Whole-Genome Sequencing Reveals the Resistome, Virulome, Pan-Genome Architecture, and Phylogenomic Diversity of Extensively Drug-Resistant Salmonella enterica Serovar Typhi in Pakistan

Abstract

Background: The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Salmonella enterica serovar Typhi has severely constrained typhoid treatment. Pakistan is a major hotspot for XDR typhoid, yet genomic evidence defining the molecular basis of antimicrobial resistance, virulence, and genomic diversity of circulating strains remains limited. Methods: A cross-sectional study was conducted from January to December 2021 involving 252 culture-confirmed S. Typhi isolates collected from patients with suspected typhoid fever in Peshawar, Pakistan. Isolates were identified by conventional microbiological methods, biochemical testing, and 16S rRNA PCR. Antimicrobial susceptibility testing (AST) was performed according to CLSI guidelines, and isolates were classified as susceptible, MDR, or XDR following WHO criteria. Fourteen non-duplicate XDR isolates were selected for Illumina whole-genome sequencing (WGS). Bioinformatic analyses included genome assembly, multilocus sequence typing (MLST), resistome and virulome characterization, mutation analysis, pan-genome analysis, and core-genome phylogenetic reconstruction. Pearson’s chi-square goodness-of-fit test was used to assess categorical distributions, with p < 0.05 considered statistically significant. Results: Among the 252 isolates, 152 (60.3%) were XDR, 73 (29.0%) were MDR, and 27 (10.7%) were susceptible. The distributions of sex, age group, and phenotypic classification differed significantly from equal expected proportions (p = 0.001, p = 0.013, and p < 0.001, respectively). Resistance was highest to ciprofloxacin, ampicillin, chloramphenicol, ceftriaxone, and co-Trimoxazole, whereas azithromycin and meropenem showed high in vitro activity. WGS showed that all sequenced isolates belonged to sequence type ST1 and had conserved genome sizes (4.84–4.95 Mb) with ~54% GC content. The resistome included blaCTX-M-15, blaTEM-1, catA1, sul1, qnrS1, aac(6′)-Iy, and dfrA7, with mutations in gyrA, parC, soxS, pmrA, and pmrB. Virulence genes associated with adhesion, type III secretion, Vi capsule biosynthesis, intracellular survival, iron acquisition, and typhoid toxin were conserved across all isolates. Pan-genome analysis indicated limited pan-genome expansion within the sequenced collection, while phylogenomic analysis showed high genetic relatedness among the sequenced isolates, indicating substantial genomic conservation within this collection. Conclusions: The high genomic relatedness and limited gene-content variation observed within the 14 sequenced XDR isolates support a genetically conserved collection, but do not by themselves establish ongoing transmission or clonal expansion. Larger and more geographically diverse genomic datasets are needed to determine the broader population structure of XDR S. Typhi in Pakistan. Continued genomic surveillance, appropriate antibiotic use, typhoid conjugate vaccination, and effective infection-control measures are needed to reduce transmission of drug-resistant typhoid.