Identification of Multiple Variant Extensively Drug-Resistant Typhoid Infections across Pakistan

Abstract

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening bacterial infection. Recently, an outbreak of a new sublineage of extensively drug resistant (XDR) S. Typhi emerged in Pakistan in the province of Sindh. This sublineage had both a composite multidrug resistance transposon integrated on the chromosome and an acquired IncY plasmid carrying the extended spectrum beta-lactamase, blaCTX-M-15, which conferred resistance to third-generation cephalosporins. We observed previously that XDR typhoid had spread beyond the originating southern Sindh Province. Thus, we sought to determine the genetic diversity of 58 ceftriaxone-resistant S. Typhi clinical isolates by whole genome sequencing collected across Pakistan from November 2018 to December 2020 to provide insights into the molecular epidemiology of the evolving outbreak. We identify multiple novel genomic integrations of the extended spectrum beta-lactamase gene into the chromosome in S. Typhi, revealing the existence of various XDR typhoid variants circulating in the country. Notably, the integration of the IncY plasmid bearing antibiotic resistance genes may allow for subsequent plasmid acquisition by these variants, potentially leading to further plasmid-borne multidrug resistance. Our results can inform containment initiatives, help track associated outcomes and international spread, and help determine how widespread the risk is.

Figure 1.

Schematic of the composite transposon resistance region in the outbreak strain. XDR = extensively drug resistant.

Figure 2.

(A) Antibiotic resistance characteristics of isolates. (B) Age distribution of Salmonella enterica serovar Typhi cases isolated. Y = years.

Figure 3.

Phylogenetic tree of extended spectrum beta-lactamase–producing isolates in this study with the outbreak reference strain (NZ_LT882486) from Klemm et al. The different genomic architecture is described fully in the text and the date of collection is annotated for each isolate in the tree. All isolates were from Punjab, unless noted otherwise. Briefly, the label consists of isolate no.-genomic architecture/integration no.-date of collection-location (if not from Punjab). Genomic architecture A contains all previously identified resistance genes and plasmid replicons of the outbreak strain. Genomic architecture B contains an ∼7.8-kb deletion of the composite multidrug resistant transposon compared with the outbreak strain. A.1 and B.1 contain a variation of plasmid p6006 (p60006v1), which contains a small deletion. There are five different integrations of blaCTX-M-15 on the chromosome, which are illustrated in Figure 5.

Figure 4.

(A) Distribution of the genomic architecture variations among all Salmonella enterica serovar Typhi isolates (N = 58). (B) Distribution of all S. enterica serovar Typhi isolates by genomic architecture and location. KPK = Khyber Pakhtunkhwa; MDR = multidrug resistant.

Figure 5.

(A) Schematic of common deletion of Tn6029 in multiple isolates. (B–F) Schematic of gene architecture of chromosomal drug resistance integrations. Insertion sequences from p6006 are illustrated in the shaded box. ATPase = adenosine triphosphatase; mRNA = messenger RNA; XDR = extensively drug resistant.