Molecular Identification and Heavy Metal Resistance Profiles of Escherichia coli from Silver Carp in the Contaminated Waters of the River Kabul, Khyber Pakhtunkhwa

Abstract

Heavy metal pollution in freshwater ecosystems presents considerable ecological and public health risks, particularly when toxic metals interact with pathogenic and metal-resistant bacteria in edible fish species. The present study investigated heavy-metal-resistant Escherichia coli isolated from silver carp obtained from the River Kabul in Pakistan. Thirty silver carp specimens were collected, and bacteria were isolated from their intestines and gills. Bacterial identification was performed using Gram staining and biochemical tests (Catalase, Oxidase, Motility, SIM, and Citrate). The Minimum Inhibitory Concentration (MIC) assay was used to assess E. coli resistance against ZnSO₄, NiCl₂, CuSO₄, and HgCl₂. Molecular identification was performed using the 16S rRNA gene sequencing and phylogenetic analysis. E. coli was isolated from 23 of 30 fish (76%), yielding four phenotypically confirmed isolates, which were further subjected to MIC testing. All isolates were Gram-negative rods with biochemical characteristics consistent with E. coli (100% concordance). MIC assays showed high resistance to ZnSO₄, NiCl₂,  and CuSO₄, while all tested isolates remained sensitive to HgCl₂. The statistical analysis revealed significant differences in resistance among the tested metals (p < 0.05). The phylogenetic analysis showed that the heavy metal-resistant isolates formed a tight clustering pattern with previously reported E. coli strains from Pakistan, suggesting possible regional adaptation in freshwater ecosystems contaminated with heavy metals. These results show that heavy metal contamination and heavy metal-resistant E. coli are both present in an important edible fish species.