4.2 Host range and gene flow
The 16S rRNA gene profile of the transconjugant communities allowed us to analyze the host ranges of R27 and pB10 in the recipient sewage community extracted from Ellinge WWTP influent. By alpha diversity measures of phylogenetic distances in the transconjugant communities, we found that both R27 and pB10 had a relatively broad potential host range (figure 3a). However it was clear that the host divergence of R27 was low, as indicated by the mean pairwise distance in contrast to pB10 (figure 3b). We found that the ASVs to which R27 and pB10 were transferred to in our conjugation assay with sewage communities, were constrained to seven families. Namely, AeromonadaceaeBurkholderiaceaeEnterobacteriaceaeFlavobacteriaceaeMoraxellaceaePseudomonadaceae and Shewanellaceae (figure 4a). R27 was predominantly transferred to members of the Enterobacteriaceae family (figure 4b). This observation justifies the low host divergence for R27 and emphasizes that this plasmid mainly is distributed within the Enterobacteriaceae family. In contrast, the transfer of pB10 is distributed more evenly among families and primarily occurring in two families i.e. Aeromonadaceae and Pseudomonadaceae (figure 4b). Other studies investigating transfer and following a similar approach have reported a far more diverse transconjugant community for the P1 plasmids. Indeed, the pool of transconjugants has been shown to include members from many phyla other than Proteobacteria, and even certain Gram positives (Klümper et al., 2015; Li et al., 2018; Musovic et al., 2014; Pinilla-Redondo et al., n.d.). This indicates that the conjugation assay used in this study is constraining the diversity of the recipient community (e.g., due to cultivation-related bias), thus limiting the maximum broadness of transfer.
Even though only a relatively low abundance of non-Enterobacteriaceae ASVs received R27, our observations expand the range of potential HI1A hosts considerably. We find transfer to genera within the families of FlavobacteriaceaeBurkholderiaceae, Moraxellaceae , Pseudomonadaceae and Shewanellaceae (figure 4A). Remarkably, this results in a grade VI host range for both R27 and pB10, according to the host range grade scale proposed by Redondo-Salvo et al. (Redondo-Salvo et al., 2020). Previous studies have so far only identified HI1A plasmids in two genera outside Enterobacteriaceae : Aeromonas and Vibrio, although both belong to the Enterobacteriales order (Maher & Taylor, 1993; Suzuki et al., 2010). Likewise, the plasmid database PLSDB (Galata et al., 2019), V2020_06_29, only contains one HI1A plasmid found in a host outside Enterobacteriaceae , a Pantoea sp. (Conlan et al., 2014). According to the NCBI taxonomy (Schoch et al., 2020), Pantoea is assigned to the Enterobacteriales family Erwiniaceae, yet according to the GTDB taxonomy (Parks et al., 2018) used in this study, it is assigned to Enterobacteriaceae . Therefore, it is possible that the majority of these broad host range events of R27 only reflect transfer host range, and are not stably maintained over time. Importantly, such short-term events may reflect crucial intermediate stepping-stones that perhaps circumvent spatial segregation of donor and suitable hosts, increasing the likelihood of reaching evolutionary stable hosts. However, these events do still raise a concern in terms of spread of ARGs. Since plasmids can integrate, fully or partially, into their hosts chromosomes, they can potentially spread ARGs to bacteria in which they are not evolutionary stable. and hence to groups of bacteria that we did not previously expect. Integrational events of plasmid into the host chromosome are well-documented and have also been found to increase plasmid susceptibility of the bacterial host (Tardif & Grant, 1983). Furthermore, many ARGs carried on plasmids are located within transposons, possibly allowing the ARGs to jump into the host chromosome or hitchhike onto new plasmids co-located in the host cell (Razavi et al., 2020). Halary et al. found that plasmids have a central role as key vectors of gene flow between bacterial genomes in nature (Halary et al., 2010). Thus, the broad plasmid transfer host range of R27, may facilitate gene flow across a surprisingly broad phylogenetic span.
Furthermore, our results indicate that HI1A plasmids may be highly involved in genetic exchange between Enterobacteriaceae, since there is a high abundance of transfer events to a relatively wide range of Enterobacteriaceae, among which several genera are known to include human pathogens. Our results thus highlight a potential link for gene flow between these pathogenic types, which requires further investigations. Other studies have likewise found proof of plasmids ensuring a high genetic interconnectedness between Enterobacteriaceae (Redondo-Salvo et al., 2020) . We found that R27 was specifically transferred to 5 out of the 9 genera with pathogenic members classified as ‘highest priority of critical pathogens’ (WHO, 2017); namely, Acinetobacter, Pseudomonas, Klebsiella, Escherichia and Serratia. This suggests that HI1A plasmids could serve as a genetic link between these genera in which resistances to cephalosporins and especially carbapenems are a major clinical issue (WHO, 2017). Antibiotic resistances, which are known to be encoded by HI1A and the closely related IncHI1B plasmids (Carattoli, 2013; Dolejska et al., 2013; Zurfluh et al., 2014).