Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

INTRODUCTION: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. RESULTS: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (Rf) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.

Identification of Mycobacteria by thin layer chromatographic analysis of mycolic acids and conventional biochemical method: four years of experience

Mycolic acids analysis by thin-layer chromatography (TLC) has been employed by several laboratories worldwide as a method for fast identification of mycobacteria. This method was introduced in Brazil by our laboratory in 1992 as a routine identification technique. Up to the present, 861 strains isolated were identified by mycolic acids TLC and by standard biochemical tests; 61 per cent out of these strains came as clinical samples, 4 per cent isolated from frogs and 35 per cent as environmental samples. Mycobacterium tuberculosis strains identified by classical methods were confirmed by their mycolic acids contents (I, III and IV). The method allowed earlier differentiation of M. avium complex - MAC (mycolic acids I, IV and VI) from M. simiae (acids I, II and IV), both with similar biochemical properties. The methods also permitted to distinguish M. fortuitum (acids I and V) from M. chelonae (acids I and II), and to detect mixed mycobacterial infections cases as M. tuberculosis with MAC and M. fortuitum with MAC. Concluding, four years experience shows that mycolic acids TLC is an easy, reliable, fast and inexpensive method, an important tool to put together conventional mycobacteria identification methods.

Testes alternativos para identificaçäo de espécies de enterobactérias. I - Verificaçäo da produçäo de Indol. II - Verificaçäo da produçäo de urease / Alternative tests for identification of enterobacteria. I - Indol production. II - Urease production

Säo propostos testes alternativos para identificaçäo de bactérias produtoras de indol e das produtoras da enzima urease. A produçäo microbiana do indol é verificada por reaçäo com nitroprussiato de sódio em meio alcalino, e o complexo formado é visualizado pelo aparecimento de cor azul com a acidificaçäo do meio. Em provas comparativas com os reagentes clássicos de Kovacs e Erlich, verificou-se concordância de respostas para espécies de enterobactérias testadas, indicando que o nitroprussiato de sódio pode ser usado como reagente para detecçäo de produçäo de indol pelas enterobactérias. O método alternativo para visualizaçäo da prova da urease consiste na inoculaçäo do microorganismo em meio líquido de Stuart (sem o indicador vermelho de fenol), seguido da identificaçäo da produçäo de amônia com o reagente de Nessler, após incubaçäo a 37ºC por tempos determinados. Em experimentos comparativos com a prova clássica, utilizando o meio de Stuart com vermelho de fenol, o método proposto mostrou-se mais sensível e mais específico