MOLECULAR CHARACTERIZATION OF SELECTED VIRULENT AND MULTIDRUG-RESISTANT GROUNDWATER BACTERIA FROM KOZHIKODE, KERALA

Abstract

Background: Groundwater serves as a major source of domestic water in Kerala; however, routine microbiological monitoring is often limited to indicator organisms and may overlook bacterial populations having clinically significant virulence traits and multidrug resistance. Objective: This study aims to check the molecular characterization selected virulent and multidrug-resistant bacterial isolates recovered from groundwater sources in Kozhikode, Kerala. Methods: A total of 392 groundwater samples were analysed using culture based microbiological methods, yielding 256 bacterial isolates. A comprehensive screening approach incorporating phenotypic identification, string test, biofilm formation assay, and antimicrobial susceptibility testing was employed to find high-risk isolates. Six representative isolates showing pronounced virulence-associated phenotypes and multidrug resistance were selected for genomic DNA extraction, 16S rRNA gene amplification, and molecular characterization. Results: Initial screening revealed the predominance of Escherichia coli and Klebsiella spp. among the recovered isolates. Klebsiella spp. showed a strong hypermucoviscosity-associated phenotype, while Klebsiella and Pseudomonas groups showed greater biofilm-forming capacity compared with other taxa. Six representative multidrug-resistant isolates, comprising GW70 and GW141 (Escherichia coli), GW109 and GW155 (Klebsiella pneumoniae), GW188 (Klebsiella oxytoca), and GW132 (Pseudomonas aeruginosa), were selected for molecular characterization based on their resistance to ceftazidime, cefotaxime–clavulanic acid, meropenem, gentamicin, and ciprofloxacin. Genomic DNA extracted from these isolates yielded concentrations ranging from 85.3 to 110.2 ng/µL, with A260/A280 purity ratios of 1.79–1.88, indicating DNA of suitable quality for downstream analysis. Subsequent 16S rRNA gene amplification produced PCR products with concentrations ranging from 37.9 to 50.1 ng/µL and A260/A280 ratios of 1.82–1.90. Molecular analysis subsequently confirmed the taxonomic identity of all six selected isolates. Conclusion: The findings suggest that groundwater sources in Kozhikode may serve as reservoirs of virulent and multidrug-resistant bacteria with potential public health significance. These results highlight the need for enhanced groundwater surveillance strategies incorporating molecular characterization alongside conventional indicator-based microbiological assessments.