Isolation and Molecular Characterization of Antibiotic-Resistant Escherichia coli in Hospital Wastewater
Keywords:
Escherichia coli, Hospital wastewater, Antimicrobial resistance, Multidrug resistance, Extended-spectrum β-lactamase (ESBL), Carbapenemase genes, Molecular characterisation.Abstract
Multidrug-resistant (MDR) pathogens are known to be spread in the environment by hospital wastewater, which is a major reservoir of antimicrobial-resistant bacteria and antimicrobial resistance genes. Escherichia coli is one of these organisms recognized as an indicator bacterium to monitor antimicrobial resistance. The objective of this study was to isolate and identify antibiotic resistant Escherichia coli (E. coli) in the hospital wastewater, understand their antimicrobial susceptibility, assess the presence of major β-lactamase and carbapenemase resistance genes and evaluate the transfer potential of these resistance genes. Samples of wastewater were collected from the main hospital effluent using a 24-hour flow-proportional sampling method. E. coli were isolated after membrane filtration on selective culture media and confirmed by MALDI-TOF mass spectrometry. The broth microdilution method was used for antimicrobial susceptibility testing to determine minimal inhibitory concentrations (MICs). The blaCTX-M, blaTEM, blaSHV, blaKPC, blaNDM and blaOXA-48 genes were identified by PCR. Geographical linkage and the mobility of resistance genes were assessed using S1-nuclease pulsed-field gel electrophoresis, Southern blot hybridisation and conjugation assays. The statistical analysis of the data was performed by ANOVA, Tukey's post hoc test and Chi-square analysis. Multidrug resistance was found at high levels and 73.1% of the isolates were multidrug resistant (MDR), meaning they were resistant to at least three antimicrobial classes. Phenotypic resistance was found against the highest levels of ampicillin, ceftriaxone and meropenem. Molecular detection revealed the presence of blaCTX-M-15 and blaCTX-M-1 in 68.0% of ESBL-producing isolates and blaNDM and blaOXA-48 in 14.5% of those with carbapenem-resistance genes. The MIC values of the isolates with multiple resistance genes were significantly higher than those of isolates with a single resistance gene (p < 0.05). Several resistance determinants were plasmid-mediated, as confirmed by conjugation assays, thus with a high potential for horizontal gene transfer. The hospital wastewater is a significant reservoir for multidrug resistant Escherichia coli and clinically relevant antimicrobial resistance genes in the environment. The presence of both ESBL and carbapenemase genes, and the ability of these genes to be transferred to other plasmids, demonstrates the possibility of environmental spread of antimicrobial resistance. To prevent the transfer of resistant bacteria and resistance genes into the environment, continuous monitoring and good wastewater-treatment strategies are necessary.
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