Septicemia is a systematic inflammatory response and can be a consequence of abdominal, urinary tract and lung infections. Keeping in view the importance of Gram-negative bacteria as one of the leading causes of septicemia, the current study was designed with the aim to determine the antibiotic susceptibility pattern, the molecular basis for antibiotic resistance and the mutations in selected genes of bacterial isolates. In this study, clinical samples (n = 3389) were collected from potentially infected male (n = 1898) and female (n = 1491) patients. A total of 443 (13.07%) patients were found to be positive for bacterial growth, of whom 181 (40.8%) were Gram-positive and 262 (59.1%) were Gram-negative. The infected patients included 238 males, who made up 12.5% of the total number tested, and 205 females, who made up 13.7%. The identification of bacterial isolates revealed that 184 patients (41.5%) were infected with Escherichia coli and 78 (17.6%) with Pseudomonas aeruginosa. The clinical isolates were identified using Gram staining biochemical tests and were confirmed using polymerase chain reaction (PCR), with specific primers for E. coli (USP) and P. aeruginosa (oprL). Most of the isolates were resistant to aztreonam (ATM), cefotaxime (CTX), ampicillin (AMP) and trimethoprim/sulfamethoxazole (SXT), and were sensitive to tigecycline (TGC), meropenem (MEM) and imipenem (IPM), as revealed by high minimum inhibitory concentration (MIC) values. Among the antibiotic-resistant bacteria, 126 (28.4%) samples were positive for ESBL, 105 (23.7%) for AmpC β-lactamases and 45 (10.1%) for MBL. The sequencing and mutational analysis of antibiotic resistance genes revealed mutations in TEM, SHV and AAC genes. We conclude that antibiotic resistance is increasing; this requires the attention of health authorities and clinicians for proper management of the disease burden.

The current study investigates the prevalence of virulence genes obtained from clinical isolates of multidrug-resistant (MDR) Klebsiella pneumoniae at Khyber Teaching Hospital Peshawar, from October 2021 to January 2023. Upon proper consent, clinical samples of suspected UTIs patients were collected and inoculated on the nutrients agar media, McConkey agar media, and Cysteine Lysine Electrolyte Deficient (CLED) agar media followed by incubation at 37°C for 24 hrs. The phenotypic and genotypic identification were employed for the bacterial isolates. The phenotypic identification includes gram staining followed by the Analytical Profile Index (API 20E). A total of 215 (3.85%) positive isolates were found with the highest prevalence observed among the female patients (4.35%) followed by male (3.26%). The highest prevalence, constituting 52.55% (n = 113), was detected in the age group of 21-40 years, followed by 31.62% (n = 68) in the 41-60 age group. Additionally, 10.23% (n = 22), 3.25% (n = 7), and 2.32% (n = 5) of cases were identified in the age groups of 01-10 years, 11-20 years, and above 60 years, respectively. Among the total positive samples, 44.65% (n = 96) were collected from the Outpatient department (OPD), while inpatient department (IPD) cases contributed 55.35% (n = 119). The antibiotic susceptibility profile of K. pneumoniae showed significant resistance to trimethoprim/Sulfamethoxazole (93%) and Colistin (79.07%). Tigecycline emerged as the most effective antibiotic with a sensitivity rate of 90%, along with Cefepime at the same level. Minimum Inhibitory Concentration (MIC) values indicated higher resistance for CTX, MEM, CN, AK, DO, CIP, and SXT in K. pneumoniae-causing UTIs from KTH, Peshawar. Molecular characterization of virulence genes reveals the highest prevalence of fimH (80%) followed by SAT (65%), papEF (49%), afa (29%), and VAT (16%). The sequencing data of the virulence genes reveals mutations in fimH and papEF, while sat, afa and vat virulence genes showed no mutations. The Chi-square test indicated a significant association between the types of bacteria, supporting our null hypothesis with a significance level of p ≤ 0.05. The current study’s finding is to evaluate the rise of antibiotic resistance in hospital settings, which highly demands the focus of health authorities and clinicians to manage the burden of the disease effectively.

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.

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.