High genetic heterogeneity of Mycobacterium intracellulare isolated from respiratory specimens.

BACKGROUND: M. intracellulare is a frequent causative pathogen of nontuberculous mycobacteria infection that causes infections in the respiratory tract, whose incidence is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity of a collection of 39 M. intracellulare human strains isolated from respiratory specimens over the last 5 years. RESULTS: The VNTR analysis showed that M. intracellulare strains displayed a high genetic diversity, indicating that the M. intracellulare genotypes are quite heterogeneous in our geographical area. Moreover, a comparison with VNTR profiles of strains from other countries confirmed that genotypes of clinical strains of M. intracellulare are not related to geographical origin. CONCLUSIONS: VNTR typing has proved to be a highly discriminatory method for better understanding the molecular epidemiology of M. intracellulare.

Comparative genomic analysis of Mycobacterium intracellulare: implications for clinical taxonomic classification in pulmonary Mycobacterium avium-intracellulare complex disease.

BACKGROUND: Mycobacterium intracellulare is a representative etiological agent of emerging pulmonary M. avium-intracellulare complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary M. avium-intracellulare complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for M. avium; however, the genomic characteristics of M. intracellulare remain to be elucidated. RESULTS: In this study, we performed comparative genomic analysis of 55 M. intracellulare and related strains such as M. paraintracellulare (MP), M. indicus pranii (MIP) and M. yonogonense. Based on the average nucleotide identity, the clinical M. intracellulare strains were phylogenetically grouped in two clusters: (1) the typical M. intracellulare (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., fadE3, fadE33), transporters (e.g., mce3), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. M. intracellulare was shown to be pan-genomic at the species and subspecies levels. The mce genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies. CONCLUSIONS: Our data suggest that genomic diversity among M. intracellulare, M. paraintracellulare, M. indicus pranii and M. yonogonense remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as mce genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for M. intracellulare and related strains.

Mycobacterium avium paratuberculosis and Mycobacterium avium complex and related subspecies as causative agents of zoonotic and occupational diseases.

Mycobacterium avium complex (MAC) and Mycobacterium avium paratuberculosis (MAP) cause zoonotic infections transmitted by birds and livestock herds. These pathogens have remained as serious economic and health threats in most areas of the world. As zoonotic diseases, the risk of development of occupational disease and even death outcome necessitate implementation of control strategies to prevent its spread. Zoonotic MAP infections include Crohn's disease, inflammatory bowel disease, ulcerative colitis, sarcoidosis, diabetes mellitus, and immune-related diseases (such as Hashimoto's thyroiditis). Paratuberculosis has classified as type B epidemic zoonotic disease according to world health organization which is transmitted to human through consumption of dairy and meat products. In addition, MAC causes pulmonary manifestations and lymphadenitis in normal hosts and human immunodeficiency virus (HIV) progression (by serotypes 1, 4, and 8). Furthermore, other subspecies have caused respiratory abscesses, neck lymph nodes, and disseminated osteomyelitis in children and ulcers. However, the data over the occupational relatedness of these subspecies is rare. These agents can cause occupational infections in susceptible herd breeders. Several molecular methods have been recognized as proper strategies for tracking the infection. In this study, some zoonotic aspects, worldwide prevalence and control strategies regarding infections due to MAP and MAC and related subspecies has been reviewed.

Mycobacterium avium in Community and Household Water, Suburban Philadelphia, Pennsylvania, USA, 2010-2012.

Attention to environmental sources of Mycobacterium avium complex (MAC) infection is a vital component of disease prevention and control. We investigated MAC colonization of household plumbing in suburban Philadelphia, Pennsylvania, USA. We used variable-number tandem-repeat genotyping and whole-genome sequencing with core genome single-nucleotide variant analysis to compare M. avium from household plumbing biofilms with M. avium isolates from patient respiratory specimens. M. avium was recovered from 30 (81.1%) of 37 households, including 19 (90.5%) of 21 M. avium patient households. For 11 (52.4%) of 21 patients with M. avium disease, isolates recovered from their respiratory and household samples were of the same genotype. Within the same community, 18 (85.7%) of 21 M. avium respiratory isolates genotypically matched household plumbing isolates. Six predominant genotypes were recovered across multiple households and respiratory specimens. M. avium colonizing municipal water and household plumbing may be a substantial source of MAC pulmonary infection.

Mycobacterium chimaera Pulmonary Disease in Cystic Fibrosis Patients, France, 2010-2017.

We report Mycobacterium chimaera pulmonary disease in 4 patients given a diagnosis of cystic fibrosis in a university hospital in Montpellier, France. All patients had M. chimaera-positive expectorated sputum specimens, clinical symptoms of pulmonary exacerbation, or a decrease in spirometry test results that improved after specific treatment.

Raman spectroscopy reveals distinct differences between two closely related bacterial strains, Mycobacterium indicus pranii and Mycobacterium intracellulare.

A common technique used to differentiate bacterial species and to determine evolutionary relationships is sequencing their 16S ribosomal RNA genes. However, this method fails when organisms exhibit high similarity in these sequences. Two such strains that have identical 16S rRNA sequences are Mycobacterium indicus pranii (MIP) and Mycobacterium intracellulare. MIP is of significance as it is used as an adjuvant for protection against tuberculosis and leprosy; in addition, it shows potent anti-cancer activity. On the other hand, M. intracellulare is an opportunistic pathogen and causes severe respiratory infections in AIDS patients. It is important to differentiate these two bacterial species as they co-exist in immuno-compromised individuals. To unambiguously distinguish these two closely related bacterial strains, we employed Raman and resonance Raman spectroscopy in conjunction with multivariate statistical tools. Phenotypic profiling for these bacterial species was performed in a kinetic manner. Differences were observed in the mycolic acid profile and carotenoid pigments to show that MIP is biochemically distinct from M. intracellulare. Resonance Raman studies confirmed that carotenoids were produced by both MIP as well as M. intracellulare, though the latter produced higher amounts. Overall, this study demonstrates the potential of Raman spectroscopy in differentiating two closely related mycobacterial strains. Graphical abstract.

Development of a LAMP assay for rapid and sensitive detection and differentiation of Mycobacterium avium subsp. avium and subsp. hominissuis.

Mycobacterium avium causes atypical mycobacterial infection in humans and animals worldwide. M. avium comprises the subspecies avium (MAA), hominissuis (MAH), silvaticum (MAS) and paratuberculosis (MAP). The M. avium complex (MAC), comprising M. avium and M. intracellulare, causes opportunistic infections of humans. M. avium subsp. avium (MAA) mainly causes avian tuberculosis while subsp. hominissuis (MAH) mainly infects pig. Distinguishing between these two subspecies is essential to the effective control of these atypical mycobacterial infections and minimization of the resulting economic loss. For this purpose, we developed a loop-mediated isothermal amplification (LAMP) assay that rapidly and sensitively detects and differentiates MAA and MAH. This MAA-LAMP assay targeting IS901 correctly detected four MAA isolates but did not detect 27 MAH and 19 non-MAA/non-MAH mycobacterial isolates. The MAAH-LAMP assay targeting IS1245 detected four MAA and 27 MAH isolates but not the other 19 mycobacterial isolates. We believe that implementation of this LAMP assay will significantly improve public health and safety. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycobacterium avium, which is pathogenic for humans and animals, represents a continuing threat to public health and safety and to food production. Therefore, improved methods are urgently required to readily and efficiently identify M. avium subspecies. Compared with conventional PCR methods, the LAMP assay herein developed more rapidly detects and better distinguishes between two major M. avium subspecies that cause disease of pig. Importantly, this highly accurate and sensitive LAMP assay detects mycobacterial DNAs using real-time fluorescence or the unaided eye with a colour-change dye, making it ideal for translation to the clinic and slaughterhouse.

Whole-genome sequence analysis of the Mycobacterium avium complex and proposal of the transfer of Mycobacterium yongonense to Mycobacterium intracellulare subsp. yongonense subsp. nov.

Bacterial whole-genome sequences contain informative features of their evolutionary pathways. Comparison of whole-genome sequences have become the method of choice for classification of prokaryotes, thus allowing the identification of bacteria from an evolutionary perspective, and providing data to resolve some current controversies. Currently, controversy exists about the assignment of members of the Mycobacterium avium complex, as is for the cases of Mycobacterium yongonense and 'Mycobacterium indicus pranii'. These two mycobacteria, closely related to Mycobacterium intracellulare on the basis of standard phenotypic and single gene-sequences comparisons, were not considered a member of such species on the basis on some particular differences displayed by a single strain. Whole-genome sequence comparison procedures, namely the average nucleotide identity and the genome distance, showed that those two mycobacteria should be considered members of the species M. intracellulare. The results were confirmed with other whole-genome comparison supplementary methods. According to the data provided, Mycobacterium yongonense and 'Mycobacterium indicus pranii' should be considered and renamed and included as members of M. intracellulare. This study highlights the problems caused when a novel species is accepted on the basis of a single strain, as was the case for M. yongonense. Based mainly on whole-genome sequence analysis, we conclude that M. yongonense should be reclassified as a subspecies of Mycobacterium intracellulareas Mycobacterium intracellularesubsp. yongonense and 'Mycobacterium indicus pranii' classified in the same subspecies as the type strain of Mycobacterium intracellulare and classified as Mycobacterium intracellularesubsp. intracellulare.

A definition of the Mycobacterium avium complex for taxonomical and clinical purposes, a review.

Nontuberculous mycobacteria, particularly the Mycobacterium avium complex (MAC) bacteria, are increasingly recognized as opportunistic pathogens of humans. As a result, studies on antibiotic treatment and taxonomy of the MAC are intensifying, but an updated definition of what constitutes the MAC, either for taxonomical studies or for clinical purposes, is lacking. On the basis of literature review and phylogenetic analyses, we propose to define the MAC as a grouping of slow-growing mycobacteria that show corresponding values in at least two of the following targets against either M. avium ATCC 25291T or Mycobacterium intracellulare ATCC 13950T: >99.4 % sequence identity for the full 16S rRNA gene, >98.7 % for the partial (5') 16S rRNA gene, >97.3 % for hsp65 and >94.4 % for rpoB region V. A >97.5 % value in concatenated analyses of >2500 bp that includes 16S rRNA, hsp65 and rpoB gene sequence data or ≥85 % average nucleotide identity to M. avium ATCC 25291T or M. intracellulare ATCC 13950T on basis of whole genome sequencing data is recommended. This molecular definition is based on the distances observed between the classical members of the MAC, M. avium and M. intracellulare. Applying this definition, the complex currently consists of 12 validly published species: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense, Mycobacterium vulneris, Mycobacterium bouchedurhonense, Mycobacterium timonense, Mycobacterium marseillense, Mycobacterium yongonense, Mycobacterium paraintracellulare and Mycobacterium lepraemurium.

PCR-Based Rapid Identification System Using Bridged Nucleic Acids for Detection of Clarithromycin-Resistant Mycobacterium avium-M. intracellulare Complex Isolates.

The nontuberculous mycobacteria (NTM) cause miscellaneous disorders in humans, especially in the lungs, which present with a variety of radiological features. To date, knowledge of the pathogenic role of the Mycobacterium avium-intracellulare complex (MAC) in the human lung and the definitive criteria for initiating multidrug therapy are still lacking. However, there is little doubt that clarithromycin is the most efficacious drug among the various treatment regimens for lung NTM. In this study, with the use of a bridged nucleic acid (BNA) probe a detection system based on a real-time PCR (BNA-PCR) for the identification of the point mutations at position 2058 or 2059 in domain V of the 23S rRNA gene responsible for clarithromycin resistance was developed and has been assessed using MAC isolates from clinical samples. Out of 199 respiratory specimens, the drug susceptibility test demonstrated 12 strains resistant to clarithromycin, while the BNA-PCR showed 8 strains carrying the point mutation at position 2058 or 2059 of the 23S rRNA gene. This system revealed that there were mycobacterial strains resistant to clarithromycin which do not carry previously identified resistance genes. This paper documents a novel system for detecting clarithromycin-resistant strains and demonstrates that although these mutations are tacitly assumed to account for >90% of the reported resistant mutants, there is a significant fraction of resistant mutants that do not harbor these mutations. Therefore, unknown mechanisms affecting clarithromycin resistance remain to be elucidated.

Mycobacterium paraintracellulare sp. nov., for the genotype INT-1 of Mycobacterium intracellulare.

Three mycobacterial strains, isolated from independent Korean patients with pulmonary infections, belonging to the Mycobacterium intracellulare genotype 1 (INT-1) were characterized using a polyphasic approach. The sequences of the 16S rRNA gene and internal transcribed spacer 1 (ITS1) of the INT-1 strains were identical to those of Mycobacterium intracellulare ATCC 13950T. However, multilocus sequence typing (MLST) analysis targeting five housekeeping genes (hsp65, rpoB, argG, gnd and pgm) revealed the phylogenetic separation of these strains from M. intracellulare ATCC 13950T. DNA-DNA hybridization values of >70 % confirmed that the three isolates belong to the same species, while the values of <70 % between one of them and the type strains of M. intracellulare and Mycobacterium chimaera confirmed their belonging to a distinct species. In addition, phenotypic characteristics such as positive growth on MacConkey agar and in acidic broth culture, unique matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS profiles of lipids, and unique mycolic acids profiles further supported the taxonomic status of these strains as representatives of a novel species of the Mycobacterium avium complex named Mycobacterium paraintracellulare. The type strain is MOTT64T (=KCTC 29084T=JCM 30622T).

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity.

BACKGROUND: A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen. RESULTS: This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes. CONCLUSIONS: Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.

Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping of mycobacterium intracellulare for strain comparison with establishment of a PCR-based database.

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the "gold standard" of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.

Absence of Mycobacterium intracellulare and presence of Mycobacterium chimaera in household water and biofilm samples of patients in the United States with Mycobacterium avium complex respiratory disease.

Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.

First case of pulmonary disease caused by a Mycobacterium avium complex strain of presumed veterinary origin in an adult human patient.

We report the first case of pulmonary disease caused by a strain of Mycobacterium avium complex of presumed veterinary origin in an elderly patient. All serial isolates were identified by multilocus sequence analysis based on rpoB, hsp65, and 16S rRNA fragments. Disease persisted despite macrolide-based combination antibiotic therapy.

Identification of potential biomarkers to distinguish Mycobacterium colombiense from other mycobacterial species.

Mycobacterium avium complex (MAC) consists of 9 species of slow-growing mycobacteria with differing degrees of pathogenicity, host preference and environmental distribution. Mycobacterium colombiense is a novel member of MAC that is responsible for disseminated infections in HIV-infected patients in Colombia and lymphadenopathy cases in Europe. At present, methods to easily differentiate novel members of MAC are lacking. In this study, we identified possible biomarkers that are potentially useful for the detection of M. colombiense by PCR or chromatography. The Randomly Amplified Polymorphic DNA (RAPD) technique was used to amplify genomic fragments of M. colombiense CECT 3035 that were subsequently used in the development of a direct colony-specific PCR assay using specific primers. The designed primers amplified a 634-bp fragment of DNA from M. colombiense, which included a 450-bp genomic region that encodes a hypothetical protein of 149 amino acids that is exclusive to M. colombiense. Bioinformatic analyses revealed that this hypothetical protein had no signal peptide, active sites or functional domains to aid its identification or classification. In addition, using thin-layer chromatography, we identified a different profile of mycolates for M. colombiense strains. The test developed in this study has potential applications in the routine identification of M. colombiense and in molecular assays designed for the surveillance of MAC strains.

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-02.

Here, we report the first complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-02, which was previously grouped in the INT2 genotype of M. intracellulare. This genome sequence will serve as a valuable reference for improving the understanding of the disparity in the virulence and epidemiologic traits between M. intracellulare genotypes.

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-64, belonging to the INT1 genotype.

Here, we report the complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-64, previously grouped into the INT1 genotype among five genotypes of M. intracellulare. This genome sequence will serve as a valuable reference for understanding the disparity in the virulence and epidemiologic traits among M. intracellulare genotypes.

Whole-genome sequence of the hypervirulent clinical strain Mycobacterium intracellulare M.i.198.

We report herein the draft genome sequence of Mycobacterium intracellulare clinical strain M.i.198, which consistently exhibits hypervirulence in human patients, human macrophages in vitro, and immunocompetent mice.

Diversity of Mycobacterium avium subsp. hominissuis mycobacteria causing lymphadenitis, France.

The knowledge of Mycobacterium avium complex (MAC) genotypes responsible for lymphadenitis is limited. We retrospectively characterized all of the MAC isolates made in our laboratory in the last 18 years by sequence-based identification and genotyping, and compared the clinical and laboratory data for lymphadenitis-associated and non-lymphadenitis-associated MAC isolates. Of 67 MAC-infected patients, 25 lymphadenitis patients were significantly younger than 42 non-lymphadenitis patients, while the male/female ratio did not significantly differ between the two groups. Cervical topography found in 76.5% of lymphadenitis patients was significantly more frequent in non-immunocompromised patients (p=0.04). M. avium subsp. hominissuis was identified in 53 patients (24 lymphadenitis, 29 non-lymphadenitis), M. colombiense in six patients (five non-lymphadenitis, one lymphadenitis), M. intracellulare in four non-lymphadenitis patients, and M. chimaera in three non-lymphadenitis patients, while negative controls remained negative. M. hominissuis was significantly associated with lymphadenitis (p=0.03). M. hominissuis isolates yielded 15 genotypes in 29 non-lymphadenitis isolates (molecular diversity, 0.622) versus 11 genotypes in 24 lymphadenitis isolates (molecular diversity, 0.578), demonstrating a non-significant lower diversity of M. hominissuis isolates cultured from lymphadenitis. The genotypes did not correlate with the clinical features. These data suggest the presence of several environmental reservoirs for M. hominissuis causing lymphadenitis in France.