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.

Distribution and outcomes of infection of Mycobacterium avium complex species in cystic fibrosis.

BACKGROUND: The majority of nontuberculous mycobacterial (NTM) pulmonary infections in people with cystic fibrosis (CF) are caused by Mycobacterium avium complex (MAC) species. Data on MAC species distribution and outcomes of infection in CF are lacking. METHODS: This was a single center, retrospective study. MAC isolates had species identification with MLSA of rpoB and the 16S23S ITS region. Clinical data were compared between species. RESULTS: Twenty-three people with CF and 57 MAC isolates were included. Infection with M. avium was the most common (65.2%). M. intracellulare was associated with higher rates of NTM disease, younger age, and steeper decline in lung function prior to infection. CONCLUSIONS: We observed worse clinical outcomes in people with M. intracellulare infection relative to other MAC species. Further investigation of clinical outcomes of MAC infection among CF patients is warranted to better define the utility of species-level identification of MAC isolates in CF.

MAC-INMV-SSR: a web application dedicated to genotyping members of Mycobacterium avium complex (MAC) including Mycobacterium avium subsp. paratuberculosis strains.

Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for surveillance of this significant veterinary pathogen. For Map, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable number variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called "MAC-INMV-SSR database" was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods.

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.

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.

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.

Investigation of non-tuberculous mycobacteria in a primary hospital from southeastern China.

INTRODUCTION: Non-tuberculous mycobacterium (NTM) can colonize the human body, leading to opportunistic infection. This study was conducted to analyze the NTM species composition in a primary hospital and investigate the potential features of the patients with different NTM species. METHODOLOGY: Mycobacterial strains were collected from the patients admitted at the hospital from January 2016 to May 2019. MPB64 assay was used to screen NTM strains and confirmed by Rv0577 amplification. The species were identified by hsp65 sequencing. The clinical records of patients with NTM were retrospectively reviewed. RESULTS: Among the 122 identified NTM isolates, the most common strains were Mycobacterium avium complex (MAC, n = 102, 83.6%), Mycobacterium abscessus (n = 9, 7.4%) and Mycobacterium lentiflavum (n = 5, 4.1%). The predominant species among MAC were Mycobacterium chimaera (n = 57, 46.7%), followed by Mycobacterium intracellulare (n = 25, 20.5%) and Mycobacterium colombiense (n = 17, 13.9%). A significantly lower percentage of positive acid-fast assay was observed in Mycobacterium colombiense positive patients than in those with Mycobacterium intracellulare and Mycobacterium chimaera. Mycobacterium intracellulare was more frequently isolated in patients from the infectious department than in other MAC members. CONCLUSIONS: A predominant prevalence of Mycobacterium chimaera in Dongyang of Zhejiang Province was different from other regions in China, indicating that its prevalence has been likely underestimated. The heterogeneity in clinical features, caused by different MAC members, required an accurate species identification of the NTM isolated in the primary hospitals.

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.

Mycobacterium avium complex infection in pigs: A review.

Mycobacterial infections in pigs are caused particularly by the Mycobacterium avium complex (MAC) and these infections lead to great economic losses mainly within the countries with high pork meat production. The importance of the MAC infections in humans is rising because of its higher prevalence and also higher mortality rates particularly in advanced countries. In addition, treatment of the MAC infections in humans tends to be complicated because of its increasing resistance to antimicrobial agents. Several studies across Europe have documented the MAC occurrence in the slaughtered pigs - not only in their lymph nodes and tonsils, which are the most frequent, but also in the diaphragmas, other organs and not least in meat. This is why we need both more specific and more sensitive methods for the MAC infection detection. Different PCR assays were established as well as advanced intravital testing by the gamma interferon release test. On the other hand, tuberculin skin test is still one of the cheapest methods of mycobacterial infections detection.

Mycobacterium avium complex bacteria remain viable in sputum during storage and refrigeration.

Diagnostic mycobacteriology often involves shipping of samples to centralized laboratories. Using two quantitative culture techniques, we show that 7 days storage of sputum samples at room temperature or 4 °C does not affect the number of viable Mycobacterium avium complex bacteria. Storage at room temperature increases the chance of contamination.

Genome sequence of Mycobacterium yongonense RT 955-2015 isolate from a patient misdiagnosed with multidrug-resistant tuberculosis: First clinical detection in Tanzania.

BACKGROUND: Mycobacterium yongonense is a recently described novel species belonging to Mycobacterium avium complex, which is the most prevalent aetiology of non-tuberculous mycobacteria associated with pulmonary infections, and poses tuberculosis diagnostic challenges in high-burden, resource-constrained settings. METHODS: Whole genome shotgun sequencing and comparative microbial genomic analyses were used to characterize the isolate from a patient diagnosed with multidrug-resistant tuberculosis (MDR-TB) after relapse. RESULTS: The genome sequence of the first case of M. yongonense (M. yongonense RT 955-2015) in Tanzania is presented. Sequence analysis revealed that the RT 955-2015 strain had a high similarity to M. yongonense 05-1390(T) (98.74%) and Mycobacterium chimaera DSM 44623(T) (98%). Its 16S rRNA showed similarity to Mycobacterium paraintracellulare KCTC 290849(T) (100%), Mycobacterium intracellulare ATCC 13950(T) (100%), M. chimaera DSM 44623(T) (99.9%), and M. yongonense 05-1390(T) (98%). The strain exhibited a substantially different rpoB sequence to that of M. yongonense 05-1390 (95.16%), but closely related to that of M. chimaera DSM 44623(T) (99.86%), M. intracellulare ATCC 13950(T), (99.53%), and M. paraintracellulare KCTC 290849(T) (99.53%). CONCLUSIONS: In light of the OrthoANI algorithm and phylogenetic analysis, it was concluded that the isolate was M. yongonense Type II genotype, which is an indication that the patient was misdiagnosed with TB/MDR-TB and received inappropriate treatment.

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.

In vitro activity of aminoglycosides, clofazimine, d-cycloserine and dapsone against 83 Mycobacterium avium complex clinical isolates.

BACKGROUND/PURPOSE: Treatment success rates for Mycobacterium avium complex (MAC) diseases range from 50% to 55% only. To explore effective antimicrobials against either Mycobacterium intracellulare or M. avium, we determined in vitro activities of five aminoglycosides, clofazimine, dapsone and d-cycloserine compared with primary (clarithromycin) and secondary (moxifloxacin and linezolid) antimycobacterial agents. METHODS: 83 non-duplicate clinical MAC isolates were collected from sputum and identified at the species level by PCR and restriction enzyme analysis of the 65 kDa hsp and rpoB genes. Drug susceptibility testing was performed using broth microdilution method. The fractional inhibitory concentration was calculated to determine synergy between isepamicin and clofazimine. RESULTS: High susceptibility rates of five aminoglycosides (isepamicin, amikacin, kanamycin, streptomycin, capreomycin, 82.7-88%), d-cycloserine (82.7%), clofazimine (97.3%) and clarithromycin (92%) against M. intracellulare, and 2 aminoglycosides (isepamicin, streptomycin, 87.5%), d-cycloserine (100%) and clarithromycin (100%) against M. avium were found. Dapsone had no inhibitory activity and moxifloxacin had little effect against both M. intracellulare and M. avium. Linezolid had modest activity whereas clofazimine had little effect against M. avium. Most MAC isolates with non-susceptibility to isepamicin were also non-susceptible to the other four aminoglycosides. Most streptomycin-susceptible MAC isolates were also susceptible to amikacin. Synergistic effect of combination of isepamicin and clofazimine was demonstrated in all (100%) M. intracellulare isolates whereas in only 50% M. avium isolates. CONCLUSION: When treating MAC diseases, species identification plays an important role in choosing treatment regimens. Combination of isepamicin and clofazimine may be a promising regimen in M. intracellulare-associated disease.

Geographic Distribution of Nontuberculous Mycobacterial Species Identified among Clinical Isolates in the United States, 2009-2013.

RATIONALE: Nontuberculous mycobacteria are an important cause of morbidity in the United States, although patient outcomes vary greatly by species. Currently, nationally representative data on the distribution of mycobacterial species from clinical isolates are limited. OBJECTIVES: Using a national hospitalization database capturing microbiologic data for nearly 6 million patient encounters, we describe the geographic distribution of, and patient demographic features associated with, clinical mycobacterial isolates in the United States. METHODS: Linked demographic and microbiologic data from the Premier Healthcare Database were extracted for all patient encounters from 2009 to 2013. Patients with at least one positive potentially pathogenic nontuberculous mycobacterial culture were identified as cases. The period prevalence was calculated, and patient-, encounter-, and hospital-level factors were analyzed. Regional differences in species distribution were analyzed; a subanalysis was conducted among patients with International Classification of Diseases, Ninth Revision, codes for pulmonary nontuberculous mycobacterial disease. Significant differences were assessed (P < 0.05). RESULTS: Of 5,928,830 unique patients included during the 5-year study period, 7,812 (0.13%) had at least one positive nontuberculous mycobacterial culture. The mean age of cases was 64 years (range, <1-89 yr), and most were female (52%) and white (70%). Hospitals with cases were more often labeled "urban" (96%), "teaching" (56%), and had at least 500 beds (78%). Species distribution differed significantly by geographic area. Mycobacterium avium complex ranged from 61 to 91% of isolates and were most frequent in the South and Northeast regions; M. abscessus/M. chelonae ranged from 2 to 18% of isolates and were most frequent in the West; and other species, including M. fortuitum and M. kansasii, ranged from 7 to 26% and were also most frequent in the West. CONCLUSIONS: Significant geographic variation exists in the distribution of nontuberculous mycobacterial species in the United States. Whereas M. avium complex was the most common species isolated in the South, M. abscessus/M. chelonae was proportionately higher in the West. Greater clinical awareness in regions with increased levels of harder-to-treat mycobacteria are needed, given differences in treatment options and implications for patient outcomes.

Accurate differentiation of Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS analysis.

PURPOSE: The increasing number of infections caused by nontuberculous mycobacteria (NTM) has prompted the need for rapid and precise identification methods of these pathogens. Several studies report the applicability of MALDI-TOF mass spectrometry (MS) for identification of NTM. However, some closely related species have very similar spectral mass fingerprints, and until recently, Mycobacterium chimaera and M. intracellulare could not be separated from each other by MALDI-TOF MS. METHODOLOGY: The conventional identification methods used in routine diagnostics have similar limitations. Recently, the differentiation of these two species within the Mycobacterium avium complex has become increasingly important due to reports of M. chimaera infections related to open heart surgery in Europe and in the USA. In this report, a method for the distinct differentiation of M. chimaera and M. intracellulare using a more detailed analysis of MALDI-TOF mass spectra is presented. KEY FINDINGS: Species-specific peaks could be identified and it was possible to assign all isolates (100 %) from reference strain collections as well as clinical isolates to the correct species. CONCLUSIONS: We have developed a model for the accurate identification of M. chimaera and M. intracellulare by MALDI-TOF MS. This approach has the potential for routine use in microbiology laboratories, as the model itself can be easily implemented into the software of the currently available systems by MALDI-TOF MS manufacturers.

Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures.

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Variable-number tandem repeat markers for Mycobacterium intracellulare genotyping: comparison to the 16S rRNA gene sequencing.

INTRODUCTION: Characterizing Mycobacterium intracellulare responsible for nontuberculous mycobacterial (NTM) infections may aid in controlling outbreaks. This study aimed to compare 16S ribosomal ribonucleic acid (rRNA) sequencing and variable-number tandem repeat (VNTR) genotyping of M. intracellulare strains isolated from clinical samples, and to characterize VNTR clusters associated with NTM infections or cavity formation. METHODOLOGY: Sputum samples were obtained from 77 HIV-negative patients with pulmonary disease between 2009 and 2013. One M. intracellulare strain was isolated from each patient and genotyped using 16S rRNA and eight loci VNTR sequencing. RESULTS: Single nucleotide polymorphism (SNP) genotyping identified seven point mutations at nucleotide positions 101, 178, 190, 252, 382, 443, and 490 in 16S rRNA, and four SNP patterns were identified: type 1 (16 strains), 2 (41 strains), 3 (11 strains), and 4 (1 strain); 5 strains had unique SNP patterns. VNTR genotyping identified VNTR12 as the most discriminating marker (allelic diversity 0.692). VNTR3 was the most homogeneous marker (allelic diversity 0.518), but each locus had high discriminating ability. The 77 strains were clustered according to the unpaired group method using arithmetic averages: cluster 1 (17 strains), 2 (43 strains), 3 (9 strains), and 4 (4 strains); 4strains had unique SNP patterns. Overall, over 90% strains were matched to similar SNP and VNTR groupings. VNTR clusters were associated with NTM infection (p =0.007) and presence of a cavity (p =0.042). Both methods distinguished four subtypes of M. intracellulare, which corresponded. CONCLUSIONS: VNTRs may represent an effective, user-friendly, low-cost typing technique.