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Motivation Wastewater treatment plants (WWTPs) harbor a dense and diverse microbial community. They constantly receive antimicrobial residues and resistant strains, and therefore provide conditions for horizontal gene transfer (HGT) of antimicrobial resistance (AMR) determinants. This facilitates the transmission of clinically important genes between, e.g. enteric and environmental bacteria, and vice versa. Despite the clinical importance, tools for predicting HGT remain underdeveloped. Results In this study, we examined to which extent water cycle microbial community composition, as inferred by partial 16S rRNA gene sequences, can predict plasmid permissiveness, i.e. the ability of cells to receive a plasmid through conjugation, based on data from standardized filter mating assays using fluorescent bio-reporter plasmids. We leveraged a range of machine learning models for predicting the permissiveness for each taxon in the community, representing the range of hosts a plasmid is able to transfer to, for three broad host-range resistance IncP plasmids (pKJK5, pB10, and RP4). Our results indicate that the predicted permissiveness from the best performing model (random forest) showed a moderate-to-strong average correlation of 0.49 for pB10 [95% confidence interval (CI): 0.44–0.55], 0.43 for pKJK5 (0.95% CI: 0.41–0.49), and 0.53 for RP4 (0.95% CI: 0.48–0.57) with the experimental permissiveness in the unseen test dataset. Predictive phylogenetic signals occurred despite the broad host-range nature of these plasmids. Our results provide a framework that contributes to the assessment of the risk of AMR pollution in wastewater systems. Availability and implementation The predictive tool is available as an application at https://github.com/DaneshMoradigaravand/PlasmidPerm., MOTIVATION: Wastewater treatment plants (WWTPs) harbor a dense and diverse microbial community. They constantly receive antimicrobial residues and resistant strains, and therefore provide conditions for horizontal gene transfer (HGT) of antimicrobial resistance (AMR) determinants. This facilitates the transmission of clinically important genes between, e.g. enteric and environmental bacteria, and vice versa. Despite the clinical importance, tools for predicting HGT remain underdeveloped. RESULTS: In this study, we examined to which extent water cycle microbial community composition, as inferred by partial 16S rRNA gene sequences, can predict plasmid permissiveness, i.e. the ability of cells to receive a plasmid through conjugation, based on data from standardized filter mating assays using fluorescent bio-reporter plasmids. We leveraged a range of machine learning models for predicting the permissiveness for each taxon in the community, representing the range of hosts a plasmid is able to transfer to, for three broad host-range resistance IncP plasmids (pKJK5, pB10, and RP4). Our results indicate that the predicted permissiveness from the best performing model (random forest) showed a moderate-to-strong average correlation of 0.49 for pB10 [95% confidence interval (CI): 0.44-0.55], 0.43 for pKJK5 (0.95% CI: 0.41-0.49), and 0.53 for RP4 (0.95% CI: 0.48-0.57) with the experimental permissiveness in the unseen test dataset. Predictive phylogenetic signals occurred despite the broad host-range nature of these plasmids. Our results provide a framework that contributes to the assessment of the risk of AMR pollution in wastewater systems. AVAILABILITY AND IMPLEMENTATION: The predictive tool is available as an application at https://github.com/DaneshMoradigaravand/PlasmidPerm.

Urban wastewater systems (UWSs) are a main receptacle of excreted antibiotic resistance genes (ARGs) and their host microorganisms. However, we lack integrated and quantitative observations of the occurrence of ARGs in the UWS to characterize the sources and identify processes that contribute to their fate. We sampled the UWSs from three medium-size cities in Denmark, Spain, and the United Kingdom and quantified 70 clinically important extended-spectrum β-lactamase and carbapenemase genes along with the mobile genetic elements and microbial communities. Results from all three countries showed that sewage-especially from hospitals-carried substantial loads of ARGs (106-107 copies per person equivalent), but these loads progressively declined along sewers and through sewage treatment plants, resulting in minimal emissions (101-104 copies per person equivalent). Removal was primarily during sewage conveyance (65 ± 36%) rather than within sewage treatment (34 ± 23%). The extended-spectrum β-lactamase and carbapenemase genes were clustered in groups based on their persistence in the UWS compartments. The less-persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while the more persistent groups appeared horizontally transferred and correlated significantly with total cell numbers and mobile genetic elements. This documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based antibiotic resistance surveillance.

Widespread use of antibiotics has enhanced the evolution of highly resilient pathogens and poses a severe risk to human health via coselection of antibiotic resistance genes (ARGs) and virulence factors (VFs). In this study, we rigorously evaluate the abundance relationship and physical linkage between ARGs and VFs by performing a comprehensive analysis of 9,070 bacterial genomes isolated from multiple species and hosts. The coexistence of ARGs and VFs was observed in bacteria across distinct phyla, pathogenicities, and habitats, especially among human-associated pathogens. The coexistence patterns of gene elements in different habitats and pathogenicity groups were similar, presumably due to frequent gene transfer. A shorter intergenic distance between mobile genetic elements and ARGs/VFs was detected in human/animal-associated bacteria, indicating a higher transfer potential. Increased accumulation of exogenous ARGs/VFs in human pathogens highlights the importance of gene acquisition in the evolution of human commensal bacteria. Overall, the findings provide insights into the genic features of combinations of ARG-VF and expand our understanding of ARG-VF coexistence in bacteria.IMPORTANCE Antibiotic resistance has become a serious global health concern. Despite numerous case studies, a comprehensive analysis of ARG and VF coexistence in bacteria is lacking. In this study, we explore the coexistence profiles of ARGs and VFs in diverse categories of bacteria by using a high-resolution bioinformatics approach. We also provide compelling evidence of unique ARG-VF gene pairs coexisting in specific bacterial genomes and reveal the potential risk associated with the coexistence of ARGs and VFs in organisms in both clinical settings and environments.

Antimicrobial resistance genes are often carried by plasmids, which greatly facilitates their spread into microbial communities. Therefore, microbial community permissiveness (the propensity of a community to take up plasmids) and the diversity of the permissive members of the community constitute key parameters to understand and predict the fate of resistance genes. This is especially true for communities across the urban water cycle. Indeed, patients under antibiotics treatment excrete resistant microbes, which are collected and transported via the sewage collection network to wastewater treatment plants (WWTP). When these resistant, host-associated bacteria mix with other bacteria better adapted to life in the environment, there is a risk of transfer of resistance plasmids. Here, we measured permissiveness towards three gfp-tagged model broad host range plasmids for communities at multiples points of the urban water cycle (hospital and residential sewers, influent of the WWTP, main WWTP reactor) in three European cities. Permissiveness to pKJK5 was highest and varied between 8.5 x 10-4 and 1.3 x 10-2 transfer per recipient, and that to RP4 was about one order of magnitude lower. Permissiveness to these two plasmids were correlated and was highest for the residential sewer samples. The cells that received the plasmids were sorted using flow cytometry and characterized by 16S rRNA gene amplicon sequencing. Preliminary analysis identified genera that are consistently capable of engaging in plasmid uptake at most points of the urban water cycle, highlighting their potential role as facilitators of antimicrobial resistance dissemination.

There is increasing concern that wastewater treatment plants (WWTPs) may contribute to the dissemination of antimicrobial resistance genes (ARGs). Indeed, there transfer of plasmid-encoded ARGs might occur as human intestinal and environmental bacteria mix. Assuming that most enteric bacteria die off, ARG survival depends on transfer to and maintenance in WWTP adapted bacteria. Here, we explore, at the community level, the notion that plasmids cannot maximize both their within-population maintenance and their transfer proficiency, but that a tradeoff exists between these two properties. We provide the first assessment of the transfer and maintenance of typical ARG-carrying plasmids in a WWTP activated sludge microbial community. This community was challenged with Escherichia coli carrying one of three GFP-tagged plasmids spanning multiple important incompatibility groups: pKJK5 (IncP), R27 (IncH) and R64 (IncI). Transconjugants were separated by fluorescence-activated cell sorting and identified by 16S rRNA gene amplicon sequencing. pKJK5 transferred to a broad phylogenetic range of bacteria, spanning 13 phyla, while transfer of R27 and R64 was limited to Enterobacteriacaea. Following initial transfer, the communities were serially propagated over at least 60 generations, and plasmid persistence monitored. For pKJK5, high plasmid-carrying fractions (up to 10%) were transiently observed, which decreased and remained above 1% by the end of the experiment. In contrast, R27 and R64 never reached high incidence. The description of the diversity of the plasmid carriers along the serial propagation experiment is pending, but will provide a unique insight into the dynamics of contrasting plasmids in complex microbial communities.

Lipoproteins of a malachite green (MG)-decolorizing bacterium Pseudomonas sp. JT-1 could bind MG to form green MG-Lipoproteins complexes, which prevented the decolorization of MG by triphenylmethane reductase.