| Campylobacterjejuni(C.jejuni)and Campylobacter coli(C.coli)are two kinds of gram-negative bacterium which are thermophilic and microaerobic.Campylobacter is usually colonized in intestinal tract of livestocks,poultry and birds.Besides,Campylobacter is a common pathogen causing foodborne disease.Severe complications such as Guillain-Barre Syndrome and other autoimmune diseases can also occur after infection.Serotyping of Campylobacter is related to its capsular(CPS)in surface,the diversity of which can be used for disease-related serotyping.Multilocus sequence typing(MLST)of Campylobacter is based on seven housekeeping gene variants of Campylobacter,which can be applied to source attribution and other related research.The method of culture-independent diagnostic tests for Campylobacter can identify the pathogens causing diarrhea and food poisoning rapidly.It can effectively carry out public health emergency work and promote the development of disease prevention and control.In this study,the results of isolation and culture of diarrhea patients in Beijing Shunyi Food-borne Disease Active Surveillance Network were compared with those of Quadruple real-time PCR.Genome-wide genetic studies of C.jejuni and C.coli can help us better understand the pathogenicity,drug resistance,transmission dynamic and evolution,which are closely related to disease control,and better carry out the control and emergency work of Campylobacter.In addition,the restriction modification system(R-M system)has an effect on the horizontal transfer of DNA fragments from various pathogenic bacteria,including Campylobacter spp.and it is important for the formation and evolution of the population structure.In this study,139 strains of C.jejuni and 77 strains of C.coli collected from different regions and hosts in China have been sequenced.The genome polymorphism and population structure of C.jejuni and C.coli were analyzed from the following aspects:genome genetic diversity and genetic characteristics of C.jejuni and C.coli;phylogenetic and recombinant characteristics of Campylobacter.The genetic characteristics of restriction modification system of C.jejuni and C.coli and the effect on population structure.Part ?:Comparison of the filtration culture and multiple real-time PCR examination for Campylobacter spp.Campylobacter is one of the most common pathogens leading to the bacterial diarrheal illness.In order to set up one effective culture independent assay for the screen of the Campylobacter infection in the diarrheal patients,the quadruple real-time PCR method comparing to the culture based on the enriched filtration method which was recognized as the most effective isolation method was assessed for 190 stool samples from the diarrheal patients collected during the Foodborne Diseases Active Surveillance Network in Beijing.This multiple real-time PCR was designed to identify the Campylobacter genus,C.jejuni,C.coli and C.lari simultaneously.With the enrichment culture method,23(12.1%,23/190)Campylobacter isolates were obtained(19 C.jejuni and 3 C.coli),however,31 samples(16.3%,31/190)were detected positively with the real-time PCR(20 C.jejuni,8 C.coli and 2 Campylobacter genus only).With the comparison,the real-time-PCR method is more sensitive than the enrichment filtration method(16.3%vs 12.1%,p=0.021).Among the culture-positive samples,95.7%(22/23)were detected positively by PCR which indicate the specificity of this method was higher.These two methods were consistent well(Kappa=0.785,p<0.05).Comparing to the culture methods,the result of the multiple real-time PCR method is sensitive,reliable and rapid.The present study indicated this multiple real-time PCR can be used both for the surveillance network and the preceding screen for bacteria isolation.This is first comparative study between the culture and multiple real-time PCR method for Campylobacter identification in stool specimens from the diarrheal patients.Part ?:Genetic characteristics of C.jejuni and C.coli in ChinaIn this study,139 strains of C.jejuni and 77 strains of C.coli were sequenced on Illumina Hiseq platform.Then the genomes were assembled by Soap denovo software and processed by Mummer,Roary and other oftware or pipeline.A total of 6294 pan genes,1011 core genes,0?162 specific genes and 112,917 SNPs were detected in C.jejuni In C.coli,6406 pan genes,1183 core genes,0?175 specific genes and 83,212 SNPs were detected.The phylogenetic tree of C.jejuni and C.coli was constructed based on SNPs with recombination removed.The whole population of C.jejuni was divided into 33 clade and 21 strains without clade aggregation,most of which were sourced from chicken(77.3%).The strains from Guillain-Barre Syndrome patients were mainly distributed in 4 clades,which were probably sourced from livestock.C.coli was divided into three main clade.Sources and regional aggregation of C.coli were miscellaneous.Through the VFBD(virulence factor database),51 and 50 conservative common virulence genes were identified in C.jejuni and C.coli,respectively.Their main functions were consist of adhesion,invasion,toxin-related genes and flagellum synthesis-related proteins.However,the genes related to bacterial surface structure CPS and LOS showed high diversity distribution,which may make the isolates better adapt to the environment and different hosts.Both C.jejuni and C.coli contained complete type ? secretory system and type VI secretory system.But type ? secretory system exsited in only 1 strain of C.jejuni and 2 strains of C.coli.Nearly half of C.jejuni(44.6%)contained VI secretion system and its distribution was aggregated,and 32.4%of C.coli strains contained VI secretion system.These strains not only have greater advantages in colonization competition,but also have higher pathogenicity.The genetic studise of secretion systems in Campylobacter provides an effective way to understand the pathogenicity,virulence and secretion mechanism of campylobacter.The drug resistance distribution of C.jejuni and C.coli was obtained by comparing the genomes with CARD(The Comprehensive Antibiotic Resistance Database)by using the Resistance Gene Identifier(RGI)software.A total of 8 classes and 18 kinds of drug resistance genes were detected.Among them,the distribution of resistance genes may also be related to the niche of the strain.The oxa-184 gene of Campylobacter jejuni is highly concentrated in the mixed strains of birds and avian strains,and a small number of other strains have been found,suggesting that birds and birds may be important vectors of Campylobacter jejuni.In addition,the distribution rate of aminoglycosides and macrolides in C.coli is much higher than that in C.jejuni,which leads to the higher drug resistance rate of C.coli population.Part ?:Genetic Characteristics of Restriction Modification Systems of C.jejuni and C.coli and their Relevance on Genome and Population StructureIn order to obtain the genetic characteristics and genomic structure of restriction modification systems(R-M systems)of C.jejuni and C.coli,the restriction endonuclease genes and methylase genes related to restriction modification systems of type ?,type ? and type ? in all genomes were extracted from the REBASE database.Then,the genetic characteristics were obtained through the distribution of various R-M systems in the population.In this study,the effects of different modification systems on population structure were evaluated from four aspects:recombination distribution,accessory genome,specific gene and exogenous gene in accessory genome.As for the endogenous and exogenous genes,we define a reference range by GC content in the core genome.The results showed that the R-M system pattern was highly conservative in most of C.jejuni phylogenetically developed within the same branch but highly different among the R-M system patterns of strains in different branches.In addition,through the analysis of accessory genes,recombination and exogenous gene acquisition,the formation and limitation of C.jejuni population structure were suggested.The R-M systems showed a high degree of correlation with the polyclonal and highly diverse population structure of C.jejuni.In terms of restriction and modification in genomes,genome difference analysis showed that type ? restriction modification system showed more "restriction",foreign DNA fragments were more easily cut off;and type ? R-M system showed more "modification",which might be more inclusive of recombinant fragments.The R-M system of C.coli is conservative in the whole species,and there is not much diversity,which may lead to the highly cloned and low diversity of C.coli population structure. |
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