Detection And Analysis Of Clostridium Difficile In Diarrhea Patients

ObjectiveTo realize the distribution, toxin’s type, antibiotic resistance and possiblemechanisms of drug resistance of Clostridium difficile from patients with diarrhea inGuangzhou, the isolation and identificaton of Colstridium difficile from stoolspecimens of clinical patients with diarrhea were carried out, and the multilocussequence typing (MLST), drug resistance rate and drug resistance-associated genesof Clostridium difficile were tested. This study will provided some laboratoryevidence for prevention and treatment of Clostridium difficile infection effectively.MethodFrom March2012to December2012and from March2013to December2013,467stool samples were collected from patients with diarrhea from The First AffiliatedHospital of Guangzhou Medical University, The Third Affiliated Hospital ofGuangzhou Medical University and The Second Affiliated Hospital of ZhongshanUniversity. To extract DNA from these stool specimens the fecal genomic DNAextraction kit was used. Using the DNA extracted from the stool specimens astemplates, the conventional PCR methods was used to detect the species-specific gene tpi, genes tcdA and tcdB of toxinA and toxinB, binary toxin genes cdtA and cdtB ofClostridium difficile(CD). Clostridium difficile were irritated to form spores and thespores were collected from the gene tpi-positive stool specimens, and the CD sporeswere innoculated on the CCFA plate and incubated in anaerobic environment.Suspicious colonies were subcultured and identified by conventional PCR methods todetect Clostridium difficile species-specific gene tpi, and toxin A, B and binary toxingenes of the isolated Clostridium difficile were detected simultaneously. Clostridiumdifficile isolates were typed by MLST. MIC values of Clostridium difficile isolates tovancomycin, metronidazole, clindamycin, ampicillin, moxifloxacin, meropenem,amoxicillin/clavulanic acid, penicillin, piperacillin, and piperacillin/tazobactam weretested by the Etest method. For the drug-resistance strains, drug resistance-associatedgenes such as clindamycin resistance-associated gene ermB and the DNA gyrase genegyrA, gyrB of quinolone resistance-associated gene were detected by PCR andsequenced to realize the drug resistance mechanisms of Clostridium difficile.ResultGene tpi was obtained from29of467stool specimens, the positive rate was6.2%(29/467). The toxin gene tcdA and tcdB were obtained from16of29specimenswith gene tpi, the positive rate was55.2%(16/29).22CD strains were isolated from29stool specimens with gene tpi, the isolation rate was75.9%(22/29). The toxin genetcdA and tcdB were positive in13of22CD strains, the positive rate was59.1%(13/22). Binary toxin gene have not found in all22CD strains. In22patients with CDstrain,6patients belong to outpatient/emergency case, the constituent radio was27.3%(6/22),3CD strains with toxin genes were isolated from6patients fromoutpatient/emergency department, the rate was50.0%(3/6) in outpatient/emergencycase;8cases belong to pediatric patients, the constituent radio was36.4%(8/22),5CDstrains with toxin genes were isolated from the8pediatric patients, the rate was62.5%(5/8); the rest patients with CD strain were all adult inpatients,5cases of them camefrom pneumology department,2cases came from oncology department.22Clostridium difficile strains can be divided into nine different ST types, ST54and ST3were relatively the dominant types, the highvirulence strains ST1(PCR-ribotyping027) and ST11(PCR-ribotyping078) have not be found.Antibiotic sensitivity test show that all22CD strains were sensitive tometronidazole, vancomycin, amoxicillin/clavulanic acid, piperacillin and piperacillin/tazobactam, none of22CD strains was resistance or intermediary to the5antimicrobial agents. But the drug-resistant rate to clindamycin was90.9%(20/22) andwas high,16of20clindamycin-resistant strains showed high-level clindamycinresistance(MIC>256μg/ml).6of22CD strains showed penicillin resistance,7of22CD strains showed penicillin intermediate, the penicillin-resistance rate was27.3%(6/22).3of22CD strains showed moxifloxacin resistance, one of22CD strainsshowed moxifloxacin intermediary, the moxifloxacin-resistance rate was13.6%(3/22),3moxifloxacin-resistance strains all showed high-level moxifloxacin resistance (MIC>32μg/ml).5of6penicillin-resistant CD strains simultaneously showed clidamycinresistance, and the3moxifloxacin-resistance strains simultaneously showedclindamycin resistance.2of22CD strains simultaneously showed drug-resistant toclindamycin, moxifloxacin and penicillin,they are multidrug resistance strains.5of22CD strains showed ampicillin intermediary (MIC=1μg/ml),1of22CD strainsshowed meropenem intermediary. The rest of CD strains have not be found toshow drug resistance. Gene ermB was obtained from8of20clindamycin-resistanceClostridium difficile strains, the positive rate was40%(8/20).3moxifloxacin-resistance strains were found to show the mutation of genes gyrA andgyrB.Conclusion1、Tthe incidence of Clostridium difficile infection in patients with diarrhea havenot be high till now in Guangzhou, the highvirulence CD strain with binary toxin havenot be found, but it is importment to detect Clostridium difficile strain in patients ofcommunity and pediatrics department.2、There are many genotypes（9kinds of sequence types）in Clostridium difficilestrains from patients with diarrhea in Guangzhou, ST54and ST3were relatively the dominant sequence types, Clostridium difficile infection are sporadic and CDinfection’s outbreak have not be found.3、 The drug-resistance rate of Clostridium difficile isolates is not high toantimicrobial agents commonly used in clinic, but there is a high drug-resistance rate toclindamycin as to CD strains, the presence of the gene ermB is one of the reasons thatthe CD strains showed drug-resistance to clindamycin, the mutation of the genes gyrAand gyrB are connected with quinolone resistance in CD strains. Multidrug resistanceClostridium difficile strains have be found.