Bacteriological quality, phenotypic virulence characterization, and antimicrobial resistance profiling of bacterial isolates from packaged drinking water in Benin city, Nigeria

Abstract

Packaged drinking water is consumed by millions of Nigerians, yet the bacteriological quality remains inadequately characterized in many localities. This study investigated the bacteriological quality, phenotypic virulence, and antimicrobial resistance profiles of isolates from packaged water sold in Benin City. Total heterotrophic bacterial counts were determined by membrane filtration for 12 packaged water brands, and bacterial identification was by conventional methods and growth on differential media. DNase activity, gelatinase, hemolysin and lipase production were determined using standard assays. Molecular characterization of representative isolates was performed using 16S rRNA gene sequencing and BLAST analysis. Antibiotic susceptibility was determined by the Kirby-Bauer disc diffusion method, and the multiple antibiotic resistance index (MAR) was calculated. Total heterotrophic bacterial counts ranged from 0.00 to 33.50 CFU/100 mL, with three bottled water brands (OC, MX, UB) and one sachet water brand (LM) exceeding the WHO/NIS permissible limit of 10 CFU/100 mL. Seven bacterial species were identified: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillus pumilus, Proteus vulgaris, Enterobacter cloacae, and Micrococcus luteus. Bacillus pumilus was the most frequently occurring species (27.91%), followed by Staphylococcus aureus (20.93%) and Pseudomonas aeruginosa (13.95%). Molecular identification confirmed all seven species with ≥95% BLAST identity. The isolates produced at least one virulence factor; Escherichia coli and Pseudomonas aeruginosa exhibited the highest overall virulence potential. The MAR index of all isolates exceeded the 0.2 threshold, with Staphylococcus aureus recording the highest index of 0.53, indicating a high-risk environmental origin. The findings reveal the urgency of enhanced water-treatment protocols.