Study On The Rapid Identification And Toxin Detection Of Clinical Clostridium Difficile Strains

BackgroundClostridium. difficile (C. difficile) is an anaerobic, Gram-positive, spore-forming, toxigenic or non-toxigenic bacillus. Now C. difficile has been recognized as an primary cause of antibiotic associated diarrhea (AAD) and Pseudo-membrane colitis (PMC). Clostridium difficile infection (CDI) which causes nosocomial infectious diarrhea has been emphasized worldwide, the morbidity and mortality in elderly hospitalized patients caused by CDI have risen up year by year. The clinical manifestations of infection with toxin-producing strains of C. difficile range from symptomless carriage, to mild or moderate diarrhea, to fulminant and sometimes fatal pseudomembranous colitis. Moreover C. difficile has two types of toxigenic strain or non-toxigenic strain. Accurate laboratory diagnosis early in the disease course is important to the successful management of CDI.ObjectivesThe purpose of this study is to establish the standard phenotypic culture method of C. difficile which is suitable for the clinical microbiological laboratory and to design a toxigenic culture approach using multiplex PCR for simultaneous identification and toxigenic type characterization of C. difficile isolates. This approach is based on specific culture of spore-forming bacteria followed by multiplex PCR targeting a species-specific internal fragment of the triose phosphate isomerase (tpi) housekeeping gene, an internal fragment of the toxin B (TcdB) gene and the toxin A (TcdA) gene.Materials and MethodsA collection of68strains were ready for the study, which were including:21standard strains (C. difficile ATCC9689, C. perfringens13124) which were normal flora or pathogens from enteric cavity;47clinical isolates of C. difficile which isolated from the552stools of patients with diarrhea in China-Japan friendship hospital from June to December2010.47clinical isolates of C. difficile were isolated and identified by the standard phenotypic culture method which were including as follows, typical colonies and odor in CCFA, yellow fluorescence under long-wavelength UV light (254nm), Gram’s staining, glutamate dehydrogenase (GDH), L-proline aminopeptidase (PRO).47isolates of C. difficile were analyzed for the charateration of toxin A and toxin B by enzyme-linked immunosorbent assay (ELISA).Three pairs of primers were designed for the amplification of (ⅰ) a species-specific internal fragment of the triose phosphate isomerase (tpi) gene,(ⅱ) an internal fragment of the toxin B gene (tcdB), and (ⅲ) an internal fragment of the toxin A gene (tcdA).21standard strains including C. difficile ATCC9689and47isolates of C. difficile were applied for the assessment of detection limit, specificity and detections of the multiplex PCR, respectively.SPSS11.5software was used for data processing and statistical analysis. Results1. The standard phenotypic culture method was established for accurate identification of C. difficile isolated in clinical microbiological laboratory.2. The strains of C. difficile were conserved in brain-heart infusion (BHI) including20%glycerol at-80℃for2years。3.47clinical isolates of C. difficile from552stools of diarrhea patients were isolated and identified by the standard phenotypic culture method.47isolates were analyzed for the charateration of toxin A and toxin B by enzyme-linked immunosorbent assay (ELISA):20strains were positive,27strains were negative.4. Multiplex PCR method was established to detect the three genes of tpi, tcdB and tcdA. The amplification products of C. difficile (ATCC9689) were consistent with the length of target fragments by electrophoresis:tpi(227bp), tcdB(144bp), tcdA(112bp). The amplified products were sequenced, the resultant sequences were completely consistent with the target sequences.5. The multiplex PCR assay evaluated in the present study had a limit of detection of5×10-4ng/μl (2×102CFU per reaction mixture) by analyzing10-fold dilutions of DNA from a strain of C. difficile (ATCC9689) with known concentrations.The specificity of the multiplex PCR assay was determined to be100%by detecting21organisms, as only C.difficile (ATCC9689) strain was positive for the respective genes (tpi, tcdA and tcdB).6.47clinical isolates of C. difficile were analyzed by Multiplex PCR. The identification results of multiplex PCR were consistent with that of the standard phynotype culture method. The toxin results of multiplex PCR:37strains were tcdA(+)/B(+),10strains were tcdA(-)/B(-), the strain with tcdA(-)/B(+) was not detected.7. It was significantly different in the two results of toxin analysis by multiplex PCR and ELISA. The positive rate of toxin by multiplex PCR was more than by ELISA. ConclusionIn this study, we successfully established the standard phenotypic culture method of C. difficile which was suitable for the clinical microbiological laboratory and a toxigenic culture approach using multiplex PCR for simultaneous identification and toxigenic type characterization of C. difficile isolates. Multiplex PCR reduced the dection procedure and shorten the detection time, which was feasible to be applied in clinical microbiological laboratory. It promoted the accuracy of strain identification and toxin analysis and contributed to the future research for strain typing, toxin typing, analyzing of antimicriobial resistance, new colone detecting and survelling nosocomial ourbreak of C. difficile.