Antimicrobial Susceptibilities, Antibiotic Resistance Genes and Virulence Factors in Gram-Negative Bacteria from Anthropogenically Impacted Aquatic Environments of North India

Introduction:: Gram-negative bacteria are developing resistance against most of the frontline anti-infective drugs. Aquatic environments act as a reservoir of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which pose a serious threat to public health, animals, and the environment. Methods:: The prevalence of antibiotic resistance in Gram-negative bacteria isolated from river water and municipal wastewater from four districts of Himachal Pradesh state, India, was determined by the disc diffusion method. Phenotypic assays were used to determine the extendedspectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), and class C cephalosporinases (AmpC). Whole genome sequencing and bioinformatics analysis of a multidrug-resistant bacterial isolate, Pseudomonas protegens BSS-21, was carried out. Results:: Bacterial isolates (n = 65) belonged to ten genera, viz. Escherichia, Klebsiella, Enterobacter, Pseudomonas, Budvicia, Buttiauxella, Citrobacter, Proteus, Serratia and Providencia. Bacterial isolates exhibited a high level of antibiotic resistance to cefazolin (69.23%), amoxycillin/ clavulanic acid (58.46%), cefoxitin (47.7%), ampicillin (41.5%), cefprozil (38.46%), aztreonam (33.85%), imipenem (29.23%), piperacillin (27.7%), cefotaxime (23.1%), ceftriaxone (21.5%) and cefuroxime (21.5%). However, resistance to other tested antimicrobials was in the lower range of 1.5% – 15.38%. Antibiotic susceptibility testing data revealed the occurrence of a multidrug-resistant phenotype in 39 isolates (60%). Phenotypic occurrence of ESBLs, MBLs, and AmpC was confirmed in 13.84%, 10.76%, and 3.07% bacterial isolates, respectively. Whole genome sequencing of P. protegens BSS-21 revealed a circular chromosome of 6251705 bp harboring ARGs (blaIMI, adeF, arnT, fosA, vanG, vanW, and yajC), virulence factors (clpV1, hsiG1, hcp1, flgC, fliA, fleN, flgI, algD, and pvdS), along with multiple transposable elements and insertion sequences. Discussion:: Multidrug-resistant bacteria exhibiting the β-lactamase phenotypes and showing the occurrence of ARGs, virulence factors, and resistance determinants were detected from river water and wastewaters of northern India. Conclusion:: This is the first report on the genomic characterization of multidrug-resistant P. protegens from the study region. These findings emphasize the need for routine monitoring and surveillance of aquatic environments as a One Health strategy for mitigating the menace of AMR. As this study has limitations of being confined to a small geographical area and whole genomic sequencing of one isolate, future explorations based on a larger study area and genomic characterization of a larger set of bacterial isolates are warranted.