Isolation, phenotyping and genotyping of Listeria monocytogenes from food and wet market in Kuching and Samarahan Divisions, Srawak

In this study, occurrence of Listeria spp., especially Listeria monocytogenes was determined in 440 food samples (poultry, beef and seafood) and 447 of tabletop samples from 11 wet markets in Kuching and Samarahan district, Sarawak. Two enrichment broths; Buffered Listeria Enrichment Broth and Fraser Broth were used in the isolation of L. monocytogenes followed by plating on selective PALCAM agar. Suspected colonies (black with grey-green zone) were picked and then streaked on the CHROMagar Listeria, a chromogenic medium for detection and isolation of L monocytogenes from other Listeria spp. A total of 82 isolates were identified as L. monocytogenes by the formation of blue colonies with white halo on this agar. All positive isolates were confirmed using species-speciff PCR. Based on the presence of the hlyA gene, the 82 isolates were confirmed as L. monocytogenes. The result showed 16 (3.6%) of the food samples and 11 (2.5%) of the tabletop samples were positive for the occurrence of L. monocytogenes. This revealed a low occurrence of L. monocytogenes in food and tabletop samples. All 82 isolates were further studied to determine their antibiotic resistance and occurrence of plasmid. L. monocytogenes isolates were resistant to most antibiotics tested (nalidixic acid (100%), streptomycin (95.1%), ceftriaxone (89%), vancomycin (36.6%), chlorampenicol (12.2%), methacillin (12.2%), gentamicin (11%), kanamycin (8.5%), cephalothin (7.3%), tetracycline (7.3%) and erythromycin (3.7%)). However, all isolates were susceptible towards 3 antibiotics; ampicillin, bacitracin and carbenicillin. None of the L. monocytogenes isolates harboured plasmid. The results of antibiotyping and plasmid profiles showed that no relationship could be established between the isolates. Screening for presence of the virulence genes (in1A gene, iap gene, prfA gene and p1cA gene), 60 isolates were selected from food (30) and tabletop (30) samples. All the isolates tested showed positive results for the presence of iap gene and prfA gene. Only 81.7% V and 91.7% of the L. monocytogenes isolates contained inlA and p1cA gene, respectively. Four different genotyping methods (random amplified polymorphic DNA (RAPD)-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, BOX-PCR, and M13 fingerprinting) were applied for characterization of 50 representative L. monocytogenes isolates. Results of all genotyping methods showed high level of diversity among the isolates. However, each method displayed different discriminatory effects for differentiation of L. monocytogenes isolates. The RAPD-PCR (using primer GEN15009) allowed for the distinguishing and grouping of intraspecies of L. monocytogenes isolated from food and tabletop samples. RAPD profiles indicated that none of the food isolates from the same market showed identical profiles with the tabletop isolates. This result revealed that the source of contamination of L. monocytogenes in food samples was not due to cross-contamination with isolates that originated from the tabletop. In this study, data collected for antibiotics resistance incidence among L. monocytogenes will provide information that is important in minimizing the possibility of listeriosis outbreak. In the event of L. monocytogenes infection in Kuching and Samarahan districts, this data will provide useful information on antibiotics that can be used for the treatment of L. monocytogenes. In addition, the data obtained from screening of the virulence genes will also contribute more information related with the presence of these genes in L. monocytogenes. For the genotyping analysis, RAPD-PCR could be a powerful tool for the investigation of L. monocytogenes in food, animal and environmental