Multidrug-resistant and virulence traits in genetically distinct vesicular Leptospira inhabiting farmland and sewage in Southern India.

The occurrence, taxonomic identity, drug resistance pattern and virulence traits of cultivable environmental Leptospira inhabiting the Indian subcontinent remain largely unexplored. Here, we describe the isolation and characterization of six novel strains of Leptospira from environmental samples and decode their drug resistance and virulence traits using in silico and in vitro approaches. Among six new isolates of Leptospira, two strains were identified to represent L. interrogans (SA-L3 and SA-L4), originating from sewage that flows proximal to a wildlife enclosure in Mangalore. We also found a strain each of L. levettii (SA-E5), L. kmetyi (SA-E7), L. koniambonensis (SA-E9) and Leptospira sp. (SA-E8, a putative L. andrefontaineae-like new species of Leptospira) within a ~ 5000 sq. ft. farmland area at Halageri, Uttara Kannada District, India. All isolates exhibited hemolysis and harbored genes encoding diverse sphingomyelinases. All strains were resistant to vancomycin, rifamycin, aztreonam, lincomycin, nalidixic acid, minocycline and troleandomycin and carried genes for a multitude of proteins conferring drug-resistant mechanisms. Leptospira sp. SA-E8 exhibited superior virulence traits and significant biofilm formation in vitro. Comparative genomics revealed remarkable interspecies variations in genome size (4.0‒4.8 Mbp), GC content (35.1‒44.8), coding sequences (3823‒5296) and RNA (39‒43). All strains exhibited the oxidation of diverse organic acids and were equipped with genetic machinery for ketone body oxidation. Our study demonstrated the occurrence of genetically distant Leptospira in environmental samples collected from India and provided evidence for conserved multidrug resistance and virulence, offering new dimensions to the theranostics of Leptospirosis. We suggest the inclusion of L. levettii SA-E5, L. kmetyi SA-E7, L. koniambonensis SA-E9 and Leptospira sp. SA-E8 in the array of leptospiral antigens being used in sero-epidemiological studies to establish their seroprevalence in humans, rodents and livestock.