Using genomics to explore the epidemiology of vancomycin resistance in a sewage system

<i>VanHAX</i>-mediated glycopeptide resistance has been consistently high in one of the three main sewer systems in Copenhagen, Lynetten, for +20 years. To explore this for other glycopeptide resistance genes, and whether the colonization has resulted in establishment of multiple bacterial taxa, we mapped 505 shotgun metagenomic data sets from the inlet of three sewage treatment plants to 831 different glycopeptide resistance genes. Only <i>vanHAX</i> and <i>vanHBX</i> genes were differentially abundant in Lynetten. Analyses of eight contigs suggested limited variations in the flanking regions. Proximity ligation metagenomic analysis of 12 samples from Lynetten identified 441 and 5 paired reads mapping to <i>vanHAX</i> and <i>vanHBX</i>, respectively. The other end of these reads was mapped to generated metagenomic-assembled genomes and NCBI using BLAST. <i>vanHBX</i> could only be linked to the phylum level (Bacillota). Plasmid analysis of <i>vanHBX</i> Hi-C contigs showed that these were mainly located on plasmids reported found in enterococci species. Most <i>vanHAX</i>-linked reads could only be linked to phylum and class level, but some reads were assigned to <i>Enterococcus faecium</i> (7 reads), <i>Enterococcus faecalis</i> (4 reads), <i>Paenibacillus apiarius</i> (2 reads), and <i>Paenibacillus thiaminolyticus</i> (27 reads). Ten of the 20 Hi-C contigs-containing <i>vanHAX</i> were annotated as plasmid, all reported found in <i>Enterococcus</i> species. This study shows that while Hi-C technology is valuable for linking antimicrobial resistance genes to bacterial taxa, it suffers from challenges in reliably mapping the linked read to a genomic region with sufficient taxonomic information. Our results also suggest that over the +20 years of colonizing a sewer system, <i>vanHAX</i> has not become widespread across multiple taxa, remaining primarily in <i>E. faecalis</i> and <i>E. faecium</i>, with the exception of <i>Paenibacillus</i>.IMPORTANCELong-term colonization of microbial communities with antimicrobial-resistant bacteria is expected to result in sharing of the resistance genes between several different bacterial taxa of the communities. We investigated microbiomes from a sewer, which have been colonized with glycopeptide-resistant bacteria harboring the mobile <i>vanHAX</i> gene cluster for a minimum of 20 years, using metagenomics sequencing and Hi-C. We found that despite the long-term presence in the sewer, the <i>vanHAX</i> genes have seemingly not disseminated widely.