Epidemiological Survey And PCR-RFLP Subtyping Of Campylobacter Jejuni Isolates From Different Origins

Campylobacter is the most common zoonotic bacteria associated with human diarrhea in both developing and developed countries. Guillain-Barre syndrome is the most common acute flaccid paralysis due to an autoimmune disorder in nature. A considerable number of GBS patients present with a prior history of Campylobacteriosis, and GBS is considered a sequela of infections caused specifically by C. jejuni. Campylobacter is normally contracted through consumption of contaminated foods including those derived from food animals. Furthermore, an emerging public health concern regarding Campylobacter is the issue of microbial food safety due to increasing numbers of antimicrobial-resistant isolates. The objective of this study is to: (i) investigate the prevalence of C. jejuni and C. coli among diarrhea patients at Yangzhou and dairy cattle in Jiangsu Province; (ii) identify the isolates by phenotypic tests and detect the antimicrobial susceptibility with K-B method recommended by Clinical and Laboratory Standard Institute in 2005; (iii) track the source of C. jejuni isolates form different origins by PCR-RFLP.1. Prevalence of C. jejuni and C. coli among diarrhea patients and dairy cattleIn this study we attempted to investigate the prevalence of C. jejuni and C. coli among diarrhea patients at Yangzhou and dairy cattle in Jiangsu Province, 2,962 of human feces samples from three different hospitals at Yangzhou and 1,531 of cloaca samples from ten cattle farms were collected for the isolation and identification of C. jejuni and C. coli. The results showed that there were 143 strains of C. jejuni and 10 strains of C. coli isolated from 2,962 human feces samples, the average positive rate was 4.83% and 0.34% respectively. Outpatients and children≤7 years old had higher prevalence rate than that of inpatients (p<0.01). The detection rate of infection during June to August had higher prevalence rate than that of other months (p<0.05). 119 strains of C. jejuni and 3 strains of C. coli were isolated from 1,531 cloaca samples of 10 different cattle farms, the average positive rate was 7.77% and 0.20%, the highest positive rate of C. jejuni and C. coli was 30.91% and 3.57% respectively. The prevalence of C. jejuni in milk cow, heifer and environment samples was 5.02%, 8.70% and 10.28% respectively, and of C. coli was 0.32%, 0.00% and 0.18%, respectively. All results obtained from this study showed that endemic of Campylobacter spp. became diverse and complication in different colonies and provided well foundation for the right evaluation and established viable measures of Campylobacter spp..2. Antibiotic resistance of C. jejuni and C. coli isolated from humans and cattleThis study was undertaken to investigate the biological properties and antibiotic resistance of C. jejuni and C. coli from diarrheal samples and cloaca samples of cattle. All the isolates were positive in oxidation tests and the hippurate hydrolase of 128 C. jejuni were also positive, the hippurate hydrolase of all other 13 C.coli were negative. The results suggested that all the isolates were Campylobacters and all the isolates of C. jejuni didn't lose the ability to hydrolyze hippurate.The 93 isolated C. jejuni from patients with sporadic diarrhea were analyzed for susceptibility to 21 kinds of antibiotics, and the results showed that these isolated strains were highly sensitive to seven kinds of antibiotics such as Gentamicin 91.40%, Azithromycin 89.25%, Erythromycin 88.46%, Amoxicillin 87.10%, Streptomycin 83.87%, Cefotaxime 81.72%, Kanamycin 81.72%, and were resistant to some frequently used medicines such as Cefoperazone 100%, Co-trimoxazole 100%, Cephalexin 97.85%, Cefaclor 93.55%, Ciprofloxacin 91.40%, Nalicixic acid 91.40%, Levofloxacin 88.17%, Oxytetracycline 87.50%. The percentage of isolates were 81.71% with resistance to 9 to 12 antimicrobials, and the multidrug resistance of isolated strains from outpatients was more severe than that of other isolated strains.Antibiotic resistance of 35 isolated C. jejuni strains from cattle were evaluated with 21 kinds of antibiotics, and the results showed that these isolated strains were highly sensitive to seven kinds of antibiotics such as Amoxicillin 100%, Azithromycin 100%, Streptomycin 97.14%, Gentamicin 94.29%, Erythromycin 91.43%, Cefotaxime 82.86%, Clindamycin 82.86%, and were resist to some frequently used medicines such as Cefoperazone 100%, Ofloxacin 100%, Ciprofloxacin 97.14%, Co-trimoxazole 97.14%, Levofloxacin 94.29%, Nalicixic acid 94.29%, Cephalexin 94.29%, Norfloxacin 91.43%, Cefaclor 88.57%. The percentage of isolates was 88.57% with resistance to 9 to 12 antimicrobials, and the multidrug resistance of isolated strains from cow was more severity than that of other isolated strains.Antibiotic resistance of 13 isolated C. coli strains were all evaluated by 21 kinds of antibiotics, and the results showed that these isolates were only sensitive to five kinds of antibiotics such as Cefotaxime 100%, Amoxicillin 92.31%, Erythromycin 92.31%, Azithromycin 92.31%, Clidamycin 84.62%, and were resistant to many frequently used antibiotics such as Cephalexin 100%, Cefoperazone 100%, Norfloxacin 100%, Ofloxacin 100%, Ciprofloxacin 100%, Levofloxacin 100%, Nalicixic acid 100%, Co-trimoxazole 100%, Tetracycline 100%, Cefaclor 92.31%, Doxycycline 92.31%, Oxytetracycline 88.89%. The percentage of isolates was 76.91% with resistance to 11, 13, 15, 16 kinds of antimicrobials, compared with the isolates of C. jejuni, the multidrug resistance of isolated C. coli was more severe.The results of this study showed that the isolated strains were resistant to a number of common antibiotics used in clinic. For instance, Cefoperazon, Cephalexin, Cefaclor, Ofloxacin, Co-trimoxazole, Ciprofloxacin, Nalicixic acid, Levofloxacin, Oxytetracycline and so on. The percentage of isolates was 92.2% with resistance to more than 9 kinds of antimicrobials. Worryingly, the multidrug resistant isolates were developed in the process of treating other kinds of infections, not Campylobacteriosis. So we should pay more attention to the development of antimicrobial resistance in Campylobacter spp. to prevent multidrug resistant isolates emerging. In a word, antimicrobial resistance of the Campylobacter spp. became more and more diverse and complicated in our country. Monitoring systems for Campylobacter spp. are important for inspecting the trends of antimicrobial susceptibility and supporting development of public policies for better prevention and treatment of Campylobacteriosis.3. Genotyping of C. jejuni isolates from different origins using PCR-fla-RFLP subtyping methodThe major objective of this work was to explore the genetic similarity between C. jejuni isolated from different sources. C. jejuni isolated from different sources (n=329) were subtyped by PCR-fla-RFLP. A flagellin gene fragment of 1.7kb was amplified from 277 C. jejuni isolates by PCR with a pair of fla gene specific primers. Digested with DdeⅠ, the PCR products showed twenty different banding profiles. Profile number 2, 5, 6, 7, 9, 11, 12, 15, 16, 18, 20 were the most frequently observed among different sources, accounting for 82.67%. In this paper, profile number 2 and 5 were predominant in the isolates from humans with sporadic diarrhea, retail poultry, bovine feces and poultry feces, which indicated that the genotype distribution of C. jejuni in poultry and bovine are similar to those of human Campylobacteriosis. We also observed that profile number 6, 11, 15, 18 were predominant in human and poultry isolates, while genotype 7 and 16 were predominant in human and bovine isolates. These findings indicated that genotype distribution in human isolates overlaps with genotype distribution in poultry and bovine isolates, presenting the possibility that bovine, as well as chickens, may serve as a source of human Campylobacteriosis. The results also show that PCR-fla-RFLP could be a powerful tool for the investigation of molecular epidemiological pattern of C. jejuni and for tracing the source of C. jejuni in clinical patients.