Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells.

BACKGROUND: Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection. RESULTS: CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h. CONCLUSION: M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae­M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA­DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA­DNA hybridization results,demonstrated that they share characteristics with M. chelonae­M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).

Demonstration of plasmid-mediated drug resistance in Mycobacterium abscessus.

Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria.

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil.

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Diversity of pulsed-field gel electrophoresis patterns of Mycobacterium abscessus type 2 clinical isolates.

An epidemic of infections by rapidly growing mycobacteria related to surgical procedures between 2004 and 2008 in Brazil was caused by a unique strain showing the Mycobacterium abscessus type 2 pattern when it was analyzed by the molecular method of PCR-restriction enzyme analysis of the hsp65 gene (PRA-hsp65). In order to investigate the diversity of M. abscessus type 2 clinical isolates and to assess whether this epidemic strain was present in specimens from nonsurgical patients, we studied 52 isolates from 38 patients showing this characteristic PRA-hsp65 pattern obtained between 2005 and 2009. All isolates were identified by sequencing of region V of the rpoB gene and typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. Seven isolates obtained from sputum, bronchoalveolar lavage fluid, and urine in three different Brazilian states showed rpoB sequences 100% similar to the rpoB sequence of epidemic strain INCQS 594 and PFGE patterns highly related to the patterns of isolates, evidencing the presence of the epidemic strain in isolates from patients not associated with the surgical epidemic. The remaining isolates showed diverse rpoB sequences, with the highest similarities being to the corresponding sequences of M. massiliense(T) CIP 108297 (21 isolates), M. bolletii(T) CIP 108541 (19 isolates), or M. abscessus(T) ATCC 19977 (5 isolates). Two additional clusters could be detected by PFGE. PFGE showed 100% typeability and reproducibility and discriminatory powers, calculated by Simpson's index of diversity, of 0.978 (DraI) and 0.986 (AseI), confirming its suitability for the discrimination of M. abscessus type 2 isolates.

Identification of the Infection Source of an Outbreak of Mycobacterium Chelonae Keratitis After Laser in Situ Keratomileusis.

PURPOSE: Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes. METHODS: In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections. RESULTS: Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes. CONCLUSIONS: Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

Draft Genome Sequence of Mycobacterium abscessus subsp. bolletii INCQS 00594.

An epidemic of surgical-site infections by a single strain of Mycobacterium abscessus subsp. bolletii affected >1,700 patients in Brazil from 2004 to 2008. The genome of the epidemic prototype strain M. abscessus subsp. bolletii INCQS 00594, deposited in the collection of the National Institute for Health Quality Control (INCQS), was sequenced.

The detection and sequencing of a broad-host-range conjugative IncP-1ß plasmid in an epidemic strain of Mycobacterium abscessus subsp. bolletii.

BACKGROUND: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,267-bp [corrected] circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1ß and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2)155 could not be demonstrated. CONCLUSIONS/SIGNIFICANCE: The occurrence of a broad-host-range IncP-1ß plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.

Correction: The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1ß Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp. bolletii.

[This corrects the article on p. e60746 in vol. 8.].

First description of natural and experimental conjugation between Mycobacteria mediated by a linear plasmid.

BACKGROUND: In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here. METHODOLOGY/PRINCIPAL FINDINGS: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau. CONCLUSIONS/SIGNIFICANCE: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.

Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil.

AIM: Our aim is to investigate if the clusters of postsurgical mycobacterial infections, reported between 2004 and 2008 in seven geographically distant states in Brazil, were caused by a single mycobacterial strain. MATERIALS & METHODS: Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison. RESULTS: All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns. CONCLUSION: A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3 region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100 per cent, 100 per cent and 92.31 per cent sensitivity and 93.06 per cent, 90.28 per cent and 100 per cent specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.(AU)

Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil

Aim: Our aim is to investigate if the clusters of postsurgical mycobacterialinfections, reported between 2004 and 2008 in seven geographically distantstates in Brazil, were caused by a single mycobacterial strain.MATERIALS & METHODS: Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison.RESULTS: All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns.CONCLUSION: A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.AU

Microsatellite variation and population genetic structure of a neotropical endangered Bryconinae species Brycon insignis Steindachner, 1877: implications for its conservation and sustainable management

Piabanha (Brycon insignis) is a freshwater fish species from the drainages in Southeastern Brazil. During the 1950s, it was an important economic and food resource for local populations, but dramatic and continuous environmental degradation seriously jeopardized the B. insignis populations in the region. Microsatellite markers were used to assess the genetic structure of wild populations of B. insignis and compare the genetic variability and integrity of the wild populations with a captive population. Samples of DNA from 208 specimens from geographically isolated populations were analyzed. Population genetic structure was investigated using F ST, R ST estimates as well as AMOVA. All five loci used in this study were polymorphic with observed heterozygosity ranging from 0.77 (± 0.15) to 0.88 (± 0.07) in the wild population and 0.90 (± 0.09) in the captive population and the allelic richness average were 7.56 (± 0.27) and 5.80 (± 1.02), respectively. Overall genetic differences were significantly partitioned among populations (F ST = 0.072, p = 0.034). Evidence of a genetic bottleneck was found in some of the wild populations, but especially in the captive population. The results showed that genetic variability still can be found in B. insignis populations which are currently structured possibly due to anthropic actions. The implications of these findings for the management and conservation of B. insignis populations are discussed.(AU)

Piabanha (Brycon insignis) é uma espécie de peixe de água doce oriunda de drenagens da região sudeste do Brasil. Durante os anos de 1950, esta espécie foi um importante recurso econômico para populações locais. Contudo, a intensa e contínua degradação ambiental afetou seriamente as populações de B. insignis na região. Marcadores microssatélites foram usados para avaliar a estrutura genética de populações selvagens de B. insignis e comparar a variabilidade genética e integridade das populações selvagens com uma população de cativeiro. Amostras de DNA de 208 espécimes de populações geograficamente isoladas foram analisadas. A estrutura populacional foi investigada usando-se estimadores de F ST e R ST bem como AMOVA. Todos os loci usados neste estudo foram polimórficos com heterozigosidades observadas variando de 0.77 (± 0.15) a 0.88 (± 0.77) em populações selvagens e 0.90 (± 0.09) na população de cativeiro e a riqueza alélica média foi de 7.56 (± 0.27) e 5.80 (± 1.02), respectivamente. A maioria das diferenças genéticas foi significativa entre populações (F ST = 0.072, p = 0.034). Evidências de efeito gargalo foram observadas em algumas populações selvagens e especialmente também na população de cativeiro. Os resultados do presente estudo mostraram que as populações de B. insignis ainda apresentam variabilidade genética e que estas populações estão atualmente estruturadas geneticamente provavelmente devido a ações antrópicas. As implicações destes achados para o manejo e conservação das populações de B. insiginis são discutidas.(AU)

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

The genus Brycon, the largest subunit of the Bryconinae, has 42 valid species distributed from southern Mexico to the La Plata River in Argentina. Henochilus is a monotypic genus, comprising a single species (H. wheatlandii) found in the upper Rio Doce basin. In the present study, partial sequences of the mitochondrial gene 16S were obtained for fifteen species of Brycon and for Henochilus wheatlandii. The results showed that the genus Brycon is paraphyletic, since Henochilus is the sister-group of B. ferox and B. insignis. The most basal species analyzed were the trans-Andean species B. henni, B. petrosus, and B. chagrensis.