Ph.D Defence of Muhammad Jawad Ullah

Mr. Muhammad Jawad Ullah, Ph.D. Research Scholar has submitted thesis on "Crispr Arrays Typing and Molecular Characterization of Salmonella enterica Isolated from Beef Samples from Peshawar" to the University of Peshawar, in partial fulfillment of the requirements for the award of degree of Doctor of Philosophy (Ph.D.) in Microbiology.

The oral examination (Public Defence) is scheduled to be held on August 04th 2026 at 10.00 p.m. in the Centre of Biotechnology & Microbiology, University of Peshawar. The abstract of the thesis is attached herewith.

All those interested in the said research work may participate in the event. They may raise relevant questions during presentation by the scholar for further evaluation.

CRISPR ARRAYS TYPING AND MOLECULAR CHARACTERIZATION OF SALMONELLA ENTERICA ISOLATED FROM BEEF SAMPLES FROM PESHAWAR

Abstract

Salmonella enterica is one of the most important causes of foodborne illnesses worldwide, with beef being one of the major sources for transmission of the pathogen, especially in low- and middle-income classes where food safety systems are still weak. Peshawar is the capital of Khyber Pakhtunkhwa (KPK), in the north of Pakistan, which is considered a high-risk environment, as conventional slaughtering exists within the city whereas cold chains are not well-maintained and antibiotics are used unregulated both for animal rearing and for humans. This study estimated the prevalence, seasonal and geographical distribution, phenotypic and genotypic characteristics of Salmonella enterica isolates from retail beef in Peshawar, Pakistan. A total of 250 beef samples were collected from February 2021 to January 2023 from 23 retail outlets, butcheries, and slaughterhouse sites. Salmonella isolation was performed using standard cultural methods (Buffered Peptone Water enrichment, XLD and SS agar), followed by biochemical identification using a 12-test panel. Of 170 confirmed isolates, 150 high-confidence isolates were selected for further characterization, including biofilm formation, antimicrobial resistance profiling, virulence and molecular resistance gene detection, CRISPR array analysis, and clonal diversity assessment. The biofilm formation was investigated by Congo red agar (CRA), test tube and microtiter plate method. Sixteen antimicrobial agents representing ten classes were tested for antimicrobial susceptibility using the disk diffusion method according to CLSI 2022 guidelines. Eight virulence genes and 13 resistance genes including genes encoding ESBL (blaCTX-M, blaCTX-M-15, blaTEM, blaOXA), PMQR (qnrS, qnrB), aadA and sul1 and QRDR mutations in gyrA and parC were targeted by PCR based screening. The selected isolates were tested for blaTEM and blaCTX-M-15 using Sanger sequencing. Five representative isolates were characterised using the CRISPR arrays identification tools: CRISPRCasFinder, CRISPRDetect, CRISPRTarget and CRISPRcasdb-BLAST. RAPD-PCR band profiles, resistance gene co-occurrence patterns and the CRISPR spacer data were integrated, and the genetic similarity was calculated using Dice's coefficient, which were then constructed as dendrogram using UPGMA of Mega 11 for inferring clonal relationships. The highest prevalence of Salmonella enterica was found in 68.0% (170/250) sample, which is highest so far reported in retail beef of any city in Pakistan. The prevalence was positively correlated with the mean monthly temperature (Spearman rs?=?0.87, p<0.001), with the highest prevalence occurring during the month of July (86.2%) and the lowest prevalence in January (43.8%). The higher contamination was observed in slaughterhouse vicinity (71.1%) and urban centres (72.9%) and significant factors were slaughterhouse proximity to live animal markets (OR?=?3.28), and lack of refrigeration (OR?=?2.64), and being a roadside stall (OR?=?2.08). The mean bacterial load obtained was 1.9 ×?10? Cfu/mL, in 44.1% of the samples there was a bacterial load level of >?10? Cfu/mL. Biochemical confirmation was done for all 170 isolates which confirmed it to be S. enterica with a conformity of ≥90% for 12 tests. Biofilm production by microtiter plate assay was found to be present in 87.3% of isolates and strong production ability was obtained by 25.3% of isolates. Higher antimicrobial resistance was found to azithromycin (87.3%), tetracycline (61.3%) and streptomycin (48.0%). Eighty-five (56.7%) and ten (6.7%) of the isolates were MDR (≥3 antimicrobial classes) or XDR (≥6 classes), respectively. (≥6 classes), respectively. The average MAR index was 0.347?±?0.206 with 72.0% of the isolates being beyond the high-risk limit of 0.2. ESBL genes were detected in 28.7% (blaCTX-M-1), 24.7% (blaTEM), 18.7% (blaCTX-M-15), and 2.7% (blaOXA) of isolates; blaSHV and blaPER-1 were not detected. The prevalence of PMQR genes qnrS and qnrB were 24.7% and 18.7%, respectively, and QRDR mutations in gyrA were 26.7%. The invA virulence gene was detected in 98.7% of isolates, with spiA (42.0%), lpfC (40.7%), prgH (34.7%), and sopB (26.7%) also prevalent. Nucleotide identity confirmed 100% of blaTEM and blaCTX-M-15 with NCBI reference sequences (NG_050145.1 and NG_048935.1 respectively) with no mutations found. CRISPR analysis of four of five representative isolates revealed the presence of Type I-E arrays, and isolate 58C1 had a different Type I-B array, with spacer homology to Salmonella plasmid pECO1114, plasmid pSLT and to a Salmonella phage Fels-2. Among 150 Salmonella enterica isolates, 58 clonal groups were identified, most of which were only found in slaughterhouse vicinity sites; two clonal groups (C6 and C27) showed the broadest resistance and virulence gene profiles and were considered XDR. This study shows an alarming association of high prevalence, high bacterial load, high level of antimicrobial resistance, widespread carriage of virulence genes and high level of biofilm formation by Salmonella enterica bacteria from retail beef in Peshawar. The study findings indicate the serious public health emergency that urgently requires coordinated, concerted, One Health interventions in slaughterhouse sanitation and waste management, cold chain system enforcement, antibiotic stewardship at human and veterinary practices, and a well-functioning national foodborne pathogen surveillance system.

Keywords: Salmonella enterica; beef; antimicrobial resistance; ESBL; MDR; XDR; biofilm; virulence genes; CRISPR; Peshawar; Pakistan; One Health.