Mycobacterium talmoniae, a Potential Pulmonary Pathogen Isolated from Multiple Patients with Bronchiectasis in the United States, Including the First Case of Clinical Disease in a Patient with Cystic Fibrosis.

We characterize three respiratory isolates of the recently described species Mycobacterium talmoniae recovered in Texas, Louisiana, and Massachusetts, including the first case of disease in a patient with underlying cystic fibrosis. The three isolates had a 100% match to M. talmoniae NE-TNMC-100812T by complete 16S rRNA, rpoB region V, and hsp65 gene sequencing. Core genomic comparisons between one isolate and the type strain revealed an average nucleotide identity of 99.8%. The isolates were susceptible to clarithromycin, amikacin, and rifabutin, while resistance was observed for tetracyclines, ciprofloxacin, and linezolid. M. talmoniae should be added to the list of potential pulmonary pathogens, including in the setting of cystic fibrosis.

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.

Emended description of Mycobacterium abscessus, Mycobacterium abscessus subsp. abscessus and Mycobacteriumabscessus subsp. bolletii and designation of Mycobacteriumabscessus subsp. massiliense comb. nov.

The taxonomic position of members of the Mycobacterium abscessus complex has been the subject of intensive investigation and, in some aspects confusion, in recent years as a result of varying approaches to genetic data interpretation. Currently, the former species Mycobacterium massiliense and Mycobacterium bolletii are grouped together as Mycobacterium abscessus subsp. bolletii. They differ greatly, however, as the former M. bolletii has a functional erm(41) gene that confers inducible resistance to macrolides, the primary therapeutic antimicrobials for M. abscessus, while in the former M. massiliense the erm(41) gene is non-functional. Furthermore, previous whole genome studies of the M. abscessus group support the separation of M. bolletii and M. massiliense. To shed further light on the population structure of Mycobacterium abscessus, 43 strains and three genomes retrieved from GenBank were subjected to pairwise comparisons using three computational approaches: verage ucleotide dentity, enome to enome istance and single nucleotide polymorphism analysis. The three methods produced overlapping results, each demonstrating three clusters of strains corresponding to the same number of taxonomic entities. The distances were insufficient to warrant distinction at the species level, but met the criteria for differentiation at the subspecies level. Based on prior erm(41)-related phenotypic data and current genomic data, we conclude that the species M. abscessus encompasses, in adjunct to the presently recognized subspecies M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a third subspecies for which we suggest the name M. abscessus subsp. massiliense comb. nov. (type strain CCUG 48898T=CIP 108297T=DSM 45103T=KCTC 19086T).

In vitro activity of amikacin against isolates of Mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rRNA gene mutation in treated isolates.

Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 µg/ml, and 86% had MICs of ≤16 µg/ml. Of the eight isolates (1.7%) with MICs of 64 µg/ml, five had an MIC of 32 µg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 µg/ml, of which seven (1.5%) had MICs of >64 µg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 µg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 µg/ml for susceptible, 32 µg/ml for intermediate, and ≥64 µg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.

Absence of transmission of NDM and OXA-48 carbapenemase genes in a chronic care unit of a long-term care facility.

Infection prevention and control measures are used to contain outbreaks of carbapenemase-producing Enterobacteriaceae. We report the absence of transmission of Klebsiella pneumoniae carrying New Delhi metallo-ß-lactamase and oxacillinase-48 genes among 19 screened contacts of an index case after 14 months of routine practices in a long-term care facility.

Insertion and deletion events that define the pathogen Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium comprises genetically related yet phenotypically distinct subspecies. Consistent with their common origin, whole-genome sequence comparisons have revealed extensive synteny among M. avium organisms. However, the sequenced strains also display numerous regions of heterogeneity that likely contribute to the diversity of the individual subspecies. Starting from a phylogenetic framework derived by multilocus sequence analysis, we examined the distribution of 25 large sequence polymorphisms across a panel of genetically defined M. avium strains. This distribution was most variable among M. avium subsp. hominissuis isolates. In contrast, M. avium subsp. paratuberculosis strains exhibited a characteristic profile, with all isolates containing a set of genomic insertions absent from other M. avium strains. The emergence of the pathogen from its putative M. avium subsp. hominissuis ancestor entailed the acquisition of approximately 125 kb of novel genetic material, followed by a second phase, characterized by reductive genomics. One genomic deletion is common to all isolates while additional deletions distinguish two major lineages of M. avium subsp. paratuberculosis. For the average strain, these losses total at least 38 kb (sheep lineage) to 90 kb (cattle lineage). This biphasic pattern of evolution, characterized by chromosomal gene acquisition with subsequent gene loss, describes the emergence of M. avium subsp. paratuberculosis and may serve as a general model for the origin of pathogenic mycobacteria.

Genomic comparison of PE and PPE genes in the Mycobacterium avium complex.

The Mycobacterium avium complex (MAC) comprises genomically similar but phenotypically divergent bacteria that inhabit diverse environments and that cause disease in different hosts. In this study, a whole-genome approach was used to examine the polymorphic PE (Pro-Glu) and PPE (Pro-Pro-Glu) gene families, implicated in immunostimulation and virulence. The four major groups of MAC organisms were examined, including the newly sequenced type strains of M. intracellulare and M. avium subsp. avium, plus M. avium subsp. paratuberculosis and M. avium subsp. hominissuis, for the purpose of finding genetic differences that could be exploited to design diagnostic tests specific to these groups and that could help explain their divergence in pathogenesis and host specificity. Unique and missing PPE genes were found in all MAC members except M. avium subsp. avium. Only M. intracellulare had a unique PE gene. Apart from this, most PE and PPE sequences were conserved, with average nucleotide sequence identities of 99.1 and 98.1%, respectively, among the M. avium subspecies, but only 82.9 and 79.7% identities with the PE and PPE sequences of M. intracellulare, respectively. A detailed analysis of the amino acid sequences was performed between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis. Most differences were detected in the PPE proteins, with amino acid substitutions and frame shifts leading to unique amino acid sequences. In conclusion, several unique PPE proteins were identified in MAC organisms next to numerous polymorphisms in both the PE and PPE gene families. These substantial differences could help explain the divergence in phenotypes within the MAC and could lead to diagnostic tests with better discriminatory abilities.

Nontuberculous mycobacteria: Insights on taxonomy and evolution.

Seventy years have passed since Ernest H. Runyon presented a phenotypic classification approach for nontuberculous mycobacteria (NTM), primarily as a starting point in trying to understand their clinical relevance. From numerical taxonomy (biochemical testing) to 16S rRNA gene sequencing to whole genome sequencing (WGS), our understanding of NTM has also evolved. Novel species are described at a rapid pace, while taxonomical relationships are re-defined in large part due to the accessibility of WGS. The evolutionary course of clonal complexes within species is better known for some NTM and less for others. In contrast with M. tuberculosis, much is left to learn about NTM as a whole.

Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium are independently evolved pathogenic clones of a much broader group of M. avium organisms.

Mycobacterium avium comprises organisms that share the same species designation despite considerable genomic and phenotypic variability. To determine the degree and nature of variability between subspecies and strains of M. avium, we used multilocus sequencing analysis, studying 56 genetically diverse strains of M. avium that included all described subspecies. In total, 8,064 bp of sequence from 10 gene loci were studied, with 205 (2.5%) representing variable positions. The majority (149/205) of these variations were found among M. avium subsp. hominissuis organisms. Recombination was also evident in this subspecies. In contrast, there was comparatively little variability and no evidence of recombination within the pathogenic subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. silvaticum. Phylogenetic analysis showed that M. avium subsp. avium and M. avium subsp. silvaticum strains clustered together on one branch, while a distinct branch defined M. avium subsp. paratuberculosis organisms. Despite the independent origin of these pathogenic subspecies, an analysis of their rates of nonsynonymous (dN) to synonymous (dS) substitutions showed increased dN/dS ratios for both: 0.67 for M. avium subsp. paratuberculosis and 0.50 for M. avium subsp. avium/M. avium subsp. silvaticum, while the value was 0.08 for M. avium subsp. hominissuis organisms. In conclusion, M. avium subsp. hominissuis represents a diverse group of organisms from which two pathogenic clones (M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum) have evolved independently.

Complete Genome Sequence of Mycobacterium chimaera SJ42, a Nonoutbreak Strain from an Immunocompromised Patient with Pulmonary Disease.

Mycobacterium chimaera, a nontuberculous mycobacterium (NTM) belonging to the Mycobacterium avium complex (MAC), is an opportunistic pathogen that can cause respiratory and disseminated disease. We report the complete genome sequence of a strain, SJ42, isolated from an immunocompromised male presenting with MAC pneumonia, assembled from Illumina and Oxford Nanopore data.

Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a containment level-III laboratory as part of a Laboratory Risk Assessment Program.

BACKGROUND: In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating/chemical fixation) may not consistently kill MTB organisms. METHODS: An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. RESULTS: Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80 degrees C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. CONCLUSIONS: This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.

A high proportion of novel mycobacteria species identified by 16S rDNA analysis among slowly growing AccuProbe-negative strains in a clinical setting.

Sequencing of the 16S ribosomal DNA (rDNA) for identification of nontuberculous mycobacteria (NTM) has contributed to the establishment of more than 35 new species during the last decade. Increasingly, NTM are accepted as potential or proven pathogens. We identified, by 16S rDNA sequence analysis, slowly growing NTM isolates negative by AccuProbe (GenProbe, San Diego, CA) that previously were identified by using conventional biochemical techniques, to determine the accuracy of reporting AccuProbe-negative NTM prior to sequence-based identification. Of 82 strains, 30 were deemed novel. An attempt was made to determine the clinical importance of previously misidentified novel species. Clinical cases are described for a number of strains previously identified as Mycobacterium terrae complex, Mycobacterium scrofulaceum, and Mycobacterium avium complex. As sequence-based identification methods become more commonplace in clinical microbiology laboratories, there is a need to understand the significance of previously undescribed species, which often mimic and subsequently are identified as well-established species.

Mycobacterium avium in the postgenomic era.

The past several years have witnessed an upsurge of genomic data pertaining to the Mycobacterium avium complex (MAC). Despite clear advances, problems with the detection of MAC persist, spanning the tests that can be used, samples required for their validation, and the use of appropriate nomenclature. Additionally, the amount of genomic variability documented to date greatly outstrips the functional understanding of epidemiologically different subsets of the organism. In this review, we discuss how postgenomic insights into the MAC have helped to clarify the relationships between MAC organisms, highlighting the distinction between environmental and pathogenic subsets of M. avium. We discuss the availability of various genetic targets for accurate classification of organisms and how these results provide a framework for future studies of MAC variability. The results of postgenomic M. avium study provide optimism that a functional understanding of these organisms will soon emerge, with genomically defined subsets that are epidemiologically distinct and possess different survival mechanisms for their various niches. Although the status quo has largely been to study different M. avium subsets in isolation, it is expected that attention to the similarities and differences between M. avium organisms will provide greater insight into their fundamental differences, including their propensity to cause disease.

Insertion sequence IS900 revisited.

Many studies investigating Mycobacterium avium subsp. paratuberculosis in Crohn's disease have used molecular detection of IS900 in clinical samples, but some have described polymorphisms in IS900 as variants of this organism. Analysis of 23 M. avium subsp. paratuberculosis isolates revealed that IS900 is highly conserved, with only two sequevars distinguishing sheep and cattle lineages. Amplification of IS900-like sequences is not sufficient as a proxy for M. avium subsp. paratuberculosis.

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease.

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.