Rapid Increase of CTX-M-Producing Shigella sonnei Isolates in Switzerland Due to Spread of Common Plasmids and International Clones

Abstract

The Swiss Centre for Antibiotic Resistance (ANRESIS) has recently noted an increase of extended-spectrum cephalosporin-resistant (ESC-R) Shigella sonnei isolates nationwide (3.8% in 2016 versus 37.5% in 2019). To understand this phenomenon, we analyzed 25 representative isolates (of which 14 were ESC-R) collected in Switzerland during 2016 to 2019. Whole-genome sequencing was achieved using both the Illumina and the Nanopore platforms. Both ESC-R and extended-spectrum cephalosporin-susceptible isolates belonged to sequence type 152 (ST152). The ESC-R isolates carried bla_CTX-M-3 in IncI1-pST57 (n = 5), bla_CTX-M-15 in IncFII (F2:A-:B-) (n = 5), bla_CTX-M-15 in IncI1-pST16, and bla_CTX-M-27, bla_CTX-M-55, or bla_CTX-M-134 in other IncFII plasmids (n = 1 each). Plasmids having the same bla and Inc group exhibited high degrees of genetic identity to each other but also to plasmids previously reported in other Enterobacterales Core-genome analysis showed that there were 4 main clusters, each of which included strains that differed by <58 single nucleotide variants (SNVs) and that consisted of both bla_CTX-M-positive and bla_CTX-M-negative isolates. Moreover, most isolates belonging to the same cluster shared an identical core-genome sequence type (cgST). For instance, cluster 1 included 4 isolates of cgST113036, of which only 3 harbored the IncI1-pST57 bla_CTX-M-3-positive plasmid. The 25 S. sonnei isolates were also subjected to phylogenetic comparison with deposited international strains. As a result, matching isolates (isolates that had the same cgST and that differed by <8 SNVs) have been reported in the United Kingdom, the United States, France, and the Netherlands. Overall, our results suggest that some common S. sonnei clusters can spread between continents and can be imported into other nations after international trips. Such clusters include, in part, isolates that do not possess bla_ESBL-harboring plasmids, indicating their tendency to acquire them from other Enterobacterales.

Keywords: CTX-M; ESBL; MLST; Shigella; WGS; cgST; core genome; plasmid; plasmid-mediated resistance.

FIG 1

BLAST comparisons of the sequences of S. sonnei bla_CTX-M-carrying plasmids against reference sequences. (A) Comparison of the sequences of five S. sonnei bla_CTX-M-3-carrying IncI1 plasmids against the sequence of the S. sonnei IncI1 plasmid pLC1477_18-1 (GenBank accession number CP035009) as a reference sequence. (B) Comparison of the sequences of five S. sonnei bla_CTX-M-15-carrying IncFII (F2:A-:B-) plasmids against the sequence of the K. pneumoniae IncFII (F2:A-:B-) plasmid pF93-2_1 (GenBank accession number CP026158) as a reference sequence. Rings were constructed using BRIG (BLAST Ring Image Generator; v0.95) software. Similarities with the reference plasmid are represented by the colored rings. Genome accession numbers are indicated in the key at the bottom. Red and blue arrows above the rings correspond to the gene features of interest. The delta symbol (Δ) next to the feature label corresponds to a partial/incomplete gene CDS. For each plasmid, we report the GenBank accession number, the species of isolation, the tree cluster from Fig. 2, the year, the plasmid name, and the plasmid size.

FIG 2

Analysis of the core-genome phylogeny of 25 S. sonnei isolates together with that of Italian strain LC-1477-18. For each strain, we show the strain, the collection year, the main β-lactamase (if present), and the cgST. The assembled WGS of strains is presented in a core-genome SNV tree. The differences in the SNV value (e.g., Δ = 1 SNV) corresponds to the number of nonidentical SNVs of the core genome between two strains. The four main clusters (gray boxes) were defined when the nucleotide identity across two or more strains was ≥97.5% of shared SNVs (difference of ≤65 SNVs). The cluster matrix shows the maximum nucleotide identity (in percent) between all strains across two clusters (top right corner) and the number of SNVs not shared among all strains compared. The scale bar (0.05) represents the average number of nucleotide substitutions per site. Asterisks represent identical cgSTs, as determined by CGE’s cgMLSTFinder (v1.1) program. ^a, the core genome represents the maximum total coverage (87%) of the alignment among all 26 S. sonnei conserved sequences, which corresponded to 2,608 SNVs; ^b, cluster 1 strains shared 98.77% SNVs; ^c, cluster 2 strains shared 98.01% SNVs; ^d cluster 3, strains shared 99.08% SNVs; ^e, cluster 4 strains shared 97.54% SNVs; ^f, in cluster 1 and cluster 2, bla_CTX-M-3 was consistently carried by the same IncI1-pST57 plasmid (Fig. 1A).

FIG 3

Enterobase SNV tree dendrogram of the Swiss S. sonnei strains (n = 25), the global lineage of strains (n = 114), strains with matching cgST (n = 16), and the Italian LC-1477-18 strain as a reference. The combined single nucleotide polymorphism profiles of all 156 strains mapped to the reference sequence are represented in a RAxML tree, corresponding to a total of 9,850 SNVs. Country labels are represented by colored circles (missing country labels correspond to strains IBESS820 from the Netherlands, Ss046 from China, and 53G from South Korea). Holt lineages I, II, III, GIII, and IV are presented in colored boxes. Dashed black brackets with lines correspond to zoom-in sections of the tree where the present study’s strains are clustered. For the Swiss isolates, we show the cluster (if any)/ESBL (if any), while for the international strains with a matching cgST, we show only the ESBL (if any). Among these strains, those detected in 2019 are indicated with an asterisk. The scale bar represents the average number of nucleotide substitutions per site. See Table S2 in the supplemental material for more information regarding the Parsnp program SNV analysis results for zoom-in sections 1 to 7: section 1, compared to the Swiss isolates possessing the same cgST, strains 811053, 266979, 175609, 152507, CFSAN091705, and 795376 show 0 to 1, 0 to 4, 1 to 5, 2, 5, and 4 SNVs, respectively; section 2, compared to the Swiss isolate, strain 526163 has the same cgST and shows 2 SNVs; section 3, compared to the Swiss isolates, strain 299890 has the same cgST and shows 2 to 3 SNVs; section 4, compared to the Swiss isolates possessing the same cgST, strains 190807, 201907857, 638735, IBESS820, 821179, and PNUSAE013040 show 6, 1, 1, 0, 8, and 1 SNVs, respectively; section 5, 61 SNVs are found between the Swiss isolate and the one isolate found in Egypt; section 6, compared to the Swiss isolate, strain 191891 has the same cgST and shows 2 SNVs; section 7, compared to the Swiss isolate, strain 524350 has the same cgST and shows 4 SNVs.