A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital

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dc.contributor.advisorAbratt, Valerie Roseen_ZA
dc.contributor.advisorReid, Sharon Jen_ZA
dc.contributor.authorBrock, Tunehafo Elisabethen_ZA
dc.date.accessioned2016-06-09T11:17:45Z
dc.date.available2016-06-09T11:17:45Z
dc.date.issued2015en_ZA
dc.description.abstractA clinical and molecular analysis of Clostridium difficile isolated from symptomatic patients at Groote Schuur Hospital was conducted in order to gain insight into the identity, epidemiology and pathogenesis of the various strains. C. difficile was detected and isolated by selective culture from stool specimens from 34 of the 162 symptomatic patients (20%). Three toxigenic-types were distinguished by PCR: A+B+ (47%), A-B+ (47%) and A-B- (6%), none of which harboured the binary toxin genes. Compared to the direct culture method, enzyme immunoassay-based detection tests (Meridian ImmunoCard and bioMérieux MiniVidas) were found to be lacking clinical sensitivity, while nucleic acid amplification tests (Hain Lifescience CDiff and Cepheid GeneXpert) were far more sensitive in the local clinical setting. PCR ribotyping identified all the A-B+ strains as PCR ribotype 017, which was the prevalent strain type (47%). Genotyping based on the tcdC gene and MLVA both grouped the ribotype 017 strains in a single clade. The antimicrobial susceptibility of all the isolates to metronidazole (MET), vancomycin (VAN), moxifloxacin (MOX) and erythromycin (ERY) were determined by the Etest method. All were sensitive to MET and VAN; however, four ribotype 017 strains displayed reduced susceptibility to MET. With regard to MOX, reduced susceptibly and full resistance were observed in 32% and 12% of the isolates, respectively, with all of these belonging to ribotype 017. MOX-resistant strains had a Thr82->Ile amino acid substitution in the GyrA enzyme and strains displaying reduced susceptibility to MOX had an Asp426->Asn amino acid substitution in GyrB. High-level resistance to ERY was observed in 47% of the isolates, which were primarily ribotype 017. ERY-resistant strains all harboured the ermB gene, suggesting that this was the genetic basis of the observed phenotype. Auto-aggregation analysis revealed that the ribotype 017 strains were significantly stronger auto-aggregators than the other ribotypes examined. Results of semi-quantitative RT-PCR analysis suggest that the expression level of the cwpV gene, encoding the CwpV protein, may play a role in auto-aggregation. In conclusion, this pilot study revealed that the GeneXpert method was the most accurate and sensitive technique for diagnosing CDI in the clinical setting at Groote Schuur Hospital. Ribotype 017 was the most prevalent strain type, and the antimicrobial resistance profile and increased auto-aggregation capacity of this ribotype may contribute to its high prevalence.en_ZA
dc.identifier.apacitationBrock, T. E. (2015). <i>A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/19966en_ZA
dc.identifier.chicagocitationBrock, Tunehafo Elisabeth. <i>"A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2015. http://hdl.handle.net/11427/19966en_ZA
dc.identifier.citationBrock, T. 2015. A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Brock, Tunehafo Elisabeth AB - A clinical and molecular analysis of Clostridium difficile isolated from symptomatic patients at Groote Schuur Hospital was conducted in order to gain insight into the identity, epidemiology and pathogenesis of the various strains. C. difficile was detected and isolated by selective culture from stool specimens from 34 of the 162 symptomatic patients (20%). Three toxigenic-types were distinguished by PCR: A+B+ (47%), A-B+ (47%) and A-B- (6%), none of which harboured the binary toxin genes. Compared to the direct culture method, enzyme immunoassay-based detection tests (Meridian ImmunoCard and bioMérieux MiniVidas) were found to be lacking clinical sensitivity, while nucleic acid amplification tests (Hain Lifescience CDiff and Cepheid GeneXpert) were far more sensitive in the local clinical setting. PCR ribotyping identified all the A-B+ strains as PCR ribotype 017, which was the prevalent strain type (47%). Genotyping based on the tcdC gene and MLVA both grouped the ribotype 017 strains in a single clade. The antimicrobial susceptibility of all the isolates to metronidazole (MET), vancomycin (VAN), moxifloxacin (MOX) and erythromycin (ERY) were determined by the Etest method. All were sensitive to MET and VAN; however, four ribotype 017 strains displayed reduced susceptibility to MET. With regard to MOX, reduced susceptibly and full resistance were observed in 32% and 12% of the isolates, respectively, with all of these belonging to ribotype 017. MOX-resistant strains had a Thr82->Ile amino acid substitution in the GyrA enzyme and strains displaying reduced susceptibility to MOX had an Asp426->Asn amino acid substitution in GyrB. High-level resistance to ERY was observed in 47% of the isolates, which were primarily ribotype 017. ERY-resistant strains all harboured the ermB gene, suggesting that this was the genetic basis of the observed phenotype. Auto-aggregation analysis revealed that the ribotype 017 strains were significantly stronger auto-aggregators than the other ribotypes examined. Results of semi-quantitative RT-PCR analysis suggest that the expression level of the cwpV gene, encoding the CwpV protein, may play a role in auto-aggregation. In conclusion, this pilot study revealed that the GeneXpert method was the most accurate and sensitive technique for diagnosing CDI in the clinical setting at Groote Schuur Hospital. Ribotype 017 was the most prevalent strain type, and the antimicrobial resistance profile and increased auto-aggregation capacity of this ribotype may contribute to its high prevalence. DA - 2015 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2015 T1 - A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital TI - A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital UR - http://hdl.handle.net/11427/19966 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/19966
dc.identifier.vancouvercitationBrock TE. A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2015 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/19966en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMolecular and Cell Biologyen_ZA
dc.titleA clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospitalen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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