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. 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. 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. 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.

Pseudomonas aeruginosa is an opportunistic pathogen causing healthcare-associated infections. Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative bacteria. Resistance arises through mutations in two-component systems (TCS) regulating the arn operon. Data on colistin resistance in P. aeruginosa from Pakistan remain limited. A total of 3189 clinical samples (urine, blood, sputum, pus, wound swabs) were cultured. P. aeruginosa was identified by Gram staining, biochemical tests (catalase, oxidase, API 20E), and oprL gene amplification. Antibiotic susceptibility was determined by disk diffusion and MIC strips. Resistance genes (PhoP, PhoQ, PmrA, PmrB, mcr-1, oprD) were detected by PCR and Sanger sequencing. Wild-type protein structures were retrieved from PDB; mutant structures were predicted using AlphaFold3. ANP (phosphoaminophosphonic acid-adenylate ester) was docked using MOE 2019.0102. Of 3189 samples, 384 (12.0%) yielded P. aeruginosa. Wound/pus (38.0%) and surgical wards (30.0%) were the predominant sources. Colistin and polymyxin B showed 99.0% susceptibility (MIC₅₀/MIC₉₀ = 1 µg/mL). High resistance was observed for Piperacillin–Tazobactam (96.4%), Aztreonam (70.6%), and Gentamicin (64.2%). oprD was the most prevalent gene (87.5%), followed by PmrB (54.0%), PhoQ (44.0%), PhoP (36.0%), PmrA (18.0%), and mcr-1 (8.0%). Docking revealed the strongest binding in wild-type PhoQ (1ID0; −12.0 kcal/mol, LYS392), wild-type PmrB (2JSO; −9.8 kcal/mol, ASP37), and wild-type PhoP (2PKX; −9.1 kcal/mol, LYS87/ARG111). Mutant proteins showed reduced binding affinities and dispersed interaction networks. Mutant PhoP formed 16 contacts (strongest −4.3 kcal/mol) versus wild-type PhoP with 13 contacts (−9.1 kcal/mol). Colistin remains highly effective against P. aeruginosa in this setting (99.0% susceptibility). The presence of mcr-1 (8.0%) and high oprD prevalence (87.5%) require continued surveillance. Mutations in TCS proteins reduce ANP binding affinity and alter interaction specificity, suggesting that ATP-competitive inhibitors targeting these kinases merit further investigation and experimental validation.

Multidrug-resistant (MDR) Klebsiella pneumoniae represents a major clinical and public health challenge worldwide, particularly in regions with limited genomic surveillance. This study investigated the clinical, phenotypic, and genomic characteristics of clinical K. pneumoniae isolates recovered from a tertiary-care hospital in Peshawar, Pakistan. A total of 2400 non-duplicate clinical specimens were processed, and antimicrobial susceptibility testing was performed according to CLSI guidelines. Whole-genome sequencing (WGS) was conducted on a purposively selected subset of 18 isolates representing diverse resistance and phenotypic profiles. Genomic analyses included multilocus sequence typing, resistome and virulome profiling, identification of resistance-associated chromosomal mutations, plasmid replicon typing, and phylogenomic comparison with publicly available international genomes. K. pneumoniae was identified in 256/2400 (10.7%) specimens, predominantly from urine samples. MDR and extensively drug-resistant (XDR) phenotypes were detected in 83.2% and 13.3% of isolates, respectively. WGS revealed substantial genomic heterogeneity, with ST147 identified as the most frequent lineage among sequenced isolates. Extended-spectrum β-lactamase genes, particularly bla_CTX-M-15, together with carbapenemase genes including bla_OXA-48-like and bla_NDM-5, were identified in multiple isolates alongside resistance-associated chromosomal alterations in gyrA, parC, ompK36, mgrB, pmrB, and ramR. Yersiniabactin-associated loci were detected in all sequenced isolates, whereas canonical hypervirulence-associated determinants (rmpA, iuc, iro) were absent. These findings highlight the complex genomic landscape of MDR K. pneumoniae in Pakistan and underscore the need for continued genomic surveillance and antimicrobial stewardship.