4.1. The plasmids transfer in the sewage community
In this study, we investigated the transfer dynamics of the HI1A plasmid R27 in the sewage communities of the influent water of three WWTPs in Sweden (Ellinge-, Klagshamn-, and Sjölunda-WWTP). We recorded transfer of R27 to the sewage community from all three WWTP, indicating the potential of HI1A plasmids as effective vectors for the spread of ARGs to the microbial communities of WWTPs (Figure 2a). Yet, we found that the transfer of the P1 plasmid pB10 to the recipient sewage community was significantly higher than that of R27 (Figure 2a). This could be a result of the broader host range of pB10 compared to R27 in the sewage community, enabling more transfer events. However, we also show that pB10 has a higher transfer than R27, in E. coli to E. coli conjugation (Figure 2b), but the relative difference between the transfer of the two plasmids is remarkably higher in the sewage community. Thus the higher transfer in the sewage community of pB10 is likely a result of both a broader host range and higher transfer efficiency. Broad host range plasmids, such as P1 plasmids are known to be common in WWTPs (Bahl et al., 2009). Furthermore, P1 plasmids have been shown several times to transfer well to WWTPs communities (Jacquiod et al., 2017; Li et al., 2018) and even increase the fitness of strains originating from WWTPs without plasmid specific selection (Li et al., 2020). Thus, we expected that pB10 would transfer well in the sewage community. However, the higher transfer of pB10 to the recipient sewage community, compared to R27, is not necessarily of high medical relevance. Since the medical relevance of a plasmid is likely dependent both on its host and accessory genetic cargo (e.g. virulence and resistance determinants). Additionally, the medical relevance also depends on the capability of the recipient sewage community to further disseminate the plasmid, potentially reaching infectious human pathogens.
The conjugative assay described in this work, mixing the enriched transconjugant sewage community harboring either R27 or pB10 and a recipient E. coli , simulated the transfer of ARGs from sewage communities to a potential pathogenic Enterobacteriaceae . The enriched transconjugant sewage communities were, for both plasmids, found to be potent plasmid donors with high transfer to the E. coli recipient, corresponding to the transfer of the respective plasmid from E. coli to E. coli (Figure 2b). Thus, the sewage community were not only recipients of R27 and pB10 but could also efficiently disseminate them further. Furthermore we found that, regardless of the donor-recipient combinations tested, R27 transferred significantly less than pB10, indicating that the difference in plasmid transfer ratios is plasmid dependent. Our results support a growing body of work exposing the sewage microbiome as a facilitator of HGT (Guo et al., 2017; Jacquiod et al., 2017; Li et al., 2018). To our knowledge, this is the first study showing that the sewage community additionally could facilitate conjugative transfer of HI1A plasmids resulting in an environmental spread of medical relevant ARGs.