Abstract

Background:
Dairy products are regularly implicated as reservoirs for various bacterial contaminants capable of harboring antibiotic resistance and virulence genes. The spread of these genes is of major concern to public health in the presence of virulent and multidrug-resistant strains that persist in various processing locations. The molecular characterization using techniques such as specific-target PCR and Random Amplified Polymorphic DNA (RAPD)-PCR genotyping are effective in determining the presence of such genes and in characterizing the bacterial diversity in the dairy processing environment.

Aim:
This study aimed to identify antibiotic resistance and virulence genes in bacterial isolates obtained from cheese, yogurt, and cream and assess their molecular diversity using RAPD-PCR.

Methods:
Samples of 290 dairy products were collected over a continuous 67-day period from various local markets and retail outlets. These included 98 cheese samples, 95 yogurt samples, and 97 cream samples. All samples were collected in sterile containers and transported to a laboratory under refrigerated conditions (4°C). To minimize bacterial overgrowth or loss of viability, samples were processed within 1-2 hours of collection. For each sample, 10g or 10 mL of each sample was added to 90 mL of sterile buffered peptone water and homogenized. These were then serially diluted (10⁻¹–10⁻⁵). Aliquots of each dilution were placed into selective and differential culture media, such as MacConkey, blood, XLD, mannitol salt, and bile esculin, to recover both gram-negative and gram-positive bacteria. They were incubated under aerobic conditions at 37 °C for 24-48 hours. Presumptive colonies were selected from the macroscopic examination and further biochemical tests. After purification, sub-culturing portions of the isolates were stored in nutrient broth containing 20% glycerol and frozen at −20 °C.

Results:
Among 290 total samples, 136 bacterial isolates were recovered and identified: 19 of Escherichia coli, 19 of Staphylococcus aureus, 16 of Listeria monocytogenes, 18 of Pseudomonas aeruginosa, and 15 of Salmonella enterica, among others. Hemolysin genes were detected among 47.79% of the isolates, with the highest rates for the cheese samples at 54.34%. Genes for adhesion were detected in 51.47% of samples, with isolates for the cream samples being the highest at 58.69%. Multiple bacterial isolates were found to demonstrate the presence of the virulence factors in combination. RAPD-PCR with 80% similarity succeeded in splitting the Gram-negative isolates into 8 and Gram-positive ones into 4 distinct groups, demonstrating significant molecular variability and likely indicating several contamination points in the dairy industry.

Conclusion:
This study clearly demonstrated the significant presence of virulence factors and antibiotic-associated genes for the dairy industry bacteria, including the genetic heterogeneous nature of the bacteria as analyzed by RAPD-PCR, calling for greater sanitary handling of dairy and ongoing molecular monitoring to reduce the presence of virulence-carrying bacteria in dairy for human consumption.

Key words: Adhesion genes; Antibiotic-resistance genes; Dairy bacteria; RAPD-PCR.