A combined metagenomics and metatranscriptomics approach to assess the occurrence and reduction of pathogenic bacteria in municipal wastewater treatment plants

The emergence and spread of pathogens, antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG) through insufficiently treated effluents from wastewater treatment plants (WWTP) pose a risk to human health and the environment. The present study focused on assessing the occurrence, prevalence and fate of dominant pathogenic bacteria, ARGs and mobile genetic element (MGE) in different WWTPs in Durban, Kwa-Zulu Natal, South Africa. The samples were taken from three wastewater treatment plants with different configurations, including trickling filter (TF), biological nutrient removal (BNR), and conventional activated sludge processes (CAS). Total genomic DNA and RNA were extracted from the samples for metagenomic and tracriptomic analysis. A total of 23 pathogenic bacterial genera, including enteric and emerging opportunistic pathogens, were detected in the samples. Acinetobacter spp. and Aeromonas spp. were the predominant pathogens in influent metagenomes, while Escherichia coli and Acinetobacter spp. dominated influent transcripts. Based on Shannon-Wiener indices, the diversity of bacteria increased from influents to final effluents in two treatment plants. ARGsthat confer resistance to aminoglycosides, beta-lactamases, tetracycline and sulfonamides were abundant in both influent and effluent samples. Results further exposed that MGE-ARG associations were the main drivers of ARG persistence to final effluents. This included 5 plasmids: R338-R151 (sulI), pRH-1238 (strB), pPM91 (aadA), pRH-1238 (aadA4-5), pRH-1238 (sulII); two class 1 integrons (aadA and arr) and 1 transposon Tn4351 (tetX). In transcripts, the MGE-ARG associations showed two plasmids: pRH-1238 (aadA) and pPM91 (aadA) and one hybrid plasmid R338-R151 (sulI). The study investigated the potential impact of operational parameters (dissolved oxygen (DO), total suspended solids (TSS), pH and temperature) on selected bacterial pathogens (Aeromonas spp, Acinetobacter spp., Pseudomonas aeruginosa and Klebsiella pneumoniae) and their fates at different stages of the three WWTPs. Principal component analysis (PCA) showed that temperature, DO, and pH were the most relevant factors influencing pathogen abundance. Among the studied pathogens, Acinetobacter spp. was the most prevalent in the influent samples, followed by Aeromonas spp. As for the aeration samples, Aeromonas spp. was dominant in WWTP1 (CAS configuration) and WWTP2 (BNR configuration), while Acinetobacter spp dominated in WWTP3 (BNR configuration). Acinetobacter spp., Aeromonas spp., and Pseudomonas aeruginosa were the dominant ones in secondary effluents, with their dominance varying across the sampling period. In the final treated effluent, Acinetobacterspp., Aeromonas spp., and P. aeruginosa were dominant, with their dominance varying from sample to sample. Additionally, free living amoebas (FLA) were also investigated for their contribution to the propagation and persistence of pathogens in secondary and final effluents. Using the conventional isolation technique, FLAs were isolated from different samples. The internalized bacteria and ARGs were further identified using metagenomic analysis. Metagenomic profiles identified nine species belonging to Acanthamoeba and two species belonging to Entamoeba. A. castellini was the most prevalent dominant species detected in effluent and final effluent samples of all three WWTPs. P. aeruginosa, S. maltophilia, A. spanius, C. testosteroni, and E. cloacae were the most dominant bacterial endosymbionts detected. Among these, S. maltophilia and P. stutzeri were detected in FLAs isolated from the final treated effluents indicating their prevalence in the chlorinated effluents. The presence of ARGs within FLAs were also ascertained. Genes conferring resistance to aminoglycosides (aadA); trimethoprim (dfrA15 and dfrA5); sulfonamides (Sul1 and SulII), macrolides (msrA, mphC); rifamycin (Arr); quinolones (qnrE1) and tetracyclines (TetA and TetG). SulI, dfrA5, AadA, dfrA15, SulII, TetA, TetG and qnrE1 were among the resistance genes that persisted into final effluents. The results of this study have contributed significantly to our current understanding of pathogens, particularly the dominant pathogens and the role of FLAs in the dispersal of pathogens and ARGs into the environment via WWTPs. The study also indicatesthat the conventionally treated effluents may still contain human pathogens, ARGs, and MGEs, which may contribute to the propagation of emerging pathogens and antibiotic resistance in the receiving environment.