Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical and isolates in Belgium.

For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated. The combination with long-read sequencing offers a potential solution, as it allows hybrid assemblies. Nevertheless, its use in surveillance has so far been limited. This study aimed to demonstrate its added value for AMR surveillance based on a case study of extended-spectrum beta-lactamases (ESBLs). ESBL genes have been reported to occur also on plasmids. To gain insight into the diversity and genetic context of ESBL genes detected in clinical isolates received by the Belgian National Reference Center between 2013 and 2018, 100 ESBL-producing and 31 ESBL-producing were sequenced with MiSeq and a representative selection of 20 and six isolates additionally with MinION technology, allowing hybrid assembly. The gene was found to be responsible for a rapid rise in the ESBL phenotype from 2017. This gene was mostly detected on multi-resistance-carrying IncFII plasmids. Based on clustering, these plasmids were determined to be distinct from the circulating plasmids before 2017. They were spread to different species and within between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing for which only clonal expansion occurred. Matches of up to 99 % to plasmids of various bacterial hosts from all over the world were found, but global alignments indicated that direct or recent ESBL-plasmid transfers did not occur. It is most likely that travellers introduced these in Belgium and subsequently spread them domestically. However, a clear link to a specific country could not be made. Moreover, integration of in the chromosome of two isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in , ESBL genes were only found on plasmids, of which and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical and isolates. The hybrid assembly data allowed us to check the accuracy of plasmid prediction tools. MOB-suite showed the highest accuracy. However, these tools cannot replace the accuracy of long-read and hybrid assemblies. This study illustrates the added value of hybrid assemblies for AMR surveillance and shows that a strategy where even just representative isolates of a collection used for hybrid assemblies could improve international AMR surveillance as it allows plasmid tracking.