Molecular Speciation of Mycobacterial Isolates from Raw Cow Milk Reveals Predominance of Mycobacterium chelonae

Abstract

Owing to the fact that raw milk intake is still practiced in selected communities globally, milk borne diseases need to be addressed as some mycobacterial species use milk as a ploy for disseminating the disease. An approach was made in the present study for screening the mycobacterial species in raw cow milk. For mycobacterial isolation, a total of 200 milk samples were collected from three districts of Northeast India—Dimapur district of Nagaland, East Khasi Hills and Ri-Bhoi districts of Meghalaya. Samples were collected by trained personnel and transported in cold chain. Following decontamination, samples were inoculated in both Lowenstein Jensen (LJ)-glycerol and LJ-pyruvate and incubated up to 12 weeks with regular interval check. Colonies were examined for acid-fastness and confirmed by genus-specific hsp65 PCR. Amplicons were sequenced, analyzed in Basic Local Alignment Search Tool (BLAST) and phylogenetically explored. A total of 22 (11%) isolates were recovered. Sequencing of genus-specific hsp65 gene identified 17 (80.95%) isolates to be Mycobacterium chelonae, 03 (14.28%) as M. lentiflavum, 01 (4.76%) M. peregrinum and 01 (0.5%) as a member of Mycobacterium tuberculosis complex. The dendrogram presented vivid species distinction as the isolates clustered into their respective phyletic line. Significant isolation of M. chelonae from raw milk in this study is noteworthy as it is an opportunistic human pathogen and one of the most dominant species isolated from human nontuberculous mycobacteria infections.