| As an important zoonotic and foodborne pathogen, Campylobacter jejuni is one of the commonest causes of gastroenteritis in humans worldwide. Specific type has been linked to Guillain-Barre syndrome. In recent years, the incidence of Campylobacter jejuni infectionshas risen. It was estimated that Campylobacter jejuni couldmake 400 to 500 million people sick worldwide each year, which has caused the widespread concern.At present, the domestic research on Campylobacter jejunimainly focused on the identification, antibiotic resistance and molecular typing of strains isolated from limited area, while lacks systematic and large-scale research and the molecular typing methods are still imperfect. In this study, we used biochemical tests, VITEK 2 compact and multiplex polymerase chain reaction to identify Campylobacter jejunifrom national chicken special monitoring network, following antibiotic resistance analysis and mol ecu larsubty ping with pulsed-field gel electrophoresis,fluorescence-labeled amplified fragment length polymorphism and polymerase chain reaction-restriction fragment length polymorphism. One database for tracing analysis of Campylobacter jejuni was established based on these datacombined with background information of strains by BioNumerics software.Part I Isolation and identification of Campylobacter jejuni.Campylobacter spp. strains were isolated according to the recommended standard method of China. Identification on 81 suspected Campylobacter jejuni strains was performed by biochemical test according to the United States Bacteriological Analytical Manual (BAM) and by commercialization of VITEK 2 compact.Multiplex PCR wasused as an assistant test simultaneously.Results: The traditional biochemical tests were entirely consistent with the results of multiplex PCR, while the coincidence rate with the VITEK 2 was 85.2%, 91.7%and 33.3% for Campylobacter jejuni and Campylobacter coli separately. Traditional biochemical tests are still the standard method to identify Campylobacter jejuni.Results of VITEK 2 were instability so it can not be used as standard identification method. Multiplex PCR reaction can be used asan assistant method to biochemical tests.Part Ⅱ Phylogenetic analysis of 16SrDNA,23SrDNA and the Internal transcribed spacerof Campylobacter jejuni.In this study, primers were designed based on the conserved region of1 6SrDNA-23SrDNA. The alignment of internal transcribed spacer ( ITS)sequences was performed by BioNumerics with reference sequences fromGenBank. Phylogenetic tree was generated with maximum parsimony method by MEGA 6.0 to study the evolutionaryrelationships between isolates and reference strains. The results showed that strains from human and those from animals showed high sequence similarity, indicating that they werehomologous in molecular biology to some extent. Isolates used in this study were identified as Campylobacter jejuni subsp. jejuni and had been conservativein the evolutionary process .Partial isolates from several regions were potentially pathogenic and had a high possibility of outbreakina wide range. It is necessary to strengthen monitoring and prevention.Part III Antibiotic resistance analysis of Campylobacter jejuni.In this study, the antibiotic susceptibilities of 72 Campylobacter jejuni isolates were tested with agar dilution method against 14 antimicrobial groups from eight classes. The results showed that the resistance to quinolones, tetracyclines and cephalosporins was high. While resistance to penicillin amoxicillin/clavulanate acid and sulfa drugs showed moderate rates.Resistance to macrolides, aminoglycosides, lincosamides and meropenem appeared as low.Chloramphenicol resistance was 0%. 72 isolates generated 31 resistance patterns, and all strains were resistance to four or more antimicrobials. The results suggest that macrolides can still be used as the first choice of clinical treatment. Meropenem, gentamicin, streptomycin,lincomycin and chloramphenicol may be considered as candidates after assessed.Part Ⅳ Molecular subtyping of Campylobacter jejuni.a) 72Campylobacter jejunistrains were subtyped by pulsed-field gel electrophoresis (PFGE). Digestion with Sma Ⅰ showed high distinguish ability than Kpn Ⅰ.Combined Sma Ⅰ with Kpn Ⅰ yielded 48 PFGE types among 72 Campylobacter jejunistrains. According to 63.9% similarity, 72 isolates were assigned to 13 Sma Ⅰ -Kpn Ⅰ clusters. Isolates from different provinces scattered in different clusters, while strains with the same PFGE type displayedcomplete geographical homology, which may help tracing food poisoning pathogens. However, there were still 51.4% of the strains could not be distinguishedby PFGE method, indicating the limited ability of PFGE.So it’s wise to interpret the results reasonably combined with epidemiological data.b) 72Campylobacter jejunistrains were subtyped by fluorescentamplified fragment length polymorphism with Hind Ⅲand Hha Ⅰ . 241 polymorphic fragments were yielded among 72 isolates, while most isolates gave 80 to 100 fragments. 72 FAFLP types were yielded among 72 strainswith a resolution of 100%. According to 70% similarity, 72 strains were assigned to 11 clusters.The lowest similarity between clusters was 56.9 %, and the highest similaritywas 94.9 %. Cluster B was the dominant groupincluding 54 strains, accounting for 75%. According to 80% similarity, cluster A could be divided into 5 sub-clusters, and cluster B was divided into 15 sub-clusters.Strains in the same sub-cluster displayedcomplete geographical homology,which may help tracing the source of poisoning isolates. FAFLP is suitable for molecular subtyping and tracing analysis of Campylobacter jejuni for its high resolution and good repeatability.FAFLP had a higher typing resolution compared with PFGE. Strains displaying the same PFGE type, could be distinguishedwith the similaritybetween 74.9% to 88.5 % by FAFLP. However, these two methods gave a good consistent on clustering results except a few isolates. FAFLP has advantages of alternative, high-throughput and automatic, but not conducive to the results comparison betweendifferent laboratoriesand requires better laboratory conditions and more experienced operator.PFGE is easy to standardized, lower requirements for laboratory conditions, facilitate to promotion, but has a lower flux which is not suitable for large sample analysis. Both methods having advantages and disadvantages, we can make a good choice according to bacterial species, sample size and laboratory conditions and so on.c) Alu I and Mnl I were chosenbased on the sequence information of 16SrDNA-23SrDNA and ITS to subtype isolates by polymerase chain-restriction fragment length polymorphism (PCR-RFLP). The cluster analysis results showed that 72 strains yielded 27 PCR-RFLP types, with the similarity ranging from 82.5 % to100 %. According to 93% similarity, 72 strains were assigned to 8 clusters. Isolates from different provinces scattered in clusters with no significant correlation.While a high correlation was observed between PCR-RFLP clusters and phylogenetic analysis to a certain extent, which may provide genotyping basis for identification of pathogenic strains and warning of food poisoning outbreaks.Part V Establishment of food traceability analysis database of Campylobacter jejuni.Isolates were identified by biochemical tests, commercialization biochemical identification card and multiplex PCR, followed by antibiotic resistanceanalysis and molecular subtyping by PFGE, FAFLP and PCR-RFLP.One database was built preliminarilyfor food traceability analysis of Campylobacter jejuni combined with the strain background information. The database based onthe foodborne disease monitoring network and supported by varioussubtyping methods, can be enriched by expanding the monitoring scope and sample types. So it can provide scientific basis forthe tracing of foodborne poisoning, antimicrobial selection, and early warning of abnormal information in routine monitoring. |
PDF Full Text Request