Investigating hospital Mycobacterium chelonae infection using whole genome sequencing and hybrid assembly.

Mycobacterium chelonae is a rapidly growing nontuberculous mycobacterium that is a common cause of nosocomial infections. Here we describe investigation of a possible nosocomial transmission of M. chelonae at the Hospital of the University of Pennsylvania (HUP). M. chelonae strains with similar high-level antibiotic resistance patterns were isolated from two patients who developed post-operative infections at HUP in 2017, suggesting a possible point source infection. The isolates, along with other clinical isolates from other patients, were sequenced using the Illumina and Oxford Nanopore technologies. The resulting short and long reads were hybrid assembled into draft genomes. The genomes were compared by quantifying single nucleotide variants in the core genome and assessed using a control dataset to quantify error rates in comparisons of identical genomes. We show that all M. chelonae isolates tested were highly dissimilar, as indicated by high pairwise SNV values, consistent with environmental acquisition and not a nosocomial point source. Our control dataset determined a threshold for evaluating identity between strains while controlling for sequencing error. Finally, antibiotic resistance genes were predicted for our isolates, and several single nucleotide variants were identified that have the potential to modulated drug resistance.

A description of Mycobacterium chelonae subsp. gwanakae subsp. nov., a rapidly growing mycobacterium with a smooth colony phenotype due to glycopeptidolipids.

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36WT and MOTT68W, were isolated from the sputa of three independent Korean patients co-infected with Mycobacterium yongonense Type II strains. The 16S rRNA gene sequences of all three strains were unique, which were closest to that of Mycobacterium chelonae subsp. bovis KCTC 39630T (99.9 % similarity). Multilocus sequence typing analysis targeting 10 housekeeping genes including hsp65 and rpoB revealed the distinct phylogenetic location of these strains, which were clustered with M. chelonae subsp. chelonae ATCC 35752T and M. chelonae subsp. bovis KCTC 39630T. Phylogenetic analysis based on whole genome sequences revealed a 95.89 % average nucleotide identity (ANI) value with M. chelonae subsp. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, phenotypic characteristics such as a smooth colony morphology and growth inhibition at 37 °C, distinct MALDI-TOF MS profiles of extracted total lipids due to surface glycopeptidolipids, and distinct drug susceptibility profiles further supported the taxonomic characterization of these strains as representing a novel subspecies of Mycobacterium chelonae. Mycobacterium chelonae subsp. gwanakae subsp. nov. is proposed and the type strain is MOTT36WT (=KCTC 29127T=JCM 32454T).

Description of Mycobacterium chelonae subsp. bovis subsp. nov., isolated from cattle (Bos taurus coreanae), emended description of Mycobacterium chelonae and creation of Mycobacterium chelonae subsp. chelonae subsp. nov.

Three rapidly growing mycobacterial strains, QIA-37T, QIA-40 and QIA-41, were isolated from the lymph nodes of three separate Korean native cattle, Hanwoo (Bos taurus coreanae). These strains were previously shown to be phylogenetically distinct but closely related to Mycobacterium chelonae ATCC 35752T by taxonomic approaches targeting three genes (16S rRNA, hsp6 and rpoB) and were further characterized using a polyphasic approach in this study. The 16S rRNA gene sequences of all three strains showed 99.7 % sequence similarity with that of the M. chelonae type strain. A multilocus sequence typing analysis targeting 10 housekeeping genes, including hsp65 and rpoB, revealed a phylogenetic cluster of these strains with M. chelonae. DNA-DNA hybridization values of 78.2 % between QIA-37T and M. chelonae indicated that it belongs to M. chelonae but is a novel subspecies distinct from M. chelonae. Phylogenetic analysis based on whole-genome sequences revealed a 95.44±0.06 % average nucleotide identity (ANI) value with M. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, distinct phenotypic characteristics such as positive growth at 37 °C, at which temperature M. chelonae does not grow, further support the taxonomic status of these strains as representatives of a novel subspecies of M. chelonae. Therefore, we propose an emended description of Mycobacterium chelonae, and descriptions of M. chelonae subsp. chelonae subsp. nov. and M. chelonae subsp. bovis subsp. nov. are presented; strains ATCC 35752T(=CCUG 47445T=CIP 104535T=DSM 43804T=JCM 6388T=NCTC 946T) and QIA-37T (=KCTC 39630T=JCM 30986T) are the type strains of the two novel subspecies.

Multilocus enzyme electrophoresis analysis of rapidly-growing mycobacteria: an alternative tool for identification and typing.

OBJECTIVES: Rapidly growing mycobacteria (RGM) have emerged as important pathogens in clinical settings, associated with esthetic procedures and postsurgical infections, pulmonary infections among cystic fibrosis patients, and other structural pulmonary diseases. Microorganisms belonging to Mycobacterium abscessus-Mycobacterium chelonae and to Mycobacterium fortuitum groups have frequently been associated with outbreaks and various epidemics. In the present study, RGM strains were characterized in order to investigate molecular markers based on proteomic analysis. METHODS: Multilocus enzyme electrophoresis (MLEE) was used for species identification and clonal analysis of RGM recovered from postsurgical wound infections during an epidemic. The study included 30M. abscessus subsp. bolletii clinical isolates, most belonging to the BRA100 clone (epidemic in Rio de Janeiro city), as well as 16 RGM ATCC reference strains. RESULTS: Molecular typing allowed the detection of diversity in the studied population and revealed species-specific isoenzymatic patterns. Additionally, the clonal relationship among M. abscessus subsp. bolletii outbreak isolates, as examined using MLEE, was markedly consistent. CONCLUSIONS: Isoenzymatic characterization was found to be a useful molecular tool to identify RGM species and to determine the relatedness among closely related M. abscessus subsp. bolletii isolates. This may be considered a powerful approach for epidemiological studies on RGM.

Risk Factors for Mycobacterium abscessus subsp. bolletii infection after laparoscopic surgery during an outbreak in Brazil.

OBJECTIVE To identify risk factors related to Mycobacterium abscessus subsp. bolletii infection during an outbreak, associated with laparoscopic surgery and to propose recommendations for preventing new cases. DESIGN A retrospective cohort study. SETTING A private hospital in Manaus, Brazil. PATIENTS A cohort of 222 patients who underwent laparoscopic surgery between July 2009 and August 2010 by a single surgical team. METHODS We collected information about the patients and the surgical procedure using a standard form. We included sex, age, and variables with P≤0.2 in the bivariate analysis in a logistic regression model. Additionally, we reviewed the procedures for reprocessing the laparoscopic surgery equipment, and the strains obtained with culture were identified by molecular methods. RESULTS We recorded 60 (27%) cases of infection. After multivariate analysis, the duration of surgery beyond 1 hour (odds ratio [OR] 2.4; 95% confidence interval [CI] 1.2-4.5), not to have been the first operated patient on a given day (OR, 2.7; 95% CI, 1.4-5.2), and the use of permanent trocar (OR, 2.2; 95% CI, 1.1-4.2) were associated with infection. We observed that the surgical team attempted to sterilize the equipment in glutaraldehyde solution when sanitary authorities had already prohibited it. Eleven strains presented 100% DNA identity with a single strain, known as BRA100 clone. CONCLUSIONS Because contaminated material can act as vehicle for infection, ensuring adequate sterilization processing of video-assisted surgery equipment was crucial to stopping this single clonal outbreak of nonturbeculous mycobacteria in Brazil.

Novel species including Mycobacterium fukienense sp. is found from tuberculosis patients in Fujian Province, China, using phylogenetic analysis of Mycobacterium chelonae/abscessus complex.

OBJECTIVE: To identify the novel species 'Mycobacterium fukienense' sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China. METHODS: Five of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. RESULTS: The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. CONCLUSION: The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex.

Detection and identification of globally distributed mycobacterial fish pathogens in some ornamental fish in India.

Mycobacteriosis is a progressive disease of a wide range of wild and captive, marine and freshwater fish species. Conventional detection of fish Mycobacteria is based on histopathology, culture, and biochemical characteristics. The present study analyzed the occurrence of Mycobacteria in clinically ill ornamental fish of different species, from different places of India. In first group, 60 fish were examined for presence of granulomatous inflammation and acid-fast bacteria. Thirty-eight (63.34 %) fish were positive for granulomatous inflammations. Presences of acid-fast bacteria were detected in 27 (45 %) fish having granulomatous inflammation and in two (3.33 %) fish without granulomatous inflammation. In total, AFB were found in 29 (48.34 %) of the 60 fish examined. In second group, 20 fish having granulomatous inflammation, 12 (60 %) samples were positive using Ziehl-Neelsen (Z-N) staining and 11 (55 %) of them were culture positive. Eight (40 %) samples were Z-N negative but two (10 %) of them were culture positive. In total, 13 (65 %) of the 20 examined fish were culture positive. On the basis of biochemical tests and 16S rRNA sequencing, 13 isolates were identified: five as Mycobacterium fortuitum, five as Mycobacterium gordonae, and three as Mycobacterium chelonae. In comparison of two decontamination methods, 2 % HCl treatment was better than 4 % NaOH treatment. Mycobacteria recovery from decontaminated samples was significantly high on Lowenstein-Jensen medium compared to Middlebrook 7H11 agar and Stonebrink (SB) media. The disease is transmissible from fish to fish and also from fish to human, so the significance of Mycobacteria in ornamental fish should not be overlooked.

The use of rpoB sequence analysis in the differentiation of Mycobacterium abscessus and Mycobacterium chelonae: a critical judgement in cystic fibrosis?

Individuals suffering from fibrocystic disease may acquire non-tuberculous mycobacteria as colonizing or infecting organisms. Mycobacterium abscessus is of particular concern because it may be very difficult to eradicate and may mitigate against lung transplantation. However, this species may be difficult to reliably differentiate from the closely related M. chelonae. We have developed a rapid, low-cost, short sequence-based technique to confirm species identity by analysis of a segment of the RNA Polymerase B (rpoB) gene.

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis for species identification and differentiation of Mycobacterium kansasii and Mycobacterium chelonae-Mycobacterium abscessus group.

OBJECTIVES: The aim of the present study was to identify and differentiate Mycobacterium kansasii and Mycobacterium chelonae-Mycobacterium abscessus group strains isolated from clinical and environmental sources in different countries. METHODS: PCR-restriction analysis of the hsp65 gene (PRA) with automated capillary electrophoresis was applied to the isolates previously identified by conventional biochemical testing and the molecular INNO-LiPA MYCOBACTERIA assay. RESULTS: PRA performed very well in comparison with the two other methods (96.4% concordance). Among 27 M. kansasii isolates, this method detected five genetic types, of which type 1 represented the most common clinical isolate, as it is worldwide. PRA differentiated 29 M. chelonae-M. abscessus group isolates into Mycobacterium immunogenum type 2 (n=13), M. chelonae (n=12), and M. abscessus types 1 (n=1) and 2 (n=1). M. immunogenum was the most frequent (69%) isolate from humans, but only one of 11 cases was clinically significant. M. chelonae was the most commonly (83%) recovered from water. PRA also identified two isolates with hsp65 alleles representing previously unreported patterns. CONCLUSIONS: PRA based on automated capillary electrophoresis is a rapid, simple, and reliable method for the identification and differentiation of both clinically relevant and environmental isolates of M. kansasii and M. chelonae-M. abscessus group.

Mycobacterium chelonae-abscessus complex associated with sinopulmonary disease, Northeastern USA.

Members of the Mycobacterium chelonae-abscessus complex represent Mycobacterium species that cause invasive infections in immunocompetent and immunocompromised hosts. We report the detection of a new pathogen that had been misidentified as M. chelonae with an atypical antimicrobial drug susceptibility profile. The discovery prompted a multicenter investigation of 26 patients. Almost all patients were from the northeastern United States, and most had underlying sinus or pulmonary disease. Infected patients had clinical features similar to those with M. abscessus infections. Taxonomically, the new pathogen shared molecular identity with members of the M. chelonae-abscessus complex. Multilocus DNA target sequencing, DNA-DNA hybridization, and deep multilocus sequencing (43 full-length genes) support a new taxon for these microorganisms. Because most isolates originated in Pennsylvania, we propose the name M. franklinii sp. nov. This investigation underscores the need for accurate identification of Mycobacterium spp. to detect new pathogens implicated in human disease.

Synchronous infection with Mycobacterium chelonae and Paecilomyces in a heart transplant patient.

A 41-year-old male who was 3 years status post heart transplant presented with a 3-month history of painful erythematous nodules and ulcers on his lower legs and right hand. First, Mycobacterium chelonae infection was revealed through several biopsies with molecular sequence analysis, and combination treatment, including clarithromycin, was started. During the treatment, lesions of the legs showed an improvement, but a fluctuant erythematous nodule on the thumb did not respond. Repetitive biopsy from the thumb ultimately identified Paecilomyces species and the patient was treated with itraconazole and terbinafine sequentially. Our case is the first report, to our knowledge, of synchronous infection with non-tuberculous mycobacteria (NTM) and Paecilomyces in a solid organ transplant recipient. Our findings highlight the importance of recognizing cutaneous NTM infections or deep mycoses, as well as the importance of choosing an appropriate treatment.

Characterization of mycobacteria from a major Brazilian outbreak suggests that revision of the taxonomic status of members of the Mycobacterium chelonae-M. abscessus group is needed.

An outbreak of postsurgical infections caused by rapidly growing mycobacteria has been ongoing in Brazil since 2004. The degrees of similarity of the rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both the Mycobacterium massiliense and the M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting identification results. Therefore, an extensive characterization of members of the M. chelonae-M. abscessus group was carried out. The M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii type strains and a subset of clinical isolates were analyzed by biochemical tests, high-performance liquid chromatography, drug susceptibility testing, PCR-restriction enzyme analysis of hsp65 (PRA-hsp65), rpoB, and hsp65 gene sequencing and analysis of phylogenetic trees, DNA-DNA hybridization (DDH), and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (RFLP-16S rRNA). The clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. The results of DDH also confirmed the >70% relatedness of the clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains; and indistinguishable RFLP-16S rRNA patterns were obtained. On the contrary, the separation of clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains from M. chelonae and M. immunogenum was supported by the results of PRA-hsp65, DDH, and RFLP-16S rRNA and by the rpoB and hsp65 phylogenetic trees. Taken together, these results led to the proposition that M. abscessus, M. massiliense, and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and these two subspecies can be distinguished by two different PRA-hsp65 patterns, which differ by a single HaeIII band, and by differences in their rpoB (3.4%) and hsp65 (1.3%) sequences.

Outbreak of nontuberculous mycobacterial subcutaneous infections related to multiple mesotherapy injections.

We describe an outbreak of severe subcutaneous infections due to nontuberculous mycobacteria following mesotherapy. Epidemiological studies and molecular comparisons of Mycobacterium chelonae strains from different patients and the environment suggested that contamination may be associated with inappropriate cleaning of the multiple-injection device with tap water.

Immunoproteomic identification of secretory and subcellular protein antigens and functional evaluation of the secretome fraction of Mycobacterium immunogenum, a newly recognized species of the Mycobacterium chelonae-Mycobacterium abscessus group.

Mycobacterium immunogenum has been associated with occupational pulmonary disease hypersensitivity pneumonitis (HP). The aim of this study was to identify immunogenic proteins (antigens) in this pathogen as a first step toward understanding its virulence factors and role in HP etiology. Immunoproteomic profiling of secreted and subcellular protein fractions using a combination of two-dimensional electrophoresis (2-DE), immunoblotting, and matrix-assisted laser desorption/ionization-Time of flight (MALDI-TOF) led to the identification of 33 immunoreactive proteins, comprising of 4 secretory, 6 cell wall-associated, 11 membranous, and 12 cytosolic proteins. Of these, eight immunoreactive proteins represented homologues of the known mycobacterial antigens, namely heat shock protein GroEL, antigen 85A, elongation factor Tu (EF-Tu), L-asparaginase, polyketide synthase, PE-PGRS, PPE, and superoxide dismutase (SOD). Global functional search revealed that the remaining 25 novel mycobacterial antigens in M. immunogenum showed homology with hypothetical proteins (11 antigens) and other bacterial proteins (14 antigens) with a role in virulence, survival, and/or diverse metabolic functions. To understand immunogenicity of the secretome in M. immunogenum, the major protein spot on the secretome 2D-gel (consisting of multiple secretory antigens such as OtsB and CtpA, among others) was eluted and subjected to functional characterization in terms of induction of innate immune response in murine alveolar macrophages. The secretome eluate caused up-regulation of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-18 and down-regulation of the anti-inflammatory cytokine IL-10, implying a potential of the secreted antigens to cause host immune response underlying the M. immunogenum-induced lung disease HP. This is the first report on identification of antigens in M. immunogenum as well as on the potential of its secretome proteins to induce host response. The identified antigens could have likely roles in virulence and/or diagnosis and serve as potential targets for drug, biocide, and/or vaccine development.

Development of a species-specific colorimetric-PCR assay for detection and species differentiation of Mycobacterium immunogenum and Mycobacterium chelonae and its comparison with quantitative real-time PCR for field metalworking fluids.

Mycobacterium immunogenum and Mycobacterium chelonae are closely related species associated with occupational hypersensitivity pneumonitis (HP) and nosocomial infections. There is a need to develop specific and readily adaptable methods for detection and speciation of these agents. Here we report development of a probe-based colorimetric-PCR assay involving heat shock protein (hsp) gene amplification (228 bp) and its detection in an ELISA-like reaction. A quantitative format of this assay was developed and validated on metalworking fluids (MWF). The assay showed a minimum detection limit of 10 fg genomic DNA or 1 mycobacterial cell, albeit with variations depending on type and composition of the MWF matrix. When applied to the field MWF samples, the developed assay was found to be comparable to the real-time PCR assay, and allowed direct speciation of MWF mycobacteria without sequencing and/or restriction pattern analysis. In conclusion, the developed colorimetric PCR allows detection and quantification of MWF mycobacteria without culturing and is the first probe-based assay for unambiguous differentiation between the two phylogenetically closely related species, M. immunogenum and M. chelonae. Considering that the assay offers high throughput format involving relatively simpler instrument infrastructure, it has a potential for applications in routine assessment of MWF mycobacteria in diagnostic and industrial laboratories.

Molecular typing of Mycobacterium chelonae isolates from a pseudo-outbreak involving an automated bronchoscope washer.

We describe a pseudo-outbreak of Mycobacterium chelonae infection in bronchoalveolar lavage fluid from 9 patients that was traced to contamination of an automated bronchoscope washer. Molecular typing using repetitive extragenic palindromic polymerase chain reaction was helpful in confirming epidemiologic and clinical findings.

Molecular systematics support the revival of Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., a species closely related to Mycobacterium chelonae.

Mycobacterial infections in fish are usually attributed to strains of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium fortuitum. Bacteria identified as M. chelonae have been isolated numerous times from salmonid fishes. Recently, this bacterium has been associated with salmon mortalities in the aquaculture industry. An M. chelonae-like species from salmon, 'Mycobacterium salmoniphilum', was described in 1960. However, the species name lost standing in nomenclature when it was omitted from the 1980 Approved Lists of Bacterial Names because the species could not be distinguished with confidence from M. fortuitum. In the 1980s, mycobacteria isolated from salmon were characterized as a distinct subspecies, 'Mycobacterium chelonae subsp. piscarium'. Again, the uncertainty of the validity of the species resulted in the subsequent withdrawal of the name. Since then, most studies have considered isolates from salmon to be M. chelonae. Nucleotide sequence analysis of the small-subunit rRNA, hsp65 and rpoB genes was used to examine the taxonomic relatedness of type cultures and authentic isolates in our culture collection available from earlier studies. The M. chelonae-like strains from salmon were phylogenetically distinct from other Mycobacterium strains and members of the M. chelonae complex. Moreover, the cell-wall-bound mycolic acids were not representative of known mycolate patterns for M. chelonae-complex organisms. These results supported the status of the species as a separate taxon and effect the valid publication of the name 'M. salmoniphilum' as Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., with the type strain SCT (=ATCC 13578T =DSM 43276T).

Multiple subcutaneous nodules associate with Mycobacterium abscessus infection following removal of an infected Groshon vascular catheter tip.

A 41-year-old man with a history of septic right leg underwent a below-the-knee amputation. One month after amputation and removal of Groshon catheter tip, he developed disseminated subcutaneous abscesses attributed to Mycobacterium abscessus.