Whole-Genome Sequences of Mycobacterium abscessus subsp. massiliense Isolates from Brazil.

The Mycobacterium abscessus complex comprises multidrug-resistant, opportunistic, and rapidly growing pathogens responsible for severe infections. Here, we report the genome composition of four Mycobacterium abscessus subsp. massiliense isolates from three sources: two from the lung of a cystic fibrosis patient, one from a mammary cyst, and one from a gutter system.

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.

Multidrug-resistant nontuberculous mycobacteria isolated from cystic fibrosis patients.

Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.

Phenotypic and molecular characterization of quinolone resistance in Mycobacterium abscessus subsp. bolletii recovered from postsurgical infections.

Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) were reported in many Brazilian states (2032 notified cases) from 2004 to 2010. Most of the confirmed cases were mainly associated with Mycobacterium massiliense (recently renamed as Mycobacterium abscessus subsp. bolletii) BRA100 clone, recovered from patients who had undergone invasive procedures in which medical instruments had not been properly sterilized and/or disinfected. Since quinolones have been an option for the treatment of general RGM infections and have been suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of all generations of quinolones for clinical and reference RGM by broth microdilution, and analysed the peptide sequences of the quinolone resistance determining regions (QRDRs) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty-four isolates of M. abscessus subsp. bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDRs, including the Ala-83 in GyrA, and Arg-447 and Asp-464 in GyrB, described as being responsible for an intrinsic low level of resistance to quinolones in mycobacteria. However, other RGM species showed distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

Mycobacterium massiliense BRA100 strain recovered from postsurgical infections: resistance to high concentrations of glutaraldehyde and alternative solutions for high level disinfection / Mycobacterium massiliense clone BRA100 associado a infecções pós-cirúrgicas: resistência a altas concentrações de glutaraldeído e produtos alternativos para desinfecção de alto nível

PURPOSE: To evaluate the minimum inhibitory concentration (MIC) of GTA against these microorganisms and alternative disinfectants for high-level disinfection (HLD). METHODS: Reference mycobacteria and clinical M. massiliense strains were included in this study. Active cultures were submitted to susceptibility qualitative tests with GTA dilutions (ranging from 1.5 percent to 8 percent), and commercial orthophthaldehyde (OPA) and peracetic acid (PA) - based solutions, during the period of exposure as recommended by National Agency of Sanitary Surveillance for HLD. RESULTS: All reference and M. massiliense non-BRA100 strains, recovered from sputum, were susceptible to any GTA concentration, OPA and PA solutions. M. massiliense BRA100 strains presented MIC of 8 percent GTA and were susceptible to OPA and PA. CONCLUSION: M. massiliense BRA100 strain is resistant to high GTA concentrations (up to 7 percent), which proves that this product is non-effective against specific rapidly growing mycobacteria and should be substituted by OPA or PA - based solutions for HLD.

OBJETIVO: Avaliar a concentração mínima inibitória (CMI) de GTA frente a M. massiliense e a susceptibilidade a produtos alternativos para desinfecção de alto nível (DAN). MÉTODOS: Cepas de M. massiliense de origem clínica e de referência foram incluídas no estudo. As culturas ativadas foram submetidas a testes qualitativos com diluições de GTA (de 1,5 por cento a 8 por cento) e com soluções comerciais de ortoftaldeído (OPA) ou ácido peracético (PA), utilizando os tempos de exposição recomendados pela Agência Nacional de Vigilância Sanitária para DAN. RESULTADOS: Todas as cepas de referência e M. massiliense não-BRA100, obtida de escarro, foram susceptíveis às concentrações de GTA, e soluções de OPA e PA. As cepas de M. massiliense BRA100 apresentaram CMI de 8 por cento para GTA e foram susceptíveis a OPA e PA. CONCLUSÃO: M. massiliense BRA100 é resistente a altas concentrações de GTA (até 7 por cento), o que demonstra que esse composto não é eficaz, e deve ser substituído por OPA ou PA nos processos de DAN.

Mycobacterium massiliense BRA100 strain recovered from postsurgical infections: resistance to high concentrations of glutaraldehyde and alternative solutions for high level disinfection.

PURPOSE: To evaluate the minimum inhibitory concentration (MIC) of GTA against these microorganisms and alternative disinfectants for high-level disinfection (HLD). METHODS: Reference mycobacteria and clinical M. massiliense strains were included in this study. Active cultures were submitted to susceptibility qualitative tests with GTA dilutions (ranging from 1.5% to 8%), and commercial orthophthaldehyde (OPA) and peracetic acid (PA)-based solutions, during the period of exposure as recommended by National Agency of Sanitary Surveillance for HLD. RESULTS: All reference and M. massiliense non-BRA100 strains, recovered from sputum, were susceptible to any GTA concentration, OPA and PA solutions. M. massiliense BRA100 strains presented MIC of 8% GTA and were susceptible to OPA and PA. CONCLUSION: M. massiliense BRA100 strain is resistant to high GTA concentrations (up to 7%), which proves that this product is non-effective against specific rapidly growing mycobacteria and should be substituted by OPA or PA-based solutions for HLD.

Epidemic of postsurgical infections caused by Mycobacterium massiliense.

An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most (n = 144; 97.2%) isolates presented a PRA-hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense. Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense. Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC(90)], 8 microg/ml) and clarithromycin (MIC(90), 0.25 microg/ml) but resistance to ciprofloxacin (MIC(90), >or=32 microg/ml), cefoxitin (MIC(90), 128 microg/ml), and doxycycline (MIC(90), >or=64 microg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.