Effect of Different Atmospheres for Isolation, Molecular Characterization, Pathogenicity Gene Screening, and Control of Campylobacter spp. from Processed Broiler Meat

Abstract

Our first experiments involved the studies of the effects of Oxyrase®, an enzyme based membrane system that selectively reduces oxygen from the medium, during the growth and isolation of Campylobacter spp. The effect of Oxyrase was tested without adding the microaerobic gas mixture for Bolton broth and an agar plate medium (modified Campy-Cefex). Based on chi-square analysis, no significant difference was found between enrichment broth cultured under microerobic conditions and enrichment broth that had Oxyrase. Comparison between plate media, however, showed that plates supplemented with Oxyrase had the lowest recovery rate (P < 0.05). The percentage of similar group of organisms after denaturing gradient gel electrophoresis (DGGE) analysis was 85% and of the different group of organisms was 15%. The analysis of Campylobacter isolates collected from broth with and without Oxyrase for restriction fragment length polymorphism (RFLP) showed similar band patterns for some strains and different band patterns for other strains. The second set of experiment examined if incubation under microaerobic conditions was indispensable for Campylobacter spp. to grow in Bolton broth. These experiments were also done with naturally contaminated broiler meat. Microscopy results from the two enrichment broth media showed no difference between the sub-samples for the growth and isolation of Campylobacter (chi square = 0.21). These results suggest that retail broiler meat enriched in Bolton broth may not need the addition of microaerobic gas mix for Campylobacter to multiply. DGGE showed the presence of different microbial niches in both the broth media. RFLP analysis showed band similarity for some samples and band differences for others. The third set of studies involved the molecular characterization of Campylobacter strains from carcasses rinses from four commercial processing plants. Three plants (A, B and C) had similar percentages of Campylobacter positive carcass rinses: 78%, 78% and 62%, respectively. Plant D, however, showed the lowest percentage (33%) of Campylobacter positive carcasses. The mean log CFU/ml was similar for all the four plants and was less than 1 CFU/ml. Pulsed-field gel electrophoresis (PFGE) analysis

revealed a considerable diversity in the band patterns among the isolates when the rinse of a single broiler carcass was direct-plated on different plate media. These results confirmed the presence of more than one strain of Campylobacter spp. on a single carcass. Campylobacter coli from different plants showed similar PFGE patterns. Next set of studies involved the screening of C. jejuni isolates for the presence of pathogenecity genes. Multiplex PCR assays were used to test for the presence of flhB, flagellar protein biosynthesis and export gene; flgB, flagellar basal body protein synthesis gene; flgE2, flagellar hook protein gene; docA, colonization and infectivity gene; and cdtA, a gene whose product makes the eukaryotic cells to be blocked in either G2 or early M phase. Multiplex PCR assays were successfully designed for the following gene combinations, fliM and docA; flgE2, cdtA and flhB; and cadF and flgB. Thirty-six C. jejuni isolates from one processing plant were positive for the presence of flhB, flgB, flgE2 and cdtA genes, but were negative for docA gene. However, 58 C. jejuni isolates from another poultry processing plant were positive for all these genes, including docA. In the last set of experiments, the effects of lemon based marinades were studied. Retail poultry meat positive for the presence of Campylobacter was used in these studies. The marinades that were used for the study included mesquite, baja chipotle, herb and garlic and real lemon. In inoculation trials, reduction in numbers of Campylobacter at the end of marination in combination with refrigeration at 0.5 h 6 h and 24 h were observed. In the case of naturally occurring Campylobacters on retail broiler meat, there was significant reduction in their numbers with all the marinades (baja chipotle, herb and garlic and real lemon) when marinated for 4 h under refrigeration was performed.