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 Shigella and Salmonella isolates in Belgium

Abstract

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 Shigella and 31 ESBL-producing Salmonella were sequenced with MiSeq and a representative selection of 20 Shigella and six Salmonella isolates additionally with MinION technology, allowing hybrid assembly. The bla CTX-M-15 gene was found to be responsible for a rapid rise in the ESBL Shigella 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 Shigella species and within Shigella sonnei between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing bla CTX-M-27 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 bla CTX-M in the chromosome of two Shigella isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in Salmonella , ESBL genes were only found on plasmids, of which bla CTX-M-55 and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical Shigella and Salmonella 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.

Genomic analysis of Shigella isolates from Lebanon reveals marked genetic diversity and antimicrobial resistance

“…CP099775.1), a plasmid from an S. sonnei isolate (S17BD05916) collected in Belgium in 2017 [53]. This alignment confirmed the presence of a complete ~111 kb IncFIB plasmid harbouring the bla CTX-M-15 gene proximal to the IS 1380 family transposase IS Ecp1 .…”

“…Circles from inside to outside indicate: (1) the nucleotide position of p4, a plasmid from S. sonnei isolate S14BD05406 collected in Belgium in 2015 [53]; (2) regions of p4 displaying high levels of sequence identity to regions from complete or draft genome assemblies of CL-011 ( S. flexneri 1c isolate, our study), CL-022 ( S.…”

“…One of the short-read assemblies (from S. boydii 10 isolate CL-045) revealed that the 111 026 bp contig carrying the IncFIB replicon was 99 % identical, over 100 % of the alignment, to p38 (GenBank accession no. CP099775.1), a plasmid from an S. sonnei isolate (S17BD05916) collected in Belgium in 2017 [ 53 ]. This alignment confirmed the presence of a complete ~111 kb IncFIB plasmid harbouring the bla CTX-M-15 gene proximal to the IS 1380 family transposase IS Ecp1 .…”

“… Circular map and comparative analysis of the IncI1 plasmids carrying the ESBL bla CTX-M-15 gene. Circles from inside to outside indicate: (1) the nucleotide position of p4, a plasmid from S. sonnei isolate S14BD05406 collected in Belgium in 2015 [ 53 ]; (2) regions of p4 displaying high levels of sequence identity to regions from complete or draft genome assemblies of CL-011 ( S. flexneri 1c isolate, our study), CL-022 ( S. sonnei isolate, our study), CL-039 ( S. sonnei isolate, our study), CL-042 ( S. sonnei isolate, our study), CL-052 ( S. sonnei isolate, our study) and CL-053 ( S. sonnei isolate, our study); and (3) antimicrobial drug resistance (AMR) genes. Asterisks indicate isolates with complete genomes.…”

ClonalTracker: a tool to elucidate dissemination patterns between vancomycin-resistantEnterococcus faeciumisolates

“…To ensure it, a good solution is to use hybrid assemblies obtained from combining both short and long-sequencing reads. This has proven to be a successful method to not only recover mobile genetic elements 40 but also to obtain more accurate and complete genomes 41 . Overall, the algorithm has been designed to assess clonal relatedness between VRE isolates.…”