Comparative Evaluation of Biochemical Methods and PCR in the Detection of Listeria monocytogenes
Comparative Evaluation of Biochemical Methods and PCR in the Detection of Listeria monocytogenes
Mahdis Mohammadjani,1,*Nasser Harzandi,2Azam Haddadi,3
1. Master's degree, Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran 2. Assistant Professor, Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran 3. Assistant Professor, Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran
Introduction: Listeria monocytogenes is a Gram-positive, non-spore-forming coccobacillus that can be found in various environments, including soil, water, and food. This bacterium is a foodborne pathogen that can resist disinfectants, form biofilms, survive under harsh conditions, and grow in different types of food. Listeria monocytogenes can cause disease in humans, especially in individuals with compromised immune systems, the elderly, infants, and pregnant women. This study was conducted due to the risk posed by diseases caused by Listeria monocytogenes and the importance of its detection in food and the environment using certain biochemical and molecular methods.
Methods: ffgIn this study, 100 food and environmental samples were collected, including 20 samples of milk, 20 samples of cheese, 10 samples of nuggets, 10 samples of leek, 10 samples of smoked fish, and 30 environmental samples, consisting of 19 soil samples and 11 water samples, from the cities of Shahriar and Andisheh during the summer and autumn seasons in 2023. For isolation and identification, two stages of enrichment and one stage of culture on selective solid medium were performed. In the first enrichment stage, 10 grams of each cheese, leek, smoked fish, chicken nugget, and soil sample, and 10 ml of milk were transferred to 90 ml of the selected liquid culture medium, Tryptic Soy Broth (TSB), and incubated at 37°C for 24 hours. For water samples, 500 ml of each was filtered through a 0.45-micron Millipore syringe filter, which was then transferred to the TSB medium. In the second enrichment stage, 0.1 ml of the cultured sample in TSB was added to 10 ml of Fraser broth and incubated at 37°C for 48 hours. Then, a culture was performed on PALCAM agar solid medium. For this, one loop of the Fraser broth culture (after 48 hours of incubation) was streaked in four stages on a PALCAM agar plate and incubated at 37°C for 48 hours. Suspected colonies were selected based on the colony morphology of the positive control grown on PALCAM agar. To separate them from other growing colonies, they were recultured on PALCAM agar. Then, biochemical tests, including catalase, CAMP, and motility tests in the SIM medium, were conducted. Finally, PCR was performed using the standard bacterial strain (ATCC19115) and primers specific to the listeriolysin O gene.fghj
Results: The preliminary results of comparing the colony morphology on PALCAM agar with the reference bacterium showed 36 suspected colonies that were morphologically identical to the reference bacterium. Seven were environmental samples, and the rest were food samples (11 milk, 11 cheese, 1 nugget, 2 smoked fish, 3 leeks, 4 water, and 4 soil). However, the results from biochemical tests varied: out of the 36 colonies analyzed, two were positive for motility, three were positive for the CAMP test, and one was positive for catalase, which in total, identified three of the 36 colonies as positive. Two colonies were both motility and CAMP positive, and one colony was both catalase and CAMP positive; all three were from food samples (two cheeses and one nugget). However, the PCR results indicated the formation of a 446-base-pair fragment, showing Listeria monocytogenes contamination in 22 of the analyzed samples, three of which were environmental samples (6 milk, 10 cheese, 1 nugget, 1 smoked fish, 1 leek, 2 water, and 1 soil).
Conclusion: The findings of this research revealed a significant discrepancy between the results obtained from biochemical and molecular tests. According to the molecular results, a considerable percentage of the samples were contaminated with Listeria monocytogenes, whereas the biochemical methods indicated a much lower percentage. This notable difference could serve as a valuable topic for future studies. Future researchers can not only repeat the experiment to investigate the reasons for the inconsistency between the two methods, but also examine clinical samples in addition to food and environmental samples.