Genotyping and Cloning of Β-Lactamase Genes (Blactx-M) From Extended Spectrum Β -Lactamase-Producing Enterobacteriaceae Wastewater Isolates from the Philippine General Hospital

Abstract

Antimicrobial resistance (AMR) continues to be one of the major public health concerns worldwide and has been exacerbated during this COVID-19 pandemic due to the overuse of antimicrobial agents. Hospital wastewaters are considered a hotspot niche of multi-drug and pathogenic bacteria such as extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae which has been reported to be ubiquitous in various ecosystems. This study detected, genotyped, and cloned the most predominant bla gene from ESBL-producing bacteria isolated from effluent wastewater of the Philippine General Hospital (PGH). Out of 100 isolates, 29% were found to be ESBL-producers based on antimicrobial susceptibility testing (AST). To further confirm ESBL production, isolates were subjected to phenotypic confirmatory disk diffusion test (PCDDT) and 15 out of 29 (51.7%) were found to be ESBL-producers. Subsequently, the genotyping of bla genes showed that 15 (100%) of the isolates carried blaCTX-M, 12 (80%) carried blaOXA, 5 (33.4%) carried blaTEM, and 3 (20%) carried blaSHV gene. Five of the isolates possessed at least three genes (blaCTX-M, blaOXA, blaTEM), seven with two genes (blaCTX-M, blaOXA or blaCTX-M, blaSHV) and two with single gene (blaCTX-M). However, one of the isolates possessed all of the bla genes tested. The blaCTX-M was found to be the most predominant β-lactamase gene as it was found in all ESBL-producing isolates. To further determine possible variants of the blaCTX-M, a gene library was constructed. The complete open reading frame (ORF) of blaCTX-M gene was amplified by Polymerase Chain Reaction and was cloned into the EcoRI and HindIII restriction sites of the pUC19 plasmid. The resulting recombinant pCTX-M plasmid was then successfully transformed in competent cells of E. coli DH5α. This study therefore provided a baseline data on the prevalence of ESBL-producing bacteria and the genotypic distribution of different bla genes among the different Enterobacteriaceae isolates from hospital wastewater of PGH which might help in the formulation of wastewater treatment plans and hospital waste management policies. Moreover, blaCTX-M gene library can be utilized to further elucidate the molecular characteristics of this gene and for diagnostics and mutagenic studies.