No abstract

Here, we report the isolation of Mycobacterium orygis from dairy cattle in Chennai, India. Spoligotyping assigned the isolate to spoligotype 587 (ST587), which belongs to M. orygis. This species was confirmed as M. orygis using whole-genome sequencing.

The major human pathogen Mycobacterium tuberculosis is rarely reported to cause disease in other animals. Cases in livestock are thought to occur through contact with infected handlers, but previous studies evaluating putative livestock-human transmission used typing techniques with limited resolution. Here, we undertook cross-sectional surveillance for tuberculosis in 271 livestock handlers and 167 cattle on three farms in Chennai, India and defined the relatedness of cultured isolates using whole genome sequencing. Humans and livestock were screened for active mycobacterial infection, and opportunistic post-mortem examination was performed on comparative intradermal test-positive cattle that died. Four cattle and 6 handlers on two farms were culture-positive for M. tuberculosis; M. bovis was not isolated. All 10 isolates (one from each case) belonged to Lineage 1. Pairwise genome comparisons of single nucleotide polymorphism (SNP) differences ranged from 1 to 600 SNPs, but 3 isolate pairs were less than 5 SNPs different. Two pairs were from handlers and the third pair were from two cattle on the same farm. The minimum pairwise SNP difference between a cattle and human isolate was >250 SNPs. Our study confirms the presence of M. tuberculosis infection in cattle in India, sequencing of which characterised relatedness between human and cattle-derived isolates.

We report the isolation of Mycobacterium orygis, a member of Mycobacterium tuberculosis complex (MTBC), from two black bucks (Antelope cervicapra) and one spotted deer (Axis axis) from the Guindy National Park forest range in Chennai, India. Lung tissue and lymph node samples collected during post-mortem examination were processed using NaOH method and cultured in solid and liquid media. DNA extracted from the cultured isolates was used to amplify the mpt64 gene by specific primers and the band visualized at 240 bps confirmed the isolates as a member of MTBC. Further examination of these isolates by spoligotyping and whole-genome sequencing confirmed the isolates as M. orygis and the phylogenetic tree revealed their well-clustered position with other M. orygis isolates around the globe. The deletion of RD7-RD10, RDOryx_1, RDOryx_4, RD12Oryx, RD301 and RD315 further substantiated these isolates as M.orygis. The exact source of infection in animals was untraceable and the pairwise comparison of the genomes based on single-nucleotide polymorphisms difference did not detect any events of transmission within the affected animals. Nevertheless, it would be wise to take into account the environment where there exists a high chance of transmission due to the increased human-animal interaction. Since it is well known that the pathogen is capable of causing infection in both human and animal hosts, systematic surveillance and screening of spotted deer, black buck as well as humans in the vicinity is essential for successful implementation of the One Health approach.

Mycobacterium tuberculosis adapts itself to various environmental stress conditions to thrive inside the phagosome for establishing a chronic infection. Serine/threonine protein kinases (STPKs) play a major role in the physiology and pathogenesis of Mycobacterium tuberculosis. Some of these STPKs are involved in regulating the growth of the mycobacterium under nutrient stress and starvation conditions. In this study, we have investigated the role of PknL, a STPK in the adaptive responses of M. tuberculosis by conditional inactivation of the gene using antisense technology. The inhibition of PknL in the knockdown strain was validated by RT-PCR. The in vitro growth kinetics of M. tuberculosis strain following inhibition of PknL was found to be bacteriostatic. The knock down strain of PknL exhibited a better survival in pH 5.5 when compared to its growth in pH 7.0. Similarly, it also exhibited more resistance to both SDS(0.01%) and Lysozyme stress (2.5mg/ml), indicating that loss of PknL enhances the growth of mycobacterium under stress conditions. SEM pictographs also represent an increase in the cell length of the knock down strain compared to Wild type stressing its role in cellular integrity. Lastly, the proteome analysis of differentially expressing PknL strains by 2D gel electrophoresis and mass spectrometry identified 19 differentially expressed proteins. Our findings have shown that PknL plays an important role in sensing the host environment and adapting itself in slowing down the growth of the pathogen and persisting within the host.