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.

Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

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.

Identificação de Mycobacterium bovis em cepas micobacterianas isoladas espécimes clínicos humanos em um complexo hospitalar na cidade do Rio de Janeiro / Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil

Entre 2005 e 2006, 8.121 espécimes clínicos enviados ao Laboratório de Micobactérias do Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax, no Rio de Janeiro, RJ, foram inoculados em meio Löwenstein-Jensen contendo glicerol e piruvato. Desses espécimes, 79 isolados de micobactérias tiveram crescimento somente em meio com piruvato, sendo selecionados para a identificação presuntiva de Mycobacterium bovis. Esses isolados foram submetidos à identificação por testes bioquímicos, amplificação por PCR com primers específicos (Rv0577 e Rv1510) e teste de suscetibilidade à pirazinamida. Todas as cepas apresentaram padrões fenotípicos e genotípicos de M. tuberculosis, não sendo detectado M. bovis.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

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.

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center, Rio de Janeiro, Brazil, in pre highly active antiretroviral therapy era.

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center, Rio de Janeiro, Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85 percent of the patients and NTM in 15 percent. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8 percent (49/83) of NTM isolates, most of them from sterile sites (75.5 percent), and in 94 percent (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99 percent of MAC are urease-negative), however IS1245 was detected in 96 percent of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2 percent), followed by M. terrae (3.6 percent), M. gordonae (2.4 percent), M. chelonae (1.2 percent), M. fortuitum (1.2 percent) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8 percent). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods

Multidrug-resistant tuberculosis among male inmates in Rio de Janeiro, Brazil

Susceptibility tests to six anti-tuberculosis drugs were performed on fifty-eight M. tuberculosis isolates obtained from tuberculous inmates in the Male Penal Sanatorium, Rio de Janeiro, Brazil. The rate of resistant tuberculosis was higher than observed in the community. The overall resistance rate was 17.2 (per cent) and 3.4 (per cent) of the isolates were multi-drug resistant.

Statins increase rifampin mycobactericidal effect

Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.

Importância do método de conservaçåo de leveduras usadas como medicamento / On the importance of the conservation method of yeasts used as medicine

Foi determinado o número de células viáveis de duas preparaçöes farmacêuticas comerciais diferentes: uma contendo Saccharomyces boulardii 17 liofiloizado e a outra contendo Saccharomyces cerevisiae FR 1972 em suspensåo em meio tam,ponado. O produto liofilizado manteve o número original de células viáveis, enquanto que a formulaçåo em meio tamponado apresentou, em média, número de células viáveis 40 vezes menor que o declarado pelo fabricante (1,6 log). Esses resultados confirmam a importância da liofilizaçåo como método de conservaçåo de formulaçöes farmacêuticas contendo microorganismos

Susceptibilidade do Vibrio cholerae a diferentes desinfetantes / Sensitivity of Vibrio cholerae to different disinfectants

A ação bactericida de diversos desinfetantes (glutaraldeído, hipoclorito de sódio, formaldeído, quaternários de amônio e fenóis sintéticos) para o Vibrio cholerae foi confirmada através das técnicas de diluição de uso (método qualitativo) e 5,5,5, (método quantitativo)

Determinaçäo da concentraçäo mínima inibitória de oito quimioterápicos para cepas do complexo M avium isoladas de pacientes portadores de SIDA no Brasil / Determination of minimal inhibitory concentration of eight chemotherapics for M avium complex strains

Neste trabalho foi testada a suscetibilidade de amostras de Mycobacterium avium isoladas de pacientes brasileiros frente a diversos agentes antimicobacterianos, de forma a determinar a mínima concentraçäo inibitória (MIC) desses quimioterápicos. Os resultados obtidos demonstram que o padräo de suscetibilidade das amostras brasileiras é similar ao do apresentado por amostras isoladas em outros países. A amostra de referência apresentou o mesmo MIC das outras amostras testadas, com exceçäo da rifampicina. Conclui-se que a resistência das micobactérias atípicas provavelmente é intrínseca, näo sujeita a mutaçöes freqüentes.

Esterilizaçäo a frio através de agentes químicos líquidos: avaliaçäo de formulaçöes à base de formaldeído / Cold sterilization with liquid chemical agents: evaluation of formaldehyde preparations

O efeito esporocida de preparaçöes contendo formaldeído nas concentraçöes de 4, 5, 6 e 8%, juntamente com álcool e quaternários de amônio, foi avaliado pela técnica da AOAC (The Association of Official Analytical Chemists). O tempo necessário para a destruiçäo dos esporos de Bacillus subtilis e Clostridium sporogenes foi, respectivamente, de 8 e 18 horas. É importante assinalar que as soluçöes contendo 4 e 5% de formaldeído näo apresentam os vapores irritantes täo característicos deste desinfetante quando utilizado em concentraçöes mais altas

Micobactérias isoladas de tatus Dasypus novemcinctus inoculados com Mycobacterium leprae / Mycobacteria isolated from armadillos Dasypus novemcinctus inoculated with Mycobacterium leprae

Os autores relatam o isolamento de duas micobactérias do fígado e do baço de tatús inoculados com o Micobacterium leprae no meio de Kato. Os resultados säo discutidos

Influencia do inóculo bacteriano no teste de susceptibilidade as quilonas pelo método de diluiçäo em agar

A influência do inóculo na atividade de suas quinolonas (Lomefloxacina e Pefloxacina) contra P. aeruginosa, E. coli, S. aureus e S. fecalis foi investigada pela técnica de diluiçäo em ágar. A açäo deste grupo de antimicrobianos foi reduzida quanto da utilizaçäo de um inóculo muito rico (10**5 e 10**6 ufc), principalmente em relaçäo aos gram negativos