Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins

Abstract

Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the bla_CTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S Typhi clone in Sindh, Pakistan. The XDR S Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S Typhi observed here adds urgency to the need for typhoid prevention measures.

Keywords: Salmonella; Typhi; antibiotic resistance; typhoid.

FIG 1

XDR isolates from Sindh, Pakistan, form a distinct cluster within the H58 phylogeny. (a) An unrooted maximum-likelihood phylogenetic tree of 98 Sindh, Pakistan, H58 isolates; 1 United Kingdom traveler isolate; and 853 global H58 isolates inferred from 1,920 SNPs using 22420_1_10_Pak60006_2016 as a reference. The XDR samples (red branches) are separated by 6 SNPs from the rest of the branch (blue), which is separated by 17 SNPs from the other H58 isolates. (b) A higher-resolution diagram of the branch from panel a rooted on nearest neighbor 10060_5_62_Fij107364_2012 displaying data for each isolate: country, sample collection (XDR or contextual organisms collected in Sindh, Pakistan, from 2016 to 2017; XDR organism from United Kingdom traveler to Pakistan; or organism from the global collection), plasmid content (p60006, described in this study, or pHCM2 cryptic plasmid), presence of chromosomal AMR cassette (integrated composite transposon), and quinolone resistance-determining region (QRDR) mutations according to the color key.

FIG 2

XDR S. Typhi isolates from Sindh, Pakistan, contain antibiotic resistance genes on the plasmid and chromosome. Genetic map of regions on the plasmid and chromosome from 22420_1_10_Pak60006_2016 containing antibiotic resistance-associated features. Chromosomal antibiotic resistance region refers to the composite transposon observed previously in H58 isolates. Regions with BLAST identity of >99% are shown in gray. The figure was made using genoPlotR (34).

FIG 3

The plasmid from XDR S. Typhi isolates from Sindh, Pakistan, is highly similar to global E. coli-associated plasmids. Plasmid p60006 was compared to representative isolates from each study described in Table S2 using BRIG (35). The innermost circle shows GC content, and the outermost circle shows the gene map, colored red for drug resistance or green for transfer activity.