Introduction and establishment of fluoroquinolone-resistant Shigella sonnei into Bhutan

Abstract

Shigella sonnei is a major contributor to the global burden of diarrhoeal disease, generally associated with dysenteric diarrhoea in developed countries but also emerging in developing countries. The reason for the recent success of S. sonnei is unknown, but is likely catalysed by its ability to acquire resistance against multiple antimicrobials. Between 2011 and 2013, S. sonnei exhibiting resistance to fluoroquinolones, the first-line treatment recommended for shigellosis, emerged in Bhutan. Aiming to reconstruct the introduction and establishment of fluoroquinolone-resistant S. sonnei populations in Bhutan, we performed whole-genome sequencing on 71 S. sonnei samples isolated in Bhutan between 2011 and 2013.We found that these strains represented an expansion of a clade within the previously described lineage III, found specifically in Central Asia. Temporal phylogenetic reconstruction demonstrated that all of the sequenced Bhutanese S. sonnei diverged from a single ancestor that was introduced into Bhutan around 2006. Our data additionally predicted that fluoroquinolone resistance, conferred by mutations in gyrA and parC, arose prior to the introduction of the founder strain into Bhutan. Once established in Bhutan, these S. sonnei had access to a broad gene pool, as indicated by the acquisition of extended-spectrum β-lactamase-encoding plasmids and genes encoding type IV pili. The data presented here outline a model for the introduction and maintenance of fluoroquinolone-resistant S. sonnei in a new setting. Given the current circulation of fluoroquinolone-resistant S. sonnei in Asia, we speculate that this pattern of introduction is being recapitulated across the region and beyond.

Keywords: Bhutan; Shigella sonnei; fluoroquinolone; resistance.

Fig. 1.

The phylogenetic structure of Bhutanese S. sonnei in the context of the global phylogeny and Central Asia clade. (A) Midpoint rooted maximum-likelihood phylogenetic tree of 183 S. sonnei strains (135 from global collection and 48 from Bhutan) reconstructed using 5393 SNPs; asterisks indicate bootstrap support values ≥ 98 % on major branches. Numbers above major branches represent ones leading to major lineages (I, II, III and IV). The light blue box highlights the Global III clade; the dark grey box overlaid on the tree highlights strains belonging to the Central Asia clade; the smaller, light grey box highlights the primarily Bhutanese Central Asia clade. (B) Magnified view of the maximum-likelihood phylogenetic tree of the Central Asia clade, including 74 S. sonnei strains (54 from Bhutan and 20 others for phylogenetic context), reconstructed using 996 SNPs; asterisks indicate bootstrap support values ≥ 80 %. The tree is midpoint rooted for purposes of clarity. Columns to the right of the phylogenetic tree show isolate metadata including: country of isolation (colours indicated in the key); the presence (dark blue) or absence (light blue) of specific genetic elements and mutations [ln2, spA, pSSE3, gyrA (S83L), gyrA (D87G), parC(S80I)], or the presence of an alternative mutation (yellow) [gyrA (D87Y)]; and resistance profiles against ciprofloxacin (CIP) and trimethoprim/sulfamethoxazole (SXT), where resistance is indicated in dark red, susceptibility in light red and missing data in grey.

Fig. 2.

Temporal phylogenetic reconstruction of Bhutanese Shigella sonnei between 2011 and 2013.Image shows a maximum clade credibility phylogenetic reconstruction of S. sonnei isolated primarily in Bhutan over the study period. Distinct subpopulations of Bhutanese S. sonnei are highlighted and indicated by letters. Black circles indicate posterior probability support ≥ 80 % on internal nodes. The asterisk indicates the branch leading to the Bhutanese S. sonnei clade, which represents 22 lineage-defining SNPs. Arrows denote branches characterized by select substitution events. Columns to the right of the phylogeny show metadata including: season (dark red, monsoon season: June–September; light red, other), and presence (dark blue) or absence (light blue) of mobile genetic elements, including specific genes (qnrS1, bla_CTXM-15, pilL–V locus) and plasmids (pHUSEC41-1-like, pSH146_65-like, pHUSEC2011-1-like, IncK). Grey bars in metadata columns indicate sequences from S. sonnei isolated outside of Bhutan.

Fig. 3.

Novel recombinant hybrid colicin biosynthesis cluster encoded on pSSE3.Schematic representation of the colicin biosynthesis cluster on plasmid pSSE3 in comparison with colicin gene clusters of pColE7-K317 and pColE6-CT14. Grey vertical bars show homology in DNA sequences (with identity >80 %). The colicin structural gene (cea), immunity gene (cei) and lysis gene (cel) are annotated as shown. The three domains of the colicin structural gene are shown the key.