Influence of the incubation conditions on culture media to optimize primary isolation of Mycobacterium bovis

Abstract

The isolation of Mycobacterium bovis is critical to a surveillance system for bovine tuberculosis based on detection of lesions in abattoirs. Thus, four solid culture media and three incubation conditions were investigated to elucidate which combination overcomes the others by assessing growth, time to the first appearance of colonies and their number. Ninety-seven samples of granulomatous lesions were submitted to the decontamination procedure by 1-hexadecylpyridinium chloride at 0.75% w/v, and inoculated on two egg-based media, Stonebrink's (ST) and Löwenstein-Jensen's with sodium pyruvate (LJp), and two agar-based media, tuberculosis blood agar (B83) and Middlebrook 7H11 medium (7H11). Each medium was incubated at 37°C for 90 days in three incubation conditions: in air, in air containing 10% carbon dioxide (CO 2 ), and in air in slopes closed with burned hydrophobic cotton and subsequently plugged with a cork to create a microaerophilic atmosphere. The colonies appeared faster and in higher number when incubated in air containing 10% CO 2 (p < 0.01), independent of media. B83 showed a faster growth and detected more isolates at 30 days of incubation, when compared to ST (0.0178), LJp (p < 0.0001) and 7H11 (p < 0.0001), though there was no difference between B83, ST and LJp at 60 and 90 days of incubation. 7H11 presented the lowest number of isolates (p < 0.0001) and a longer period for the appearance of the first colony (p < 0.001). According to our findings, the concomitant use of ST and B83 media incubated in air containing 10% CO 2 increases the isolation of M. bovis in a shorter period of time, which improves bovine tuberculosis diagnosis. Key words: Primary isolation. Bovine tuberculosis. Mycobacterium bovis. Culture media. Incubation conditions. ResumoO isolamento do Mycobacterium bovis é fundamental para um sistema de vigilância para tuberculose bovina baseado na detecção de lesões em abatedouro. Assim, quatro meios de cultura sólidos e três condições de incubação foram investigados para elucidar qual combinação supera as outras através da avaliação de crescimento, tempo para o aparecimento da primeira colônia e número de colônias. Noventa e sete amostras de lesões granulomatosas foram submetidas ao processo de descontaminação por cloreto de 1-hexadecilpiridínio a 0,75%, e inoculadas em dois meios a base de ovo, Stonebrink (ST) e Löwenstein-Jensen com piruvato de sódio (LJp), e dois meios a base de ágar, ágar sangue tuberculose (B83) e Middlebrook 7H11 (7H11). Cada meio foi incubado a 37°C por 90 dias, em três condições de incubação: em atmosfera normal, em atmosfera com acréscimo de 10% de dióxido de carbono (CO 2 ), e em atmosfera normal em tubos fechados com algodão hidrófobo queimado e subsequentemente fechado com rolha para criar uma atmosfera microaerófila. As colônias apareceram mais rapidamente e em maior número quando incubadas em atmosfera com 10% de CO 2 (p < 0,01), independente dos meios. As micobactérias cresceram em maior abundância e mais rapidamente no meio B83 aos 30 dias de i...

Complete Genome Sequencing of Mycobacterium bovis SP38 and Comparative Genomics of Mycobacterium bovis and M. tuberculosis Strains

“…This strain was originally isolated from a granulomatous lesion collected from a bovine in a slaughterhouse located in the State of São Paulo, Brazil in 2010 (Morato et al, 2016 ). The lesion was cultured in Stonebrink medium as described previously (Ikuta et al, 2016 ), following standard procedures (Centro Panamericano de Zoonosis, 1985 ), and the isolate was stored at −20°C with no further passages. For the purpose of extracting DNA for this study, this isolate was reactivated in Stonebrink medium.…”

Isolation of potential zoonotic Mycobacterium spp. from diseased freshwater angelfish (Pterophyllum scalare) from an aquarium

“…For mycobacteria isolation, all samples were decontaminated with 0.75% cetylpyridinium chloride (Ikuta et al, 2016). Thereafter samples were inoculated in duplicate into Stonebrink and Löwenstein-Jensen media and incubated at 25 °C and 37 °C for up to 60 days (CPZ, 1995).…”