Quantitative Analysis Of Campylobacter Spp. On Retail Chicken And Their Decontamination Treatment Based On Organic Acid

Campylobacter spp. is the most common zoonosis pathogen of foodborne infection reported after Salmonella and Shigella. Campylobacter can not only cause human gastroenteritis and food poisoning, but also keep closely contact with Guillian-Barr syndrome. Many poultries serve as reservoirs of Campylobacter, especially chickens. Commercial broiler chicken has been identified as an important vehicle, and a link between human cases of campylobacteriosis and the prevalence of Campylobacter infection in chicken was identified in a risk assessment.It is a key route to protect human from Campylobacter hazard that we should fully recognize its harmful impact to animal-derived food safety and take timely effective measures to control it. Microbiological quantitative testing provides information on consumer exposure to pathogens on raw meat just prior to potential outbreak in developed countries, and it can decrease the risk of cross-contamination. In developing countries, information on foodborne pathogens and disease is insufficient due to the inadequate data provided by the passive surveillance systems. To date, few quantitative analysis and surveillance data about the contamination level of retail meats have been available in China. The lack of available information on pathogen concentrations has been identified as an important data gap with respect to evaluating the effectiveness of intervention and control measures in primary poultry production and processing.This study was designed and performed to accurately obtain the baseline data of Campylobacter spp. on whole chickens sold in supermarkets and wet markets in Yangzhou city. Furthermore, the conditions for reducing Campylobacter load were optimized by response surface analysis method with Box-behnken software. On the other hand, the flgS mutant strain was constructed, and its acid-tolerance characteristics was identified. The results lay the foundation for further study of regulation mechanism and effective decontamination strategy of Campylobacter spp. on retail chicken.1. Quantitative analysis of the contamination level of Campylobacter on retail chickenTo assess the risks to consumers from Campylobacter spp. in whole raw chickens sold in Yangzhou city, a 12-month quantitative survey was performed in succession. In this study,480 samples were collected from supermarkets and wet markets from 2012 to 2013. The level of Campylobacter contamination was determined using the direct plating method and molecular biology method.The results of quantitative detection showed that the positive rate of all 240 samples was 51.3%, the mean value of Campylobacter enumeration was 1473.49 CFU/g. Among the 123 Campylobacter positive samples, the enumeration of 78.86% samples was higher than 20CFU/g. The positive rate of samples from supermarkets and open fairs was 35.8% and 66.7% respectively, and the corresponding enumeration of Campylobacter spp.was 152.3CFU/g and 2183.6CFU/g. Acordingly, the Campylobacter contamination level of wet markets was greater compared to that of supermarkets. Seasonality was observed in both qualitative and quantitative studies for both pathogens, with summer being the high-incidence season. Diversity of Campylobacter isolates demonstrated that C. jejuni (45.5%) and C. coli (30.9%) were the most common species, except for the mixed contamination. Survey results indicated that there was a need for more interventions to minimize the exposure of consumers to Campylobacter.2. Development and application of decontamination treatment based on organic acid for the control of Campylobacter spp. on broiler chickenAccording to the<GB 2760-2011 National Food Safety Standards of Food Additives>, screened the lactic acid as the most effective bactericide from the permissible acids. After the single factor analysis of Campylobacter sterilization effect and evaluation of 3 factors (concentration, time, temperature), the best interval was identified. To obtain the maximum of bacteriostasis and IMP, response surface analysis was conducted with 3 independent variables and 2 dependent variables by Box-Benhnken design (BBD). Low-concentration, long-time processing mode and high-concentration, short-time processing mode were constructed, the most optimal values were validated and applied in the chicken processing production line.According to the experimental results and the fitted quadratic polynomial model, the best parameters for low-concentration, long-time processing mode were selected as 0.19%,17min, 20.9℃. In this condition, the predicted value of bacteriostasis and IMP was 98.62%and 1.8678mg/g。And the best parameters for high-concentration, short-time processing mode were selected as 0.82%,6.86s, 7.97℃. In this condition, the predicted value of bacteriostasis and IMP was 98.81% and 1.8775mg/g。The error of the value in verification experiment was less than ±1%, which indicated that parameters were reliable. Applied it to the chicken processing production line, the positive rate of Campylobacter didn’t decrease sharply. Regarding to the number of Campylobacter and total bacteria, there is no significant difference in the two modes, and both are less effective than traditional method. What’s more, there is no significant difference in the combination of the two modes and traditional method.3. Construction of the flgS mutant strain of Campylobacter jejuni and its acid-tolerance characteristicsAccording to the C. jejuni NCTC11168 whole genome sequence published in GenBank and the map of plasmid pRY107, primers were designed to amplify flgS gene and the kanamycin resistance gene cassette. The target gene was ligated into the cloning vector pMD-19T and verified by sequencing respectively. By enzyme digestion and ligation reaction, the kanamycin resistance gene was inserted into the flgS gene to construct the suicide vector pMD19T-flgS-kanr. The suicide vector was introduced into C. jejuni NCTC11168 competent cells by electroporation, and the flgS mutant strain was selected through allelic recombination. The positive transformed colonies were selected on ager plate with antibiotics, and identified by polymerase chain reaction using specific primer.The acid-tolerant ability of the mutant strain and wild strain was evaluated. Confronted with the stress of both high or low concentration, resistance of mutant strain was significantly lower than the wild strain, which showed that the flgS gene plays an important role in the acid tolerance. Since the flgS gene was a regulation gene, the influence of inactivation to other stress genes was studied using real-time PCR. The results showed the expression of stress genes would increase in acidic environment due to the inactivation of flgS gene, indicating the flgS gene has a role in regulating stress gene expression, and the regulation is similar with rpoN (σ54) as one exception in two different acidic environment. The expression level of rpoN increased obviously in long-term acid stress, but not in short-term acid stress. Although the compensation exist, the survival rate of mutant strain decreased sharply in acidic environment. It was speculated that a complex network coordinate the stress genes of Campylobacter jejuni, and flgS play an irreplaceable role in acid-stress sense and confrontation.