| The disease caused by Campylobacter jejuni is one of the major challenges for public health through the world.In the past few decades,the prevalence of C.jejuni and campylobacteriosis have increased rapidly in both developed and developing countries,especially in North America,Europe and Australia.Studies in Africa,Asia and the Middle East also confirmed the widespread of C.jejuni.C.jejuni not only threatened human health,but also complicated clinical treatment,which has attracted a significant public concern to its prevention and control.However,the potential pathogenicity and antibiotic resistance among C.jejuni strains are different,also the distribution of virulence genes and antibiotic resistance genes are diverse.Therefore,the precise prevention and control of C.jejuni can be achieved by typing at first,and then analyzing the characteristics of virulence,antibiotic resistance and related genes of each type.In the current study,C.jejuni strains were firstly typed by molecular method;Secondly,the antibiotic resistance and the ability of adhesion and invasion to Caco-2 cells were determined,moreover the antibiotic resistance and virulence genes in the strains were identified;Finally,the ability of adhesion,invasion and antibiotic resistance between different types was compared,and the correlation between types and virulence,antibiotic resistance was explored.The main research contents and conclusions are as follows:Firstly,the DNA of 100 C.jejuni strains was extracted,and the whole genome sequence(WGS)was obtained by sequencing.Data of WGS was upload to MLST(Multilocus Sequence Typing)and CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)database to obtain the results of STs(Sequence Types)and CCTs(Conventional CRISPR Types)of all strains.The results showed that a total of 29 STs were identified by MLST,of which 8 STs were firstly reported in the Pub MLST database.The dominant ST was ST583(21%,21 strains),followed by ST42(15%,15 strains),ST61(12%,12 strains)and ST2276(10%,10 strains).A total of 42 CCTs were obtained by CRISPR typing.The dominant CCT were CCT1 and CCT29(12%,12 strains),followed by CCT5(9%,9 strains)and CCT6(7%,7 strains).NCTC11168 was identified as ST43 and CCT17.Diversity of typing results were calculated with the Simpson’s index,the D value of MLST and CRISPR typing were 0.908 and 0.953 respectively,which showed the high resolution of the two typing methods and the feasibility of CRISPR in typing.Secondly,the results of antimicrobial susceptibility testing showed that the overall antibiotic resistance rate of C.jejuni was 88%.The resistance rate to fluoroquinolones was more than 75%,which was the highest,followed by tetracycline(59%,59 strains),whereas all the isolates were susceptible to erythromycin and telithromycin.18 C.jejuni strains were detected as multidrug resistant,and a total of 14 drug resistance patterns were observed among the 100 strains.The most frequent drug resistance pattern was CIP-TETNAL(38%,38 strains).The prevalence of tet(O)gene was low(36%),but gyr A was high(81%)in C.jejuni,and the prevalence of efflux pump genes cme A,cme B and cme C were high as well,indicating that efflux pump system is common in C.jejuni strains.A great difference in the adhesion and invasion ability of C.jejuni strains to Caco-2 cells were observed.The highest and lowest adhesion rate was 28.48% and 0.02% respectively,the average adhesion rate was 1.89%,and 0.1%-1.0% was the main concentration range of adhesion rate;the invasion rate ranged from 0.00% to 6.26% with an average of 0.22%,0.01% to 0.10% was the mainly invasion rate range.Adhesion related genes cad F and peb1 A were both detected in 88 strains(88%).The prevalence rates of invasion related genes cia B and cia C were 80% and 50% respectively,and flagella movement related genes fla A and fli G were 54% and 64% respectively.More than half strains were detected with virulence related genes,suggesting that these six genes played an important role in the process of strains adhesion and invasion to Caco-2 cells.The adhesion and invasion rates of gentamicin and CIP-GEN-TET-NAL were significantly higher than others(p ≤0.05),but there was no correlation between antibiotics,drug resistance patterns and virulence.Thirdly,based on the results above,the correlation between typing and virulence,antibiotic resistance phenotypes were analyzed.The results showed that there was a significant difference(p ≤ 0.05)in antibiotic resistance among STs and CCTs.Strains of ST61 and CCT1 were the most sensitive to fluoroquinolones,but CCT5 and CCT6 were the opposite.CCT29 was sensitive to tetracycline,but ST354 and ST6175 had higher resistance rate to tetracycline,and ST11326 had the most resistance to gentamicin.The prevalence of gyr A and tet(O)in STs and CCTs were significantly different(p ≤ 0.05).Correlation between the two genes and ST42,ST354,ST2276,ST6175,ST11326,CCT5 and CCT6 were found,while ST583,CCT14 and CCT29 were only correlated with gyr A.There was significant difference(p ≤ 0.05)in the adhesion and invasion rates among different STs,the adhesion and invasion rates of ST11326 were higher than other STs,but there was no significant difference among CCTs.The analysis results of types and virulence related genes showed that the prevalence of peb1 A was no difference among types.In addition,significant difference(p ≤ 0.05)was observed between the other five virulence genes of STs and CCTs.ST6175 and CCT14 had high correlation with cad F,cia B,cia C and fli G.ST583,ST2276,ST11326 and CCT21 correlation with cad F and cia B.ST42 correlation with fli G,cad F and cia C.Phenotypes and genotypes of virulence was no corresponding among CCTs,indicating that some virulence genes were not expressed and phenotype were determined by multiple genes.In conclusion,MLST and CRISPR were performed on C.jejuni,100 strains were subgrouped into 29 STs and 42 CCTs.The ability of adhesion,invasion and antibiotic resistance were analyzed,also,the related genes were detected.Statistical analysis showed that there was no significant correlation between antibiotic resistance and virulence.Significant difference(p ≤ 0.05)was observed in antibiotic resistance,adhesion,invasion and the distribution of related genes among STs.The strains of ST11326 were resistant to ciprofloxacin,nalidixic acid,tetracycline,and hold the most resistance to gentamicin,the adhesion and invasion rates were higher than other STs as well.Results of CCTs were similar to STs except adhesion and invasion.The ability of CCT5 and CCT6 resistant to ciprofloxacin,nalidixic acid and tetracycline were higher compared with other CCTs.And the adhesion and invasion rates of CCT43 were higher.This study can provide reference for the epidemiological study,prevention and control of C.jejuni. |
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