Biology subjects

Kumaraswamy, J.

Publications and source records attributed to Kumaraswamy, J..

2 recordsLinked to original sources

bioRxiv2025

Preliminary genomic data on five Orientia tsutsugamushi strains isolated in Vellore, India

IntroductionOrientia tsutsugamushi, the causative agent of scrub typhus, can be isolated in Vero or L929 cells and has a small genome (2-2.5 Mb). However, genome assembly is challenging due to the presence of host DNA contamination and a high proportion of repeat regions (up to 51%). Current global data includes 11 fully annotated genomes, with none from India. Here, we present the first whole-genome sequences of O. tsutsugamushi from India. MethodsFive O. tsutsugamushi strains were cultured in Vero cells and confirmed by 47kDa real-time PCR. Genomic DNA was extracted after removal of host DNA and sequencing libraries were prepared. Whole-genome sequencing was performed using the PacBio Sequel II system in CCS/HiFi mode. The raw reads were assembled using Flye, and genome completeness was assessed with QUAST and BUSCO. Annotation was performed using the NCBI PGAP pipeline and comparative genome analysis by Roary. Phylogenetic analysis was based on the full-length 56kDa gene, which contains four variable domains. ResultsWe report five complete genomes of O. tsutsugamushi, four of which are circular and one linear. Genome sizes range from 2.1 to 2.4 Mb. The total number of predicted genes falls between 2,379 and 2,715, with an average of 1,824 coding genes and 613 pseudogenes. Repeat regions constitute 53-59% of the genome, a higher proportion than previously reported. All five genomes have been submitted to NCBI GenBank (Accession Numbers: CP166954-58). Phylogenetic analysis based on the full-length 56kDa gene revealed that two strains belong to the Karp genogroup, two to Kato, and one to TA763. ConclusionThis study presents the first whole-genome sequencing data of O. tsutsugamushi from India. Notably, the repetitive regions in these genomes are more extensive than previously reported. Further analyses with additional isolates are necessary to validate this observation. Comprehensive phylogenomic studies, particularly to elucidate evolutionary dynamics and potential recombination events will provide further information.

microbiology↗

bioRxiv2023

First isolate of Orientia tsutsugamushi from Vellore, South India

BackgroundScrub typhus a common cause of acute febrile illness in India caused by Orientia tsutsugamushi an obligate intracellular bacterium requiring cell culture for isolation. Cell lines like Vero and L929 are most suitable for isolating and maintaining this organism. This study was undertaken to isolate and characterize of Orientia tsutsugamushi from whole blood samples at a tertiary care centre in Southern India. MethodsThe PBMCs (peripheral blood mononuclear cells) collected from scrub typhus positive (47kDa qPCR positive) patients were inoculated into Vero and L929 cell line at 80% confluence for primary isolation. The inoculated flasks were incubated at 37{degrees}C with 5% CO2 for 30 days and examined for presence of Orientia tsutsugamushi on the day 10, 15, 20 post-inoculation and everyday thereafter for a maximum of 30 days post inoculation. The scrapings were subjected to Giemsa staining, IFA, 47kDa qPCR and transmission electron microscopy (TEM). The isolates were passaged 3-4 times to ensure viability and then stored in DMEM with 10% FBS (-80{degrees}C). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene. ResultsAmongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR at 24th day of inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology with Orientia tsutsugamushi MW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype. ConclusionsWe have successfully isolated and characterised the Orientia tsutsugamushi for the first time at our centre from PBMCs. Based on the partial TSA56 gene sequence our isolates belongs to TA763 and Gilliam genotype. More number of samples are being processed for identifying further isolates followed by genomic analysis.

microbiology↗