Prevalence of Milk-Borne Bacterial Pathogens via Touchdown Multiplex PCR to Unravel Incidence trends and Associated Risk Factors
Main Article Content
Keywords
Milk-borne pathogens, Conventional isolation, Characterization, TD-PCR, Antibiotic sensitivity, Prevalence, Risk factors
Abstract
Foodborne illnesses represent a substantial challenge for global public health. Milk, being a fundamental component of our dietary intake, offers crucial nutrients such as lipids, proteins, and various vitamins and minerals, notably calcium and phosphorus. However, milk also holds the potential to harbor harmful bacteria such as Staphylococcus aureus and Salmonella etc. posing significant health threats to consumers. This project aims to establish a reliable and sophisticated detection method to identify diverse pathogens in milk samples, along with focusing particularly on their prevalence rate in district Faisalabad, of province Punjab, Pakistan. Additionally, the study seeks to assess the antibiotic susceptibility profiles of these pathogens and some of the potential risk factors (such as animal herd size at the farm, age of animal, type of floor of the farm, farm hygiene, milking type i.e., manual or machine, udder hygiene, animal’s health condition and farm type etc.) that might affect the prevalence of food-borne pathogens. Milk samples (n=381) were procured from five distinct subdivisions (Faisalabad City, Chakk Jhumra, Jaranwala, Samundri and Tandlianwala) of district Faisalabad.. Initially, all the pathogens were isolated using traditional methods (bacterial culturing on media), and their characteristics were determined using several biochemical techniques, in silico analysis and individual PCR analysis. The accuracy of TD-PCR was evaluated based on its specificity, sensitivity, and validity, demonstrating robust performance with 100% specificity and sensitivity. Analysis of the samples unveiled the presence of pathogens including E. coli, Salmonella, L. monocytogenes, S. aureus, and Campylobacter species, confirmed through in silico assessment via the Vitek-2 system, alongside DNA extraction and TD-PCR. 52% samples showed positive results for bacterial growth. Antibiotic susceptibility testing revealed varying levels of resistance among the isolates. Staph. Aureus and Campylobacter spp showed highest resistance (71% & 67%, respectively) to tetracycline, E. coli O157:H7 (78%) to streptomycin, Listeria monocytogenes (32%) to -Trimethoprim and Salmonella enterica (81%) to kanamycin. Through an exhaustive risk analysis, eight potential risk factors associated with milk sources and dairy farms were identified. Following the optimization of PCR assay, an examination of 231 raw milk samples was conducted from diverse sources disclosed the highest prevalence in Faisalabad city, reaching 57%. Risk factor analysis revealed that herd size, age, type of floor, farm hygiene, milking type udder hygiene, and animal’s health condition were found statistically in-significant with a p value higher than 0.05 whereas the risk factor of farm type was found statistically highly significant with a p value of 0.004. These findings carry significant implications for food safety protocols, commercial practices, and economic evaluations. The advancement of TD multiplex PCR as a dependable tool for scrutinizing raw milk samples marks substantial progress in bolstering food safety efforts in Pakistan
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