates in fish products after hot smoking and frying

SummaryFish mycobacteriosis should be considered as one possible means of transmission of mycobacterial infections to humans. In our study, we examined the survival of three field Mycobacterium avium subsp. hominissuis (MAH) isolates in fish meat during thermal processing technologies using culture and quantitative real‐time PCR (qPCR) methods. Minced carp meat mixture was artificially contaminated with known amount of MAH cells and survival and absolute numbers of MAH was monitored after hot smoking and frying. The viability of MAH was significantly lower after the frying process, whereas in response to hot smoking viable MAH cells could still be cultured even after 10 min at 70 °C. Significant differences in thermal stability were also observed among the three different MAH isolates. The human isolate was the most resistant, whereas the environmental isolate was the least resistant, which is probably due to host adaptability. In this work we confirmed that MAH can survive temperatures of 70 °C and thus can pose a risk to consumers.

Different immune response of pigs to Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis infection

Mycobacterium avium subsp. avium (MAA) and Mycobacterium avium subsp. hominissuis (MAH) are the most common mycobacterial species isolated from granulomatous lesions in swine in countries with controlled bovine tuberculosis. This study is focused on the immunological aspect of MAA and MAH infection in pigs. We detected induction of humoral and cell-mediated immunity in experimentally infected pigs. Specific antibodies were analyzed in serum by ELISA and the IFN-γ release assay was used for evaluation of cell-mediated immunity. While MAA induced a significant increase of both types of immune responses, MAH-infected pigs had an unvarying level of specific antibodies and showed low cell-mediated immunity with high individual variability. The subsequent in vitro experiment confirmed the lower immunogenicity of the MAH strain in comparison to MAA. MAH-infected porcine monocyte-derived macrophages showed a weaker induction of pro-inflammatory mediators in comparison to MAA, which included mRNA for IL-1β, TNF-α, IL-23p19, IL-18 and chemokines CCL-3, CCL-5, CXCL-8 and CXCL-10. Additionally, qualitative proteomic analysis revealed 28 proteins exclusively in MAA and 7 proteins unique to MAH. In conclusion, closely related M. avium subspecies MAA and MAH showed different capacities to stimulate the porcine immune system. From a diagnostic point of view, the IFN-γ release assay showed higher sensitivity than the detection of specific antibodies by ELISA and seems to be an effective tool for discrimination of MAA-infected pigs. In the case of MAH infection, the IFN-γ release assay could fail because of the low immunogenic capacity of the MAH strain.

Survival of Mycobacterium avium subsp. hominissuis in homemade smoked pork sausages

We assessed the survival of Mycobacterium avium subsp. hominissuis (MAH) in artificially contaminated homemade smoked sausages prepared from pork meat according to traditional recipes, the effect of storage of such sausages at –20°C for three months on MAH viability and to compare assessment of MAH viability/presence by culture and qPCR. Three isolates of MAH were inoculated into the sausage mixture at concentrations of 10<sup>6</sup> CFU per gram of meat and cold smoked at 40°C for 12 h or hot smoked at 70°C for 6 hours. MAH survived the cold smoking procedure without any significant decrease in viable MAH CFU counts; no viable MAH were detected in the hot smoked sausages. The storage of sausages at –20°C caused a decrease in viable MAH counts of about 1 to 3 log<sub>10</sub>. Absolute MAH counts determined by qPCR were not significantly reduced by the storage or smoking. The presence of viable MAH in sausages after the cold smoking should be considered as a risk for immunodeficient individuals and children.  

Genetic diversity of Mycobacterium avium subsp. hominissuis strains isolated from humans, pigs, and human living environment

Mycobacterium avium subsp. hominissuis (MAH) strains are genetically diverse and cause infections in pigs and humans. To elucidate the geographical and host-dependent variations in the genetic diversity of MAH, we performed variable numbers of tandem repeat (VNTR) analysis targeting 19 loci for MAH samples from humans (n=146), bathroom environments (n=37), and pigs (n=75) in Japan; these data were then compared with previously reported VNTR data from other countries. The minimum spanning tree (MST) and the multi-dimensional scaling (MDS) analyses based on the VNTR data indicated a high degree of genetic relatedness between isolates from humans and bathrooms in Japan, but a low degree of similarity with the isolates from France and Finland. Moreover, the comparison showed a higher similarity of isolates from Japanese pigs with those from French humans and pigs and Finnish humans and pigs than with other isolates from humans and bathrooms in Japan. The singularity of the Japanese MAH was characterized as the prevalence of hsp65 sequevar code 15 and ISMav6 for the human and bathroom isolates; however, none of the isolates obtained from the pigs belonged to the code 15 or possessed ISMav6. The genetic diversity of MAH and its regional variations imply a possible regional or local specific source of infection and route of transmission of MAH for humans.

6 - Porphyrias in Animals

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic pathogen that causes infections in man and animals. In this study, 18 goat kids were inoculated orally with a high dose of MAH. One group of goats (n = 9) developed severe clinical disease for up to 2–3 months post inoculation (mpi). At necropsy examination, there were ulcerative and granulomatous lesions in gut-associated lymphoid tissue and granulomas with extensive necrosis in the lymph nodes (LNs) of the cranial mesenteric lymphocentre (CMLNs). Culture revealed growth of MAH in all lesions with systemic spread. A second group of goats were healthy at the end of the trial (13 mpi); however, all had extensive granulomas in the CMLNs, but no extra-intestinal spread of bacteria. Moderate faecal shedding occurred in all goats up to 2 mpi. Microscopical characterization of the granulomas revealed solid non-necrotic, necrotic, calcified and fibrocalcified granulomas with resemblance to those seen in human and bovine tuberculosis. The two different courses of disease, with highly heterogenic lesions, systemic spread in goats with severe clinical disease and the development of granulomas of all stages in the surviving goats, makes the experimental infection of goats with MAH a valuable model for tuberculosis research. This model might allow new insights into host–pathogen interaction and anti-mycobacterial compound testing.