Mapping bacterial diversity and antibiotic resistance across wastewater treatment plant stages: Insights from high-resolution 16S rRNA sequencing of the V3-V4 regions to detection of multi-drug resistant bacteria

Abstract

Previous studies on the exponential increase of bacterial drug resistance in wastewater treatment plants have reinforced the need for continuous, targeted surveillance to further map the prokaryotic community structure. To this aim, samples of wastewater influent and effluent from three local wastewater treatment plants (WWTPs) were collected. The investigation was narrowed to focus on Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE), and prevalent non-ESKAPE drug-resistant pathogens. High-resolution 16S rRNA sequencing of the V3-V4 hypervariable regions was performed to characterize bacterial communities across wastewater stages collected from the three local WWTPs. Taxonomic profiling demonstrated a 52 %–78 % reduction in bacterial diversity at the species level in effluent samples, with a significant decrease in Shannon diversity index post-treatment. Results elucidated the following in effluents: Escherichia coli (2.7 %), Pseudomonas spp. (0.71 %), Aeromonas spp. (91 %), Salmonella spp. (0.06 %), and Paenibacillus spp. (40.68 %). A broad spectrum of drug resistance was noted, with effluents resistant to multiple antibiotics: E. coli showed resistance to 13 antibiotics, Pseudomonas spp. to 10, Klebsiella spp. to 14, and Salmonella, Aeromonas, and Paenibacillus spp. to 15 antibiotics each. Despite a significant reduction in bacterial abundance and diversity in treated effluent, the persistence of certain pathogens suggests the urgent need for continuous wastewater surveillance and additional safeguards in interventions.