Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA+B+ isolates with known ribotypes (RTs). Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene. Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene. The present study demonstrates that the majority of clinical tcdA+B+ isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/toxinotype 0 and the RT 126/toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence.

• Klíčová slova
• Clostridioides (clostridium) difficile, Iran, PaLoc, Ribotype, Toxinotype, tcdA(+)B(+) isolates, tcdC,
• MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální toxiny genetika   MeSH
• Clostridioides difficile klasifikace   genetika   patogenita   MeSH
• dítě MeSH
• DNA bakterií MeSH
• dospělí MeSH
• feces mikrobiologie   MeSH
• genetická variace * MeSH
• genotyp MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• molekulární typizace MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• represorové proteiny genetika   MeSH
• ribotypizace MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• virulence genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Írán epidemiologie   MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální toxiny MeSH
• DNA bakterií MeSH
• represorové proteiny MeSH
• TcdC protein, Clostridium difficile MeSH Prohlížeč

We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert®C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes' detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.

• Klíčová slova
• MLVA, Slovakia, Thr82Ile, binary toxin, ribotyping, tcdC, Δ117,
• Publikační typ
• časopisecké články MeSH

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, 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 Δ117 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 Δ117 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.

• Klíčová slova
• Capillary-electrophoresis ribotyping, Clostridium difficile, MLST, MLVA, Moxifloxacin resistance,
• MeSH
• ADP-ribosatransferasy genetika   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• DNA bakterií analýza   MeSH
• DNA gyráza genetika   MeSH
• dospělí MeSH
• enterotoxiny genetika   MeSH
• fluorochinolony farmakologie   MeSH
• genotyp MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• minisatelitní repetice genetika   MeSH
• mladý dospělý MeSH
• molekulární epidemiologie MeSH
• moxifloxacin MeSH
• multilokusová sekvenční typizace metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• represorové proteiny genetika   MeSH
• ribotypizace metody   MeSH
• sekvence nukleotidů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• techniky typizace bakterií * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Finsko MeSH
• Názvy látek
• actin-specific ADP-ribosyltransferase, Clostridium MeSH Prohlížeč
• ADP-ribosatransferasy MeSH
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• bakteriální toxiny MeSH
• DNA bakterií MeSH
• DNA gyráza MeSH
• enterotoxiny MeSH
• fluorochinolony MeSH
• moxifloxacin MeSH
• represorové proteiny MeSH
• tcdA protein, Clostridium difficile MeSH Prohlížeč
• TcdC protein, Clostridium difficile MeSH Prohlížeč
• toxB protein, Clostridium difficile MeSH Prohlížeč

Clostridium difficile is a leading nosocomial pathogen and molecular typing is a crucial part of monitoring its occurrence and spread. Over a three-year period (2013-2015), clinical C. difficile isolates from 32 Czech hospitals were collected for molecular characterisation. Of 2201 C. difficile isolates, 177 (8%) were non-toxigenic, 2024 (92%) were toxigenic (tcdA and tcdB) and of these, 677 (33.5%) carried genes for binary toxin production (cdtA, cdtB). Capillary-electrophoresis (CE) ribotyping of the 2201 isolates yielded 166 different CE-ribotyping profiles, of which 53 were represented by at least two isolates for each profile. Of these, 29 CE-ribotyping patterns were common to the Leeds-Leiden C. difficile reference strain library and the WEBRIBO database (83.7% isolates), and 24 patterns were recognized only by the WEBRIBO database (11.2% isolates). Isolates belonging to these 53 CE-ribotyping profiles comprised 94.9% of all isolates. The ten most frequent CE-ribotyping profiles were 176 (n=588, 26.7%), 001 (n=456, 20.7%), 014 (n=176, 8%), 012 (n=127, 5.8%), 017 (n=85, 3.9%), 020 (n=68, 3.1%), 596 (n=55, 2.5%), 002-like (n=45, 2.1%), 010 (n=35, 1.6%) and 078 (n=34, 1.6%). Multi-locus sequence typing (MLST) of seven housekeeping genes performed in one isolate of each of 53 different CE-ribotyping profiles revealed 40 different sequence types (STs). We conclude that molecular characterisation of Czech C. difficile isolates revealed a high diversity of CE-ribotyping profiles; the prevailing RTs were 001 (20.7%) and 176 (027-like, 26.7%).

• Klíčová slova
• Capillary electrophoresis ribotyping, Clostridium difficile, MLST, Molecular typing, Toxin genes, tcdC,
• MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• dítě MeSH
• dospělí MeSH
• genetická variace * MeSH
• genotyp MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• molekulární epidemiologie MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ribotypizace MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• bakteriální toxiny MeSH

Clostridium difficile is a major nosocomial pathogen of present times. The analysis of 624 C. difficile strains from 11 hospitals in the Czech Republic in 2013 revealed that 40% of isolates belonged to ribotype 176. These results suggest that the incidence of CDI (C. difficile infection) in the Czech Republic has increased probably in connection with C. difficile ribotype 176. The molecular systems Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA) diagnoses toxigenic strains and supports C. difficile ribotype 027 determination based on three specific target places in the toxigenic C. difficile genome. Twenty-nine strains cultivated from stool specimens were evaluated by the Xpert systems as presumed C. difficile PCR ribotype 027 were confirmed as a C. difficile ribotype 176 based on ribotyping. A further 120 C. difficile strains of ribotype 176 were examined for the presence of genes tcdB, cdtB and deletion in position 117 in the tcdC gene. Our experience shows that due to the correspondence of the target places, C. difficile ribotype 176 may be interpreted as ribotype 027 by Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA). Further molecular analysis as ribotyping based on capillary electrophoresis is needed to differentiate between C. difficile ribotypes 027 and 176 for appropriate epidemiological situation control on local and national levels.

• MeSH
• bakteriální proteiny genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• delece genu MeSH
• feces mikrobiologie   MeSH
• infekce spojené se zdravotní péčí mikrobiologie   MeSH
• lidé MeSH
• nemocnice statistika a číselné údaje   MeSH
• ribotypizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• bakteriální proteiny MeSH

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• diskové difúzní antimikrobiální testy MeSH
• incidence MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• minisatelitní repetice MeSH
• molekulární typizace * MeSH
• nemocnice MeSH
• ribotypizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální toxiny MeSH

Clostridioides difficile is the most common causative agent of healthcare-associated diarrhea. C. difficile strains produce a crystalline surface layer protein (SlpA), encoded by the slpA gene. Previous studies have shown that SlpA varies among C. difficile strains. In this study, we used the SlpA sequence-based typing system (SlpAST) for the molecular genotyping of C. difficile clinical isolates identified in Iran; the PCR ribotypes (RTs) and toxin profiles of the isolates were also characterized. Forty-eight C. difficile isolates were obtained from diarrheal patients, and characterized by capillary electrophoresis (CE) PCR ribotyping and the detection of toxin genes. In addition, the genetic diversity of the slpA gene was investigated by Sanger sequencing. The most common RTs were RT126 (20.8%), followed by RT001 (12.5%) and RT084 (10.4%). The intact PaLoc arrangement representing cdu2+/tcdR+/tcdB+/tcdE+/tcdA+/tcdC+/cdd3+ profile was the predominant pattern and cdtA and cdtB genes were found in one-third of the isolates. Using the SlpA genotyping, 12 main genotypes and 16 subtypes were identified. The SlpA type 078-1 was the most prevalent genotype (20.8%), and identified within the isolates of RT126. The yok-1, gr-1, cr-1 and kr-3 genotypes were detected in 14.5%, 12.5%, 12.5% and 8.3% of isolates, respectively. Almost all the isolates with the same RT were clustered in similar SlpA sequence types. In comparison to PCR ribotyping, SlpAST, as a simple and highly reproducible sequenced-based technique, can discriminate well between C. difficile isolates. This typing method appears to be a valuable tool for the epidemiological study of C. difficile isolates worldwide.

• Klíčová slova
• Clostridioides difficile, Iran, PaLoc, Ribotypes, SlpA genotyping, SlpAST,
• MeSH
• bakteriální proteiny genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• dítě MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• fylogeneze * MeSH
• genetická variace MeSH
• klostridiové infekce mikrobiologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• techniky typizace bakterií MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Írán MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• surface layer protein A, Bacteria MeSH Prohlížeč

BACKGROUND: Clostridium difficile is currently a significant cause of nosocomial diarrhea. For several years, the number of infectious cases in the community has also been increasing. Since the beginning of 2010, quite a large increase in the number of Clostridium difficile infections (CDIs) has been noted in Pardubice Regional Hospital (PRH). The objectives of this study were to describe and evaluate the methods used in the laboratory diagnosis of CDIs in PRH, and to describe the laboratory diagnostic algorithm used here. MATERIAL AND METHODS: Samples of stools were taken from symptomatic patients hospitalized or examined in the outpatient departments of PRH from 1 July 2010 to 31 December 2012. For the detection of glutamate dehydrogenase (GDH) and toxin A/B, the dual test based upon the principle enzyme immunoassays C. Diff Quik Chek Complete, Techlabo (D-EIA) was used. The system GeneXpert PCR Cepheid (PCR) was used for confirmation of laboratory findings. Since the beginning of 2011, all the GDH-positive samples were cultured. RESULTS: A total of 2,040 samples were examined. The D-EIA test was used for examination of 2,014 samples. Of those, 1,373 (68.2 %) samples were GDH- and toxin A/B-negative. In 359 (17.8 %) samples, both GDH and toxin A/B were detected. The D-EIA sensitivity and specificity for detecting toxigenic strains in stool samples were 21.8% and 97.2%, respectively. The PPV and NPV rates calculated for the populations with prevalence rates of disorders of 5%, 10%, 20% and 50 % were 0.29, 0.46, 0.66, 0.88 and 0.96, 0.92, 0.83, 0.55, respectively. The sensitivity and specificity of GDH for the detection of Clostridium difficile in stools were 100.0% and 96.2%, respectively. PCR examination was carried out in 140 samples. Of those, 82 samples were PCR-positive. The gene for the production of toxin B was detected in 47%, the finding suspected for ribotype 027 (gene for toxin B, binary toxin and deletion of tcdC) in 48%. In 5% of the samples, the gene for toxin B and the gene for the binary toxin were detected. CONCLUSION: Considering the low sensitivity of the D-EIA test for detecting the toxigenic strain of Clostridium difficile, if used as the only one, a two-step algorithm was introduced for routine laboratory examination of infections with Clostridium difficile in the Clinical Microbiology Department of PRH. In the first step, the D-EIA test diagnosed 86 % of examined samples in 30 minutes as positive (GDH +; toxin A/B +) or negative (GDH -; toxin A/B -). The examination with PCR in the second step increased the number of patients diagnosed with CDI. The test results are available within two hours. This enables quick introduction of isolation measures in the departments of PRH and appropriate antibiotic treatment of the patients.

• MeSH
• algoritmy MeSH
• azurová mořidla MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bakteriální toxiny genetika   metabolismus   MeSH
• Clostridioides difficile enzymologie   genetika   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• glutamátdehydrogenasa genetika   metabolismus   MeSH
• imunoenzymatické techniky metody   MeSH
• lidé MeSH
• methylenová modř MeSH
• polymerázová řetězová reakce metody   MeSH
• pseudomembranózní enterokolitida diagnóza   mikrobiologie   MeSH
• senzitivita a specificita MeSH
• xantheny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• azurová mořidla MeSH
• bakteriální proteiny MeSH
• bakteriální toxiny MeSH
• Diff Quik MeSH Prohlížeč
• glutamátdehydrogenasa MeSH
• methylenová modř MeSH
• xantheny MeSH