Purpose: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B,... Show morePurpose: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods.Methods: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test.Results: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Delta 117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA.Conclusion: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Delta 117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes. Copyright (C) 2017, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. Show less
Andres-Lasheras, S.; Martin-Burriel, I.; Mainar-Jaime, R.C.; Morales, M.; Kuijper, E.; Blanco, J.L.; ... ; Bolea, R. 2018
This thesis describes the application of molecular typing methods to study the epidemiology and evolution of Clostridium difficile. C. difficile is a gut bacterium that is a major cause of... Show moreThis thesis describes the application of molecular typing methods to study the epidemiology and evolution of Clostridium difficile. C. difficile is a gut bacterium that is a major cause of antibiotic associated diahrrea in the developed world. The emergence of the __hypervirulent__ types causing C. difficile infections (CDI) with more severe complications has driven the demand for rapid molecular assays that can detect these types. We aimed to identify novel molecular markers for __hypervirulent__ C. difficile and use these markers to develop new rapid assays. This thesis describes the development and evaluation of a new (multiplex) real-time PCR assay to diagnose CDI. It also provides an overview of the currently applied methods to study CDI. We report on an internationally-standardized protocol for high resolution, capillary gel-based electrophoresis, PCR-ribotyping. Furthermore, we describe two novel genetic markers that are linked to two C. difficile phylogenetic lineages. We describe the application of multilocus sequence typing to study the population structure of C. difficile. We report on whole genome sequencing (WGS) and the related single nucleotide polymorphism (SNP) analysis, a recently emerged typing method that we used to study transmission of C. difficile type 078 between animals (pigs) and humans (farmers). Show less
