Ph.D Defence of Ms. Faryal Khattak
Ms. Faryal Khattak, Ph.D. Research Scholar has submitted thesis on "Phylogenetic Analysis and molecular Profiling of Escherichia coli Recovered from Raw Milk Collected 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 July 8th, 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.
Abstract
PHYLOGENETIC ANALYSIS AND MOLECULAR PROFILING OF ESCHERICHIA COLI RECOVERED FROM RAW MILK COLLECTED FROM PESHAWAR
E. coli is a known food-borne pathogen responsible for many infections ranging from mild diarrhea to severe hemolytic uremic syndrome, a life-threatening infection. These bacteria are transmitted to humans through contaminated food such as raw milk, undercooked meat, and vegetables. Milk and other dairy products are considered excellent sources of energy and nutrients. Milk can be easily contaminated with E. coli during processing from the environment (water, soil, air) and cattle. Improper storage of milk results in the proliferation of these bacteria in milk. Intake of milk contaminated with E. coli is one of the main causes of food-borne illness. The contamination of milk with E. coli has not been frequently reported in Pakistan. The study focused on examining the presence, antibiotic resistance pattern, pathogenic potential, phylogenetic, and clonal relatedness of milk-contaminating E. coli. A total of 300 milk samples were collected from local shops in Peshawar in sterile Falcon tubes and screened for the existence of E. coli using differential and selective media. All the isolates were phenotypically identified using microscopy and biochemical tests. Disc diffusion procedure was carried out for antibiotic susceptibility testing using sixteen antibiotics. For phenotypic detection of extended-spectrum beta-lactamases, the double disc synergy test was used. Molecular detection of ESBL genes (blaTEM, blaSHV, blaCTX-M, blaOXA-10, and blaCTX-M15) and MBL genes (blaVIM, blaIMP, and blaNDM-1) was performed for all isolates using specific primers and PCR conditions. The presence of class I integron (int-I), class II integron (int-II), and integrase (int) genes was analyzed through PCR for all the isolates. Specific antibiotic-resistant genes (sul, aadA, and tetM) were analyzed in phenotypically resistant strains. All the isolates were analyzed for the occurrence of intestinal pathogenic E. coli by amplifying specific marker genes. Phylogenetic grouping of isolated E. coli was carried out with previously reported primers using the triplex PCR approach to classify all the isolates into specific phylogroups. Detection of mutations in the quinolone resistance-determining region of gyrA and parC genes in phenotypically quinolone-resistant isolates was carried out through MAMA PCR. For the estimation of genetic variability among isolates, ERIC-PCR was utilized. Mutational analysis of the blaCTX-M-15 gene in three ceftazidime-resistant isolates and the blaTEM gene in five intestinal pathogens and multidrug-resistant isolates was carried out using the Sanger sequencing method. Out of 300 raw milk samples analyzed 65.66% (n=197) were contaminated with E.coli. A total of 200 isolates were recovered. Antibiotic resistance pattern observed was: azithromycin 63.5% (n= 125), Cefoxitin 39% (n=78), Augmentin 23.5% (n=47), Streptomycin 19.5% (n=39), Ciprofloxacin 14.5% (n=29), Sulphamethaxazole/ trimethoprim 13% (n=26), Doxycycline 9.5% (n=19), Moxifloxacin 8% (n=16), Levofloxacin and Ceftazidime 6.5% (n=13), Colistin 6% (n=12), Gentamicin 1.5% (n=3), Imipenem and Fosfomycin 1% (n=2), Piperacillin tazobactam 0.5% (n=1), while no isolate was resistant to Meropenem. Phenotypically 3% (n= 6) isolates showed ESBLs production. Prevalence of ESBLs and MBL genes was blaCTX-M, blaTEM, blaCTX-M15 and blaNDM-1 51% (n=101), 66% (n=133), 11% (n=22), and 31% (n= 62) respectively. However, blaSHV, blaOXA-10, blaIMP and blaVIM were not identified in any isolate. The prevalence of int-I gene was highest that was detected in 98.5 % (n=197) isolates, followed by int gene 76.5% (n=153) and then int-II in 16% (n=32) isolates. The results showed that sul, aadA, and tetM genes were present in 26.93% (n=7), 57.14% (n=24) and 63.15% (n=12) isolates respectively. Among all isolates 7.5% (n=15) were identified as intestinal pathogenic E.coli. Triplex PCR for phylogenetic grouping revealed that 60.5 % (n=121) isolates belonged to group A, 35.5% (n=71) isolates belonged to group B1 and 4% (n=8) belonged group D. The results of MAMA PCR revealed that total 93% (n=26/28), 39% (n=11/28), 86% (n=25/29) and 97% (n=28/29) isolates had mutation at gyrA 87, gyrA 83, parC 80 and parC 84 respectively. ERIC PCR result showed high genetic diversity among isolates. All the isolates were grouped into 98 clusters based on similarity coefficient ≥ 85%. Mutational analysis results revealed no mutations in blaCTMX-15 genes in sequenced samples, while three silent mutations along with three novel mutations were spotted in blaTEM gene at position A107-G107, G344-A344 and C645-T645 that caused amino acid changes in peptide sequence at position 3 from Isoleucine toValine, at 92 fromValine to Isoleucine and at position 182 from Alanine to Valine respectively.
The study revealed a significant prevalence of bacterial contamination in milk, as well as multidrug-resistant and ESBL-producing E. coli, which suggests inadequate sanitation and hygiene. In order to control the spread of pathogenic bacteria, laws should be implemented to monitor milk regularly. Appropriate use of antibiotics, good sanitation conditions, and proper handling procedures are key factors in improving the microbiological quality and safety of milk.