Laboratory Detection,Molecular Epidemiology And Virulence Difference Study Of Clostridium Difficile

Objective:Study the diagnostic methods of Clostridium difficile infections(CDI).Investigate the molecular epidemiology,antimicrobial susceptibility and drug resistance mechanism of C.difficile in Beijing.Investigate the virulence difference among the major clones spread in Beijing.Methods:A total of 804 fecal samples and 348 toxigenic C.difficile isolates from Peking Union Medical College Hospital,Beijing Chao-Yang Hospital,Aerospace Center Hospital,and General Hospital of Coal were involved in the study.The isolation and identification ability of Chrome ID C.difficile agar(CDIF),and the diagnostic sensitivity and specificity of glutamate dehydrogenase(GDH)assay were evaluated and compared with culture-based method.The diagnostic sensitivity and specificity of Clostridium difficile toxins A/B(CDAB)assay and GeneXpert assay were evaluated and compared with toxigenic culture-based method.Multilocus sequence typing(MLST)and PCR-Ribotying were performed to analyze the molecular characteristics.Agar dilution method was performed to investigate the in vitro activity of 11 antimicrobial agents against the 348 toxigenic C.difficile isolates.GyrA,GyrB and RpoB amino acid variation were analyzed to study the relationship between drug resistance phenotype and genotype of quinolones and rifampin against C.difficile isolates.In terms of virulence difference study,mRNA,toxin protein,cytotoxicity test and animal test were performed on the representative C.difficile strains mainly spreading in Beijing,China.Results:In terms of laboratory diagnosis,the CDIF agar with the identification ability showed rapid,accurate and easy and simple to handle features.GDH assay revealed high sensitivity(100%)and specificity(92.8%).The positive results indicated the existence of C.difficile in the samples but did not reveal the toxigenic ablility of C.difficile isolates.The toxin in fecal samples could be detected directly by CDAB assay,however,the assay showed low sensitivity(47.5%)but high specificity(96.4%),which was not recommended to diagnose CDI alone.The toxin genes of fecal samples could be detected directly by GeneXpert assay,which showed high sensitivity(99%)and specificity(89.7%),but the cost was also higher.The two-step or three-step flows were recommended to diagnose CDI.In terms of molecular epidemiology,three hypervirulent ST1/RT027 strains were isolated,and the two strains were first reported in north China.The third one caused serve CDI.The study found ST81/PU09,ST37/RT017,ST3/RT001 and ST54/RT012 were the major clones spreading in Beijing.Eight sequence types(STs)including ST382,ST383,ST3 84,ST385,ST410,ST411,ST413 and ST414 were first identified and named in the study.The prevalent clones varied a lot among different hospitals.70%of the strains from Beijing Chao-Yang Hospital were ST81/PU09 strains.In additional,we firstly established an easy and accurate Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass(MALDI-TOF MS)based method to identify MLST clade 4 C.difficile isolates,which were mainly composed by ST81/PU09 and ST37/RT017 highly resistant isolates.The five characteristic peak markers including 2691.43 Da,2704.91 Da,2711.93 Da,3247.27 and 3290.76 Da can accurately predict clade 4 clones.In terms of antimicrobial susceptibility test,all the 348 C.difficile isolates were sensitive to metronidazole,vancomycin,meropenem and piperacillin/tazobactam,but showed high resistance rates against c.lindamycin(93.1%),erythromycin(69.5%),tetracycline(42.5%),ciprofloxacin(88.8%),levofloxacin(47.4%)and moxifloxacin(40.2%).The drug resistance rate of rifampin was 8.3%and most of the resistant strains were ST37/RT017 isolates.The drug resistance rates varied a lot among different types of strains,and the isolates of ST81/PU09 and ST37/RT017 revealed the highest drug resistance rates.GyrA,GyrB and RpoB mutations were the responsible resistance mechanism of the quinolones and rifampin resistant strains.The prevalence rates and molecular types of the drug resistance genes varied a lot among different types of C.difficile strains,and the isolates of ST81/PU09 and ST37/RT017 revealed the highest drug resistance gene mutation rates.In terms of virulence study,the strains of ST1/RT027 showed the highest virulence on three levels including toxin protein,cytotoxicity and animal test,followed by ST3/RT001 strains.The ST54/RT012,ST37/RT017 and ST81/PU09 strains showed lower virulence.The relative quantities of tcdA and tcdC genes of ST3/RT001 and ST54/RT012 strains were significantly higher but the tcdB genes of ST3/RT001 and ST54/RT012 strains were significantly lower than that of ST37/RT017 and ST81/PU09 strains.Conclusions:The now available diagnostic methods had distinctive features,and we recommended Chinese laboratories to combine multiple methods to diagnose CDI.The hypervirulent ST1/RT027 strains had been existing in Beijing,and we need to enhance the surveillance system to avoid the outbreak of the strains.The major clones spreading in Beijing were ST81/PU09 and ST37/RT017 strains,which showed higher drug resistance rates but lower virulence.The study will provide significant supporting and reference data to improve the CDI diagnostic ability,master the molecular characteristics,drug resistance and virulence status of C.difficile isolates spreading in Beijing.