Characterization of antibiotic-resistant bacteria in two non-integrated fish farming sites

Abstract

Antibiotic resistance genes (ARGs) are emerging pollutants that pose a significant threat to public health. The utilization of antibiotics in aquaculture fosters both the rise of antibiotic-resistant bacteria and the dissemination of antibiotic resistance genes (ARGs) in the fish farming environment. The present investigation centered on the prevalence of antimicrobial resistance genes (ARGs) within Gram-negative bacteria and their corresponding susceptibility to antimicrobial agents. A total of thirty-five samples were collected from fish seed hatcheries located in Islamabad and Faisalabad, Pakistan. These samples consisted of eighteen water samples and seventeen sediment samples. The samples were subjected to plating on various selective media and media supplemented with antibiotics, after which they were incubated at 37ºC for 24 hours. A total of 992 bacterial isolates were obtained from a set of thirty-five water and sediment samples. Among them, a subset of 15.5% (n=143) were identified as Gram-negative bacteria, while 12.6% (n=116) were Gram-positive bacteria. Additionally, 8.1% (n=75) of the isolates belong to Enterococci, whereas a significant majority of 63.8% (n=588) were antibiotic-resistant Gram negative bacteria. The highest CFU count was reported in sediment samples which were 1x107 CFU/mL for Gram-negative bacteria. The conventional PCR was used to screen for genes encoding extended-spectrum beta-lactamase (ESBLs), carbapenemases, tetracycline resistance, polymyxin resistance, and plasmid-mediated quinolone resistance (PMQR). Of the 35 samples, fourteen types of ARGs were identified within 79 isolates. The isolates correspond to Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Acinetobacter, baumannii, E. coli, Serratia marcescens, Citrobacter freundii, Proteus mirabilis, Edwardsiella tarda, and Klebsiella oxytoca. Among the PMQR genes, aac(6')-Ib-cr was found as the most common gene associated with ciprofloxacin resistance, accounting for 11.11%, followed by qnrB in 7.9% of isolates. The presence of qnrS was observed in 2.3% of isolates, Conversely, found no prevalence of qnrA, qnrC, and qnrD. Moreover, a coexistence of more than one PMQR genes were detected in 1.6% of the isolates. The prevalent gene in ESBL-producing isolates was blaCTX-MU, found in 6.1% of the isolates, followed by blaSHV in 3.8% of cases. Furthermore, blaCTX M9 was found positive in 2.2% of isolates, but blaTEM was reported in just 0.7% of isolates. Regarding polymyxin resistance genes, mcr-1 was found in only 0.9% of the isolates, with no prevalence of other polymyxin resistance genes. Among the carbapenem resistance genes, blaOXA viii Chapter 1 Abstract 48 was found in 7.2% of isolates, followed by 4.5% carrying blaVIM and 2.7% carrying blaNDM. From oxytetracycline-resistant isolates, tetA was found, the most prevalent, appearing in 10.3% of isolates, followed by tetB in 6% and tetE in 5.1% of isolates. Furthermore, a co-occurrence of more than one tetracycline resistance gene was detected in 3.4% of the isolates. The multiple antibiotic resistance (MAR) index for isolates positive for antibiotic resistance determinants were found to vary from 0.2 to 1. These findings highlight the prevalence of multidrug-resistant bacteria and antimicrobial resistance genes in fishponds, posing a substantial threat to both human and animal health, as well as the environment. As a result, comprehensive surveillance measures are required to halt the spread of antibiotic resistance.