Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan;9(1):mgen000925.
doi: 10.1099/mgen.0.000925.

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

Affiliations

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

Bas Berbers et al. Microb Genom. 2023 Jan.

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.

Keywords: Salmonella; Shigella; antimicrobial resistance; extended spectrum beta-lactamase; plasmids; whole genome sequencing.

PubMed Disclaimer

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
ESBL genes determined from short-read assemblies of 100 Shigella (a) and 31 Salmonella (b) isolates included in this study.
Fig. 2.
Fig. 2.
ProgressiveMauve alignment of the IncFII plasmids reconstructed with hybrid assemblies. The colours indicate locally collinear blocks (LCBs), which are homologous regions without rearrangements between two or more sequences. The p-numbers are the ESBL plasmids of the corresponding i-number isolates.
Fig. 3.
Fig. 3.
Accuracy of prediction by blast using the NCBI nt database, Mlplasmid, MOB-suite and plasflow on the Shigella hybrid assemblies. A prediction was considered correct when Mlplasmid and plasflow indicated with at least 70 % certainty that the ESBL contig was part of the correct structure as confirmed by the hybrid assemblies, for blast if >70 % of hits out of 100 matched it was considered a correct prediction and mob-suite did not give certainty values so the output was used as such.
Fig. 4.
Fig. 4.
Context of the ESBL genes (green), transposases (blue), hypothetical genes (grey) and other genes (purple) for chromosomal integration of ESBL genes in Shigella S17BD05200 (i36) and S17BD06357 (i41). The direction of the arrow displays the orientation of the genes. For i41 the closest gene is yhbX (not shown). Red bars indicate the locations of the inverted repeat right (IRR) and inverted repeat left (IRL).
Fig. 5.
Fig. 5.
Genetic context of the ESBL gene in isolate S17BD04134 (i34). Each arrow represents a gene and the direction of the arrow displays the orientation of the genes. Blue=transposase, green=antimicrobial resistance gene, grey=hypothetical gene.
Fig. 6.
Fig. 6.
Neighbour-joining tree based on a Plasmid pubMLST analysis on the short-read assemblies of Shigella isolates indicating that blaCTX-M-15 and blaCTX-M-27 isolates harboured different plasmids after 2017 compared to previous years. Each node represents at least one of the 100 Shigella isolates, where a bigger node represents multiple isolates. The distance between nodes corresponds to the number of different allele identifiers divided by the number of shared allele identifiers. Colours indicate the isolation year of isolates (a) or the blaCTX-M gene variant (b).
Fig. 7.
Fig. 7.
Clonal and interspecies transmission within bla CTX-M-15 and bla CTX-M-27 clusters in Shigella . Neighbour-joining tree based on ESBL plasmid alignments with filtered short-read assemblies showing more precise clustering on ESBL plasmid diversity that also separates bla CTX-M-15 and bla CTX-M-27 isolates. Each node represents at least one of the 100 Shigella isolates, where a bigger node represents multiple isolates. The distance between nodes is based on the average nucleotides aligned between genome pairs and then normalized to a value between 0 and 1. Colours indicate the isolation year of isolates (a) or the blaCTX-M gene variant (b) or the genotype (c). or the species (d). The clusters of bla CTX-M-15 and bla CTX-M-27 isolates are indicated with a blue and purple circle, respectively.

Similar articles

Cited by

References

    1. O’Neill J. Antimicrobial resistance: tackling a crisis for the health and wealth of nations. Rev Antimicrob Resist. 2014:1–16.
    1. World Health Organization Global action plan on antimicrobial resistance. 2015. - PubMed
    1. Puttaswamy S, Gupta SK, Regunath H, Smith LP, Sengupta S. A comprehensive review of the present and future antibiotic susceptibility testing (AST) systems. Arch Clin Microbiol. 2018;09:03. doi: 10.4172/1989-8436.100083. - DOI
    1. Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J Antimicrob Chemother. 2006;57:154–155. doi: 10.1093/jac/dki412. - DOI - PubMed
    1. Simonsen GS. Antimicrobial resistance surveillance in Europe and beyond. Euro Surveill. 2018;23:1800560. doi: 10.2807/1560-7917.ES.2018.23.42.1800560. - DOI - PMC - PubMed

Publication types

Substances

LinkOut - more resources