Developing Tadpole Xenopus laevis as a Comparative Animal Model to Study Mycobacterium abscessus Pathogenicity.

Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus.

Identification of Mycobacterium abscessus species and subspecies using the Cas12a/sgRNA-based nucleic acid detection platform.

The rapidly growing mycobacterium Mycobacterium abscessus is a clinically important organism causing pulmonary and skin diseases. The M. abscessus complex is comprised of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Here, we aimed to develop a Cas12a/sgRNA-based nucleic acid detection platform to identify M. abscessus species and subspecies. By designing specific sgRNA probes targeting rpoB and erm(41), we demonstrated that M. abscessus could be differentiated from other major mycobacterial species and identified at the subspecies level. Using this platform, a total of 38 clinical M. abscessus isolates were identified, 18 as M. abscessus subsp. abscessus and 20 as M. abscessus subsp. massiliense. We concluded that the Cas12a/sgRNA-based nucleic acid detection platform provides an easy-to-use, quick, and cost-effective approach for identification of M. abscessus species and subspecies.

Outcome according to subspecies following lung transplantation in cystic fibrosis pediatric patients infected with Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus infection has been associated with variable outcomes following lung transplantation. M abscessus comprises three subspecies (M abscessus subsp abscessus, M abscessus subsp massiliense, and M abscessus subsp bolletii). We investigated whether lung transplantation outcome in cystic fibrosis (CF) patients in a single center was related to the M abscessus subspecies and genetic cluster. METHODS: CF patients with chronic M abscessus infection transplanted at Great Ormond Street Hospital between 2004 and 2017 were retrospectively examined. All M abscessus isolates were identified to subspecies level by polymerase chain reaction and sequencing. Genetic cluster was determined by variable number tandem repeat profiling and whole-genome sequencing (WGS), and sequence type inferred from WGS. RESULTS: Thirteen patients with chronic M abscessus infection underwent heart/lung or lung transplantation. Subspecies identification showed n = 1 with M abscessus bolletii, n = 5 with M abscessus massiliense, and n = 7 with M abscessus abscessus infection. Eight (62%) patients (one with M abscessus massiliense and seven with M abscessus abscessus) died post-lung transplant. The patient with M abscessus bolletii and three patients with M abscessus massiliense did well post-transplant. One patient with M abscessus massiliense is receiving ongoing treatment. CONCLUSIONS: Dramatically worse outcomes are observed in patients infected with M abscessus subspecies abscessus, the majority of whom were infected with ST-1 and ST-26 strains. Patients infected with other M abcsessus strains can have acceptable outcomes.

[Mycobacterium abscessus complex representatives in patients with bronchopulmonary pathology: prevalence, peculiarities of cultivation and identification.]

More and more publications appear in the modern literature on the increase in the prevalence of non-tuberculous mycobacteria (NTMs), in particular, representatives of M. chelonae / Mycobacterium abscessus complex (MABSc). The paper presents data on the current classification of M. chelonae / Mycobacterium abscessus complex and its main representatives. The main data on the possible sources and ways of infection of MABSc patients in hospital are presented. The main features of cultivation on various nutrient media and their possible identification using modern methods are also indicated. The main risk factors for the development of mycobacteriosis in patients and the possible clinical picture are described. The prevalence of MABSc representatives in the structure of non-tuberculous mycobacteria isolated from clinical material from 483 patients from the Samara region was assessed for examination for tuberculosis, and the prevalence from 933 patients with cystic fibrosis (CF) from 55 regions of the Russian Federation from 2016 to 2019 was estimated. In total, as a result of the study, 316 NTM strains (65.4%) were isolated and identified in the first group of patients. M.abscessus was isolated and identified 10 strains and 5 strains - M.chelonae, which amounted to 3.2% and 1.6%, respectively, of all NTMs. In general, MABSc representatives were isolated in 3.1% of the examined patients. As a result of a screening study of patients with CF, 14194 microorganism strains from 933 patients were isolated and identified. Altogether M. abscessus was isolated and confirmed from 14 patients of different ages. Thus, the prevalence of MABSc among the examined patients with CF in the Russian Federation was 1.5%.

Thiostrepton: A Novel Therapeutic Drug Candidate for Mycobacterium abscessus Infection.

Mycobacterium abscessus is a rapid-growing, multidrug-resistant, non-tuberculous mycobacterial species responsible for a variety of human infections, such as cutaneous and pulmonary infections. M. abscessus infections are very difficult to eradicate due to the natural and acquired multidrug resistance profiles of M. abscessus. Thus, there is an urgent need for the development of effective drugs or regimens against M. abscessus infections. Here, we report the activity of a US Food and Drug Administration approved drug, thiostrepton, against M. abscessus. We found that thiostrepton significantly inhibited the growth of M. abscessus wild-type strains, subspecies, clinical isolates, and drug-resistant mutants in vitro and in macrophages. In addition, treatment of macrophages with thiostrepton significantly decreased proinflammatory cytokine production in a dose-dependent manner, suggesting an inhibitory effect of thiostrepton on inflammation induced during M. abscessus infection. We further showed that thiostrepton exhibits antimicrobial effects in vivo using a zebrafish model of M. abscessus infection.

Two-Phase Hospital-Associated Outbreak of Mycobacterium abscessus: Investigation and Mitigation.

BACKGROUND: Nontuberculous mycobacteria (NTM) commonly colonize municipal water supplies and cause healthcare-associated outbreaks. We investigated a biphasic outbreak of Mycobacterium abscessus at a tertiary care hospital. METHODS: Case patients had recent hospital exposure and laboratory-confirmed colonization or infection with M. abscessus from January 2013 through December 2015. We conducted a multidisciplinary epidemiologic, field, and laboratory investigation. RESULTS: The incidence rate of M. abscessus increased from 0.7 cases per 10000 patient-days during the baseline period (January 2013-July 2013) to 3.0 cases per 10000 patient-days during phase 1 of the outbreak (August 2013-May 2014) (incidence rate ratio, 4.6 [95% confidence interval, 2.3-8.8]; P < .001). Thirty-six of 71 (51%) phase 1 cases were lung transplant patients with positive respiratory cultures. We eliminated tap water exposure to the aerodigestive tract among high-risk patients, and the incidence rate decreased to baseline. Twelve of 24 (50%) phase 2 (December 2014-June 2015) cases occurred in cardiac surgery patients with invasive infections. Phase 2 resolved after we implemented an intensified disinfection protocol and used sterile water for heater-cooler units of cardiopulmonary bypass machines. Molecular fingerprinting of clinical isolates identified 2 clonal strains of M. abscessus; 1 clone was isolated from water sources at a new hospital addition. We made several water engineering interventions to improve water flow and increase disinfectant levels. CONCLUSIONS: We investigated and mitigated a 2-phase clonal outbreak of M. abscessus linked to hospital tap water. Healthcare facilities with endemic NTM should consider similar tap water avoidance and engineering strategies to decrease risk of NTM infection.

Antimicrobial resistance in Mycobacterium abscessus complex isolated from patients with skin and soft tissue infections at a tertiary teaching hospital in Taiwan.

Background: Mycobacterium abscessus complex (MABC) is the most common non-tuberculous mycobacterium that causes complicated skin and soft tissue infections (cSSTIs). The selection of antimycobacterial agents for successful treatment of such infections is a critical issue. Objectives: To investigate the antimicrobial susceptibility patterns of MABC isolates from skin and soft tissue to a variety of antimycobacterial agents. Methods: Sixty-seven MABC isolates were collected and partial gene sequencing of secA1, rpoB and hsp65 was used to classify them into three subspecies: M. abscessus subsp. abscessus (MAB), M. abscessus subsp. massiliense (MMA) and M. abscessus subsp. bolletii (MBO). The MICs of 11 antimycobacterial agents for these 67 isolates were determined using a broth microdilution method and commercial Sensititre RAPMYCOI MIC plates, as recommended by CLSI. Results: In total, 28 MAB, 38 MMA and 1 MBO were isolated from patients with cSSTIs at our hospital. Most MABC strains were resistant to ciprofloxacin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin and trimethoprim/sulfamethoxazole. In addition, most MABC strains were intermediately susceptible or resistant to cefoxitin. Eighteen of the 28 MABs and 1 MBO isolate harboured the T28 polymorphism in the erm(41) gene. Two of the 38 MMA isolates had an rrl A2059G point mutation. Most of the MABC strains were susceptible to amikacin and tigecycline. Conclusions: In Taiwan, amikacin, clarithromycin and tigecycline have good activity against MMA and MAB erm(41) C28 sequevar isolates, whereas amikacin and tigecycline, rather than clarithromycin, have good activity against both MBO and MAB erm(41) T28 sequevar isolates. Clinical trials are warranted to correlate these data with clinical outcomes.

Dissemination of Mycobacterium abscessus via global transmission networks.

Mycobacterium abscessus, a multidrug-resistant nontuberculous mycobacterium, has emerged as a major pathogen affecting people with cystic fibrosis (CF). Although originally thought to be acquired independently from the environment, most individuals are infected with one of several dominant circulating clones (DCCs), indicating the presence of global transmission networks of M. abscessus. How and when these clones emerged and spread globally is unclear. Here, we use evolutionary analyses of isolates from individuals both with and without CF to reconstruct the population history, spatiotemporal spread and recent transmission networks of the DCCs. We demonstrate synchronous expansion of six unrelated DCCs in the 1960s, a period associated with major changes in CF care and survival. Each of these clones has spread globally as a result of rare intercontinental transmission events. We show that the DCCs, but not environmentally acquired isolates, exhibit a specific smoking-associated mutational signature and that current transmission networks include individuals both with and without CF. We therefore propose that the DCCs initially emerged in non-CF populations but were then amplified and spread through the CF community. While individuals with CF are probably the most permissive host, non-CF individuals continue to play a key role in transmission networks and may facilitate long-distance transmission.

Genome reorganization during emergence of host-associated Mycobacterium abscessus.

Mycobacterium abscessus is a rapid growing, free-living species of bacterium that also causes lung infections in humans. Human infections are usually acquired from the environment; however, dominant circulating clones (DCCs) have emerged recently in both M. abscessus subsp. massiliense and subsp. abscessus that appear to be transmitted among humans and are now globally distributed. These recently emerged clones are potentially informative about the ecological and evolutionary mechanisms of pathogen emergence and host adaptation. The geographical distribution of DCCs has been reported, but the genomic processes underlying their transition from environmental bacterium to human pathogen are not well characterized. To address this knowledge gap, we delineated the structure of M. abscessus subspecies abscessus and massiliense using genomic data from 200 clinical isolates of M. abscessus from seven geographical regions. We identified differences in overall patterns of lateral gene transfer (LGT) and barriers to LGT between subspecies and between environmental and host-adapted bacteria. We further characterized genome reorganization that accompanied bacterial host adaptation, inferring selection pressures acting at both genic and intergenic loci. We found that both subspecies encode an expansive pangenome with many genes at rare frequencies. Recombination appears more frequent in M. abscessus subsp. massiliense than in subsp. abscessus, consistent with prior reports. We found evidence suggesting that phage are exchanged between subspecies, despite genetic barriers evident elsewhere throughout the genome. Patterns of LGT differed according to niche, with less LGT observed among host-adapted DCCs versus environmental bacteria. We also found evidence suggesting that DCCs are under distinct selection pressures at both genic and intergenic sites. Our results indicate that host adaptation of M. abscessus was accompanied by major changes in genome evolution, including shifts in the apparent frequency of LGT and impacts of selection. Differences were evident among the DCCs as well, which varied in the degree of gene content remodelling, suggesting they were placed differently along the evolutionary trajectory toward host adaptation. These results provide insight into the evolutionary forces that reshape bacterial genomes as they emerge into the pathogenic niche.

A novel DNA chromatography method to discriminate Mycobacterium abscessus subspecies and macrolide susceptibility.

BACKGROUND: The clinical impact of infection with Mycobacterium (M.) abscessus complex (MABC), a group of emerging non-tuberculosis mycobacteria (NTM), is increasing. M. abscessus subsp. abscessus/bolletii frequently shows natural resistance to macrolide antibiotics, whereas M. abscessus subsp. massiliense is generally susceptible. Therefore, rapid and accurate discrimination of macrolide-susceptible MABC subgroups is required for effective clinical decisions about macrolide treatments for MABC infection. We aimed to develop a simple and rapid diagnostic that can identify MABC isolates showing macrolide susceptibility. METHODS: Whole genome sequencing (WGS) was performed for 148 clinical or environmental MABC isolates from Japan to identify genetic markers that can discriminate three MABC subspecies and the macrolide-susceptible erm(41) T28C sequevar. Using the identified genetic markers, we established PCR based- or DNA chromatography-based assays. Validation testing was performed using MABC isolates from Taiwan. FINDING: We identified unique sequence regions that could be used to differentiate the three subspecies. Our WGS-based phylogenetic analysis indicated that M. abscessus carrying the macrolide-susceptible erm(41) T28C sequevar were tightly clustered, and identified 11 genes that were significantly associated with the lineage for use as genetic markers. To detect these genetic markers and the erm(41) locus, we developed a DNA chromatography method that identified three subspecies, the erm(41) T28C sequevar and intact erm(41) for MABC in a single assay within one hour. The agreement rate between the DNA chromatography-based and WGS-based identification was 99·7%. INTERPRETATION: We developed a novel, rapid and simple DNA chromatography method for identification of MABC macrolide susceptibility with high accuracy. FUNDING: AMED, JSPS KAKENHI.

Clinical Whole Genome Sequencing for Clarithromycin and Amikacin Resistance Prediction and Subspecies Identification of Mycobacterium abscessus.

Mycobacterium abscessus infections are an emerging health care concern in patients with chronic pulmonary diseases, leading to high morbidity and mortality. One major challenge is resistance to clarithromycin, a cornerstone antibiotic with high efficacy. Therefore, treatment is primarily guided by phenotypic susceptibility results of clarithromycin, which requires extended incubation to assess for inducible resistance. Resistance mechanisms for clarithromycin include induction of erm(41) and mutations in the 23S rRNA gene (rrl). In addition, mutations in the 16S rRNA encoding gene (rrs) can confer high-level amikacin resistance, another essential drug in the treatment of M. abscessus infections. Herein, we developed a clinical whole genome sequencing (WGS) assay for clarithromycin resistance based on rrl and erm(41) gene sequences and amikacin resistance based on the rrs sequence in M. abscessus, as well as subspecies identification. Genotypic-based predictions were determined for 104 isolates from 68 patients. The overall accuracy of genotypic prediction for clarithromycin compared with phenotypic susceptibility results was 100% (95% CI, 96.45%-100%). For amikacin, we also obtained 100% accuracy (95% CI, 96.52%-100%). The high concordance between the genotypic and phenotypic results demonstrates that a WGS-based assay can be used in a clinical laboratory for determining resistance to clarithromycin and amikacin in M. abscessus isolates. WGS can also provide subspecies identification and high-definition phylogenetic information for more accurate M. abscessus strain typing.

Global phylogenomic analyses of Mycobacterium abscessus provide context for non cystic fibrosis infections and the evolution of antibiotic resistance.

Mycobacterium abscessus (MAB) is an emerging pathogen that leads to chronic lung infections. To date, the global population structure of non-cystic fibrosis (CF) MAB and evolutionary patterns of drug resistance emergence have not been investigated. Here we construct a global dataset of 1,279 MAB whole genomes from CF or non-CF patients. We utilize whole genome analysis to assess relatedness, phylogeography, and drug resistance evolution. MAB isolates from CF and non-CF hosts are interspersed throughout the phylogeny, such that the majority of dominant circulating clones include isolates from both populations, indicating that global spread of MAB clones is not sequestered to CF contexts. We identify a large clade of M. abscessus harboring the erm(41) T28C mutation, predicted to confer macrolide susceptibility in this otherwise macrolide-resistant species. Identification of multiple evolutionary events within this clade, consistent with regain of wild type, intrinsic macrolide resistance, underscores the critical importance of macrolides in MAB.

The Prophage and Plasmid Mobilome as a Likely Driver of Mycobacterium abscessus Diversity.

Mycobacterium abscessus is an emerging pathogen that is often refractory to antibiotic control. Treatment is further complicated by considerable variation among clinical isolates in both their genetic constitution and their clinical manifestations. Here, we show that the prophage and plasmid mobilome is a likely contributor to this variation. Prophages and plasmids are common, abundant, and highly diverse, and code for large repertoires of genes influencing virulence, antibiotic susceptibility, and defense against viral infection. At least 85% of the strains we describe carry one or more prophages, representing at least 17 distinct and diverse sequence "clusters," integrated at 18 different attB locations. The prophages code for 19 distinct configurations of polymorphic toxin and toxin-immunity systems, each with WXG-100 motifs for export through type VII secretion systems. These are located adjacent to attachment junctions, are lysogenically expressed, and are implicated in promoting growth in infected host cells. Although the plethora of prophages and plasmids confounds the understanding of M. abscessus pathogenicity, they also provide an abundance of tools for M. abscessus engineering.IMPORTANCEMycobacterium abscessus is an important emerging pathogen that is challenging to treat with current antibiotic regimens. There is substantial genomic variation in M. abscessus clinical isolates, but little is known about how this influences pathogenicity and in vivo growth. Much of the genomic variation is likely due to the large and varied mobilome, especially a large and diverse array of prophages and plasmids. The prophages are unrelated to previously characterized phages of mycobacteria and code for a diverse array of genes implicated in both viral defense and in vivo growth. Prophage-encoded polymorphic toxin proteins secreted via the type VII secretion system are common and highly varied and likely contribute to strain-specific pathogenesis.

Genomic characterization of sporadic isolates of the dominant clone of Mycobacterium abscessus subspecies massiliense.

Recent studies have characterized a dominant clone (Clone 1) of Mycobacterium abscessus subspecies massiliense (M. massiliense) associated with high prevalence in cystic fibrosis (CF) patients, pulmonary outbreaks in the United States (US) and United Kingdom (UK), and a Brazilian epidemic of skin infections. The prevalence of Clone 1 in non-CF patients in the US and the relationship of sporadic US isolates to outbreak clones are not known. We surveyed a reference US Mycobacteria Laboratory and a US biorepository of CF-associated Mycobacteria isolates for Clone 1. We then compared genomic variation and antimicrobial resistance (AMR) mutations between sporadic non-CF, CF, and outbreak Clone 1 isolates. Among reference lab samples, 57/147 (39%) of patients with M. massiliense had Clone 1, including pulmonary and extrapulmonary infections, compared to 11/64 (17%) in the CF isolate biorepository. Core and pan genome analyses revealed that outbreak isolates had similar numbers of single nucleotide polymorphisms (SNPs) and accessory genes as sporadic US Clone 1 isolates. However, pulmonary outbreak isolates were more likely to have AMR mutations compared to sporadic isolates. Clone 1 isolates are present among non-CF and CF patients across the US, but additional studies will be needed to resolve potential routes of transmission and spread.

A single-gene approach for the subspecies classification of Mycobacteroides abscessus.

The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.

Serum immunoglobulin a antibodies to glycopeptidolipid core antigen for Mycobacteroides abscessus complex lung disease.

Background: Mycobacteroides abscessus complex (MABC) exhibits smooth morphotypes, expressing glycopeptidolipid (GPL), and rough morphotypes, expressing diminished GPL, on the MABC cell wall. Few reports have focused on the relationship between anti-GPL-core immunoglobulin A (IgA) antibody and colony morphology in MABC lung disease. Methods: This study aimed to test GPL core antigen in patients with MABC lung disease to investigate the relationship between coinfection/contamination in other nontuberculous mycobacteria species and colony morphology variant in MABC isolates. Patients with MABC lung disease and contamination diagnosed between 2012 and 2017 at our hospital were enrolled retrospectively. Results: Of the assessed patients, 43 patients with MABC lung disease and 13 with MABC contamination were included. There was a significant difference in anti-GPL-core IgA antibody levels between them (P = 0.02). Forty-three patients with MABC lung disease were divided into two groups as positive and negative antibodies groups. A significant increase in the positive anti-GPL-core IgA antibody was observed in coexistence with both Mycobacterium avium complex (MAC) (P = 0.02) and the isolate of the smooth variant (P = 0.03) in MABC. Conclusions: Anti-GPL-core IgA antibodies in patients with MABC are greatly influenced by MAC coexistence, and colony morphology variant of the MABC isolate.

Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in Mycobacterium abscessus.

Background: Mycobacterium abscessus is notorious for being intrinsically resistant to most antibiotics. Antibiotic efflux is one of the mechanisms used by M. abscessus to pump out antibiotics from their cells. Inhibiting efflux pumps (EPs) can be an attractive strategy to enhance the activity of drugs. The objective of this study is to determine the activity of EP inhibitors (EPIs) to enhance the efficacy of the new drug bedaquiline against M. abscessus clinical isolates. Methods: A total of 31 phenotypically and genotypically identified M. abscessus subsp. abscessus, M. abscesss subsp. massiliense, and M. abscessus subsp. bolletii clinical isolates were studied. The contribution of EPs was determined by investigating the minimum inhibitory concentration (MIC) levels of bedaquiline reduction in the absence and presence of EPIs verapamil and reserpine using the resazurin microtiter assay. Results: The observed bedaquiline MIC reduction by verapamil was observed in 100% isolates and by reserpine in 54.8% isolates. Bedaquiline MIC was 4-32-fold using verapamil with M. abscessus subsp. bolletii showing the highest fold change and between 2- and 4-fold using reserpine. Conclusions: The results obtained in this study confirm that bedaquiline MIC decreased in the presence of EPIs verapamil and reserpine in clinical isolates of M. abscessus. Verapamil was the most effective EPI. As shown in previous studies, verapamil may have clinical potential as adjunctive therapy to enhance the effect of bedaquiline.

A multilocus sequence typing scheme for Mycobacterium abscessus complex (MAB-multilocus sequence typing) using whole-genome sequencing data.

Background: Mycobacterium abscessus is a rapid growing nontuberculous mycobacteria (NTM) and a clinically significant pathogen capable of causing variable infections in humans that are difficult to treat and may require months of therapy/surgical interventions. Like other NTMs, M. abscessus can be associated with outbreaks leading to complex investigations and treatment of affected cases. Typing schemes for bacterial pathogens provide numerous applications; including identifying chain of transmission and tracking genomic evolution, are lacking or limited for many NTMs including M. abscessus. Methods: We extended the existing scheme from PubMLST using whole-genome data for M. abscessus by extracting data for 15 genetic regions within the M. abscessus genome. A total of 168 whole genomes and 11 gene sequences were used to build this scheme (MAB-multilocus sequence typing [MLST]). Results: All seven genes from the PubMLST scheme, namely argH, cya, gnd, murC, pta, purH, and rpoB, were expanded by 10, 14, 13, 10, 13, 10, and 9 alleles, respectively. Another eight novel genes were added including hsp 65, erm(41), arr, rrs, rrl, gyrA, gyrB, and recA with 16, 16, 25, 7, 32, 35, 29, and 15 alleles, respectively, with 85 unique sequence types identified among all isolates. Conclusion: MAB-MLST can provide identification of M. abscessus complex to the subspecies level based on three genes and can provide antimicrobial resistance susceptibility prediction based on results from seven genes. MAB-MLST generated a total of 85 STs, resulting in subtyping of 90 additional isolates that could not be genotyped using PubMLST and yielding results comparable to whole-genome sequencing (WGS). Implementation of a Galaxy-based data analysis tool, MAB-MLST, that simplifies the WGS data and yet maintains a high discriminatory index that can aid in deciphering an outbreak has vast applicability for routine diagnostics.

New Mycobacteroides abscessus subsp. massiliense strains with recombinant hsp65 gene laterally transferred from Mycobacteroides abscessus subsp. abscessus: Potential for misidentification of M. abscessus strains with the hsp65-based method.

It has been reported that lateral gene transfer (LGT) events among Mycobacteroides abscessus strains are prevalent. The hsp65 gene, a chronometer gene for bacterial phylogenetic analysis, is resistant to LGT events, particularly among mycobacterial strains, rendering the hsp65-targeting method the most widely used method for mycobacterial detection. To determine the prevalence of M. abscessus strains that are subject to hsp65 LGT, we applied rpoB typing to 100 clinically isolated Korean strains of M. abscessus that had been identified by hsp65 sequence analysis. The analysis indicated the presence of 2 rough strains, showing a discrepancy between the 2 typing methods. MLST analysis based on the partial sequencing of seven housekeeping genes, erm(41) PCR and further hsp65 PCR-restriction enzyme and polymorphism analysis (PRA) were conducted to identify the two strains. The MLST results showed that the two strains belong to M. abscessus subsp. massiliense and not to M. abscessus subsp. abscessus, as indicated by the rpoB-based analysis, suggesting that their hsp65 genes are subject to LGT from M. abscessus subsp. abscessus. Further analysis of these strains using the hsp65 PRA method indicated that these strains possess a PRA pattern identical to that of M. abscessus subsp. abscessus and distinct from that of M. abscessus subsp. massiliense. In conclusion, we identified two M. abscessus subsp. massiliense rough strains from Korean patients with hsp65 genes that might be laterally transferred from M. abscessus subsp. abscessus. To the best of our knowledge, this is the first demonstration of possible LGT events associated with the hsp65 gene in mycobacteria. Our results also suggest that there is the potential for misidentification when the hsp65-based protocol is used for mycobacterial identification.

Current significance of the Mycobacterium chelonae-abscessus group.

Organisms of the Mycobacterium chelonae-abscessus group can be significant pathogens in humans. They produce a number of diseases including acute, invasive and chronic infections, which may be difficult to diagnose correctly. Identification among members of this group is complicated by differentiating at least eleven (11) known species and subspecies and complexity of identification methodologies. Treatment of their infections may be problematic due to their correct species identification, antibiotic resistance, their differential susceptibility to the limited number of drugs available, and scarcity of susceptibility testing.