RESUMO

PURPOSE: Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine licensed against tuberculosis. Despite the protection offered by the vaccine, in some circumstances children and immunocompromised individuals can develop associated infections, known as BCGitis. Drug susceptibility patterns of BCG clinical strains have rarely been described. We aimed to describe the susceptibility pattern of BCG clinical strains isolated in two different countries. METHODS: We performed culture-based drug susceptibility testing of thirty one BCG strains isolated from patients in Brazil (n=5, 16%) and Argentina (n=26, 84%) using the broth micro-dilution method (phenotypic method). Final antibiotic concentrations for susceptibility testing ranged from 0.5 to 16 mg/L for amikacin, 0.3125 to 10 mg/L for ethambutol, 0.05 to 1.6 mg/L for isoniazid and 0.25 to 8 mg/L for rifampicin, streptomycin and ofloxacin. Additionally, we compared the results with genetic data obtained by whole genome sequencing. RESULTS: By using the phenotypic method we detected one strain resistant to ethambutol, three strains resistant to rifampicin and one resistant to isoniazid. Additionally, two strains that were phenotypically resistant to both isoniazid and rifampicin carried mutations in the katG and rpoB genes simultaneously. CONCLUSION: There is evidence of the emergence of BCG-resistant strains isolated from vaccine-related complications. We recommend drug susceptibility testing of the BCG strain causing the infection in order to prevent treatment failure.

RESUMO

Mycobacterium bovis is a member of the M. tuberculosis complex, a group composed by species with high genetic homology. The pathogen is the etiological agent of bovine tuberculosis, an important zoonosis that is mainly transmitted by inhalation of infectious droplet nuclei or by ingestion of milk and crude milk derivative products from tuberculosis cows. The definitive identification of M. bovis, up to species level, is time consuming and difficult. In this work, the objective was to standardize a polymerase chain reaction followed by an enzyme restriction analysis in order to identify the M. tuberculosis complex in milk, without a microbiological isolation step. Reference strains and raw milk seeded with M. Bovis, were used as the starting material.  A 441pb fragment of the hsp65 gene was amplified and digested by two restriction enzymes BstEII and HaeIII. The obtained profile was used to identify the M. tuberculosis complex in milk. The minimum limit of detection of M. bovis in milk was 10CFU/mL. PRA methodology proved to be a specific and sensible method. It can be used to assist the microbiological and biochemical methods commonly used to identifying the bacilli in clinical samples, as milk  Key word: Detection limit (PRA), Mycobacterium tuberculosis complex, milk Mycobacterium bovis, Restriction Enzyme Analysis (PCR),

Mycobacterium bovis é membro do complexo Mycobacterium tuberculosis (MTBC), grupo este composto por espécies com grande homologia genética. É o agente etiológico da tuberculose bovina, importante zoonose transmissível ao homem, principalmente através da inalação do bacilo e/ou pelo consumo de leite e derivados não-pasteurizados provenientes de vacas tuberculosas. O objetivo deste estudo foi padronizar a identificação de micobactérias do complexo M. tuberculosis presentes no leite, por metodologia molecular. Fez-se a extração de DNA diretamente do leite contaminado e realizou-se a identificação molecular pela reação em cadeia da polimerase seguida de análise de restrição do fragmento amplificado (PRA). Utilizaram-se inhagens de referência e leite cru artificialmente contaminado com M. bovis IP. Um fragmento de 441pb do gene hsp65 foi amplificado, tratado com BstEII e HaeIII e empregou-se o perfil de restrição enzimática obtido para identificar o complexo M. tuberculosis no leite. Com a PRA foi possível detectar com especificidade e sensibilidade a presença de M. bovis em até 10 UFC/mL de leite. A metodologia padronizada poderá auxiliar os métodos microbiológicos e bioquímicos tradicionalmente usados na identificação do bacilo em alimentos suspeitos de contaminação, como, por exemplo, o leite proveniente de animais suspeitos de infecção por M. bovis.Palavras-chaves: Análise de pe

RESUMO

Mycobacterium bovis is a member of the M. tuberculosis complex, a group composed by species with high genetic homology. The pathogen is the etiological agent of bovine tuberculosis, an important zoonosis that is mainly transmitted by inhalation of infectious droplet nuclei or by ingestion of milk and crude milk derivative products from tuberculosis cows. The definitive identification of M. bovis, up to species level, is time consuming and difficult. In this work, the objective was to standardize a polymerase chain reaction followed by an enzyme restriction analysis in order to identify the M. tuberculosis complex in milk, without a microbiological isolation step. Reference strains and raw milk seeded with M. Bovis, were used as the starting material.  A 441pb fragment of the hsp65 gene was amplified and digested by two restriction enzymes BstEII and HaeIII. The obtained profile was used to identify the M. tuberculosis complex in milk. The minimum limit of detection of M. bovis in milk was 10CFU/mL. PRA methodology proved to be a specific and sensible method. It can be used to assist the microbiological and biochemical methods commonly used to identifying the bacilli in clinical samples, as milk  Key word: Detection limit (PRA), Mycobacterium tuberculosis complex, milk Mycobacterium bovis, Restriction Enzyme Analysis (PCR),

Mycobacterium bovis é membro do complexo Mycobacterium tuberculosis (MTBC), grupo este composto por espécies com grande homologia genética. É o agente etiológico da tuberculose bovina, importante zoonose transmissível ao homem, principalmente através da inalação do bacilo e/ou pelo consumo de leite e derivados não-pasteurizados provenientes de vacas tuberculosas. O objetivo deste estudo foi padronizar a identificação de micobactérias do complexo M. tuberculosis presentes no leite, por metodologia molecular. Fez-se a extração de DNA diretamente do leite contaminado e realizou-se a identificação molecular pela reação em cadeia da polimerase seguida de análise de restrição do fragmento amplificado (PRA). Utilizaram-se inhagens de referência e leite cru artificialmente contaminado com M. bovis IP. Um fragmento de 441pb do gene hsp65 foi amplificado, tratado com BstEII e HaeIII e empregou-se o perfil de restrição enzimática obtido para identificar o complexo M. tuberculosis no leite. Com a PRA foi possível detectar com especificidade e sensibilidade a presença de M. bovis em até 10 UFC/mL de leite. A metodologia padronizada poderá auxiliar os métodos microbiológicos e bioquímicos tradicionalmente usados na identificação do bacilo em alimentos suspeitos de contaminação, como, por exemplo, o leite proveniente de animais suspeitos de infecção por M. bovis.Palavras-chaves: Análise de pe

RESUMO

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 that observed in the community. The overall resistance rate was 17.2% and 3.4% of the isolates were multi-drug resistant.

Testes de suscetibilidade a seis drogas anti-tuberculose foram realizados em 58 amostras de M. tuberculosis isoladas de pacientes do Sanatório Penal Masculino do Rio de Janeiro, Brasil. Foi observada uma alta taxa de resistência quando comparada com as taxas encontradas na comunidade. A resistência total (a qualquer droga) foi de 17,2% e a multi-resistência de 3,4%.