Study On The Molecular Epidemiology And Antibiotic Resistance Of Clostridium Difficile Isolates

Objective： Clostridium difficile is a Gram-positive, anaerobicspore–forming Bacillus commonly found in the environment and human gut.The entero pathogenicity of Clostridium difficile is associated with theproduction of enterotoxin A and cytotoxin B. Overuseing of antibiotics，immunosuppressive agents and chemotherapy drugs may lead to overgrowthof the toxic strain of C. difficile with high resistance，which are supposed to bethe major contributing factors leading to Clostridium difficile associateddiarrhea(CDAD). The globally increasing incidence of CDAD，especially theoutbreak of nosocomial infection caused by the hypervirulent strain in NorthAmerica，arouse worldwide attention. In order to understand the popularsituation of toxic Clostridium difficile in ShiJiaZhuang of China, we have a aseries studies on the molecular characteristics of virulence, PCR-ribotypingand antibiotic resistance of Clostridium difficile isolates. provide the basis foreffectively preventing the outbreak of Clostridium difficile.Method: A total of235stool samples were collected during July2010-December2011in The Second Hospital of Hebei Medical University,The Fourth Hospital of Hebei Medical University, The First Hospital of HebeiMedical University and Hebei People’s Hospital. Firstly, Toxins A and Bgenes of C. difficile were detected by conventional Polymerase chain reaction(PCR). Then, the toxin-positive samples were incubated by Enrichment SporeCulture Method.Finally, The isolated C. difficile were detected tcdA,tcdBgenes of toxin A and B, cdtA,cdt B genes of binary-toxin by a multiplex PCR.PCR-ribotyping of toxic Clostridium difficile were conducted by means ofanalysis of16s-23s internal spacer region polymorphism with conventionalPCR assay．The bands were compared and analyzed using Quantity Onesoftware. Minimum inhibitory concentrations (MICs) of levofloxacin, vancomycin, metronidazole, clindamycin, and tigecycline against Clostridiumdifficile were determined by the agar dilution technique.The ermB gene ofclindamycin resistance were detected by conventional PCR.Result: A total of37positive samples were collected from235stoolsamples by conventional PCR for direct detection of fecal Clostridium difficiletoxins A and B genes, the positive rate was15.7%. A total of32Clostridiumdifficile were incubated from the37toxin-positive samples by EnrichmentSpore Culture, the culture positive rate was86.5%. Using multiplex PCRmethod to detect the tcdA and tcdB genes of toxin A and B, cdtA、cdt B genesof binary-toxin and16SrDNA gene, found that the tcdA、tcdB genes werepositive in all the strains while the binary-toxin genes were negative.32toxicisolates were identified that included12gene types by PCR-ribotyping，thedominant type was SI，accounting for28％．No isolates belonged to ribotype027or078．All strains were highly susceptible to metronidazole, vancomycinand tigecycline. But high-level resistance to clindamycin (MIC>128mg/l) andlevofloxacin (MIC>64mg/l) was found, the resistance rate was100%and87.5%, respectively. Resistance to meropenem was18.8%lower thanclindamycin and levofloxacin．Clindamycin resistance associated with geneermB was positive in18out of32Clostridium difficile strains，accounting for56.25％.Conclusion:1Using conventional PCR method to detect Clostridiumdifficile toxins A and B gene, In order to have an early diagnosis ofClostridium difficile infection.2Enrichment Spore Culture can increasing thepositive rate of Clostridium difficile, thereby can be used being an effectivemethod for Clostridium difficile culture in clinical.3The primary toxinClostridium difficile was tcdA+and tcdB+in ShiJiaZhuang of China, didn’tfound the strains with tcdA-and tcdB+, neither did the strains producingbinary toxin.412different gene types were identified by PCR-ribotyping inthis area，the dominant type was SI，accounting for28％, didn’t found thestrains belonging to ribotype027or078．5In this study, by measuring theMIC values of a variety of antibiotics for Clostridium difficile,found that the strains separated from ShiJiaZhuang have a high-level resistance toclindamycin and levofloxacin,and some strains were resistant to meropenem.Not found the resistance strain for Metronidazole, vancomycin and tigecycline.Metronidazole and vancomycin can be regard as the first-line antibiotics intreatment of CDAD in this area.