The Assessment of the Risk Ranking and Mobility Potential Associated with Environmental Resistomes in Wastewater Using Metagenomic Assembly

The environmental pollution of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) is a growing public health concern. In the current study, de novo metagenomic assembly and bioinformatics analysis approaches were utilized to estimate the quantitative risk index of the environmental resistomes in wastewater influent (INF) and effluent (EFF) of a conventional wastewater treatment plant (WWTP) in Egypt. Furthermore, the risk indices of the local INF and EFF resistomes were compared to those calculated for the selected publicly available wastewater datasets from eight countries worldwide. Additionally, a classification framework prioritizing the public health hazard level of the discharged non-redundant highly mobilized ARGs was introduced. This integrative outline considered the estimated mobility potential percentage, host pathogenicity, and annotation category (perfect, strict, and loose) of the detected ARGs on their assembled contigs. Moreover, high-quality metagenome-assembled genomes (MAGs) were extracted and the putative genome bins with acquired ARGs were determined. The comprehensive resistome risk scores of the local WWTP showed that INF resistome had a slightly higher risk index (47.87) compared to the average score of the other examined counterparts (41.06). However, the estimated risk value of EFF resistome (26.80) was ranked within the global average (26.06) of the selected international WWTPs. Furthermore, the determination of the samples’ risk ranking showed that most of the effluent resistomes were clustered in a lower risk rank compared to the other selected samples for raw sewage, influent, and hospital wastewater, indicating the impact of the wastewater treatment process on reducing the ARG mobilization potential in downstream environments. The evaluation of the ARGs’ genetic context in their ARG-carrying contigs (ACCs) indicated that a total of 161/648 (25%) non-redundant ARGs were co-located with sequences of mobile genetic determinants on the same ACC in both the INF and EFF assemblies. These ARGs comprised the pan mobile resistome of the studied WWTP. Of them, 111 ARGs with a mobility potential percent (M%) less than 95% were grouped at the least risk level 5. The remaining 50 highly mobilized ARGs (M% ≥ 95%) were extracted and classified into four higher risk levels. Those of risk levels 1 and 2 (39 ARGs) represented the current ARG dissemination threats for further monitoring in downstream environments, where they were all carried by pathogenic hosts and annotated to the perfect and strict categories by the resistance gene identifier software (RGI). A total of 10 highly mobilized ARGs were assigned to risk rank 3, as they comprised the loose hits of the RGI analysis. Finally, the risk level 4 ARGs constituted genes that co-existed with the non-pathogenic sequence on the ACCs and were represented by one gene in the current analysis framework. The two previous categories constituted new highly mobilized ARGs of emergent threat to public health. On the other hand, a total of 35 and 118 MAGs were recovered from INF and EFF assembled metagenomes, respectively, using selection cutoff thresholds of a minimum completeness of 70% and a maximum contamination of 10%. While none of the INF MAGs carried any acquired ARGs, six EFF genome bins (5%) were associated with ten acquired ARGs, as indicated by the ResFinder software. These results suggest that potential horizontal gene transfer (HGT) events have evolved among the community members of the studied EFF samples.