BackgroundOn 29 January 2024, the European Centre for Disease Prevention and Control distributed an alert about a metronidazole-resistant Clostridioides difficile outbreak of PCR ribotype (RT) 955 in England.AimWe aimed to investigate the presence of RT955 in Czech, Slovak and Polish C. difficile isolates and evaluate different culture media for detecting its metronidazole resistance.MethodsIsolates with binary toxin genes identified as 'unknown' by the WEBRIBO PCR ribotyping database up to 2023 were re-analysed after adding the RT955 profile to the database. The RT955 isolates were characterised by whole genome sequencing and tested for susceptibility to 15 antimicrobials.ResultsWe did not find RT955 in Czech (n = 6,661, 2012-2023) and Slovak (n = 776, 2015-2023) isolates, but identified 13 RT955 cases (n = 303, 2021-2023) in three hospitals in Poland. By whole genome multilocus sequence typing, 10 isolates clustered into one clonal complex including a sequence of United Kingdom strain ERR12670107, and shared similar antimicrobial resistance genes/mutations. All 13 isolates were resistant to ciprofloxacin/moxifloxacin, erythromycin/clindamycin and ceftazidime. All isolates had a mutation in the nimB gene promoter and in NimB (Tyr130Ser and Leu155Ile). The metronidazole resistance was detected in all isolates using brain-heart-infusion agar supplemented with haemin and Chocolate agar. Results were discrepant with the European Committee on Antimicrobial Susceptibility Testing-recommended Fastidious anaerobe agar and Brucella blood agar.ConclusionThe identification of clonally related haem-dependent metronidazole-resistant C. difficile RT955 in multiple hospitals indicates a need for prospective surveillance to estimate its prevalence in Europe.

• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Drug Resistance, Bacterial * genetics   MeSH
• Clostridioides difficile * genetics   drug effects   isolation & purification   classification   MeSH
• Disease Outbreaks MeSH
• Clostridium Infections * epidemiology   microbiology   drug therapy   MeSH
• Humans MeSH
• Metronidazole * pharmacology   MeSH
• Microbial Sensitivity Tests MeSH
• Multilocus Sequence Typing MeSH
• Polymerase Chain Reaction MeSH
• Ribotyping * MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH
• Poland MeSH
• Slovakia MeSH

OBJECTIVES: To analyse characteristics of Clostridioides difficile PCR ribotype 176 clinical isolates from Poland, the Czech Republic and Slovakia with regard to the differences in its epidemiology. METHODS: Antimicrobial susceptibility testing and whole genome sequencing were performed on a selected group of 22 clonally related isolates as determined by multilocus variable-number tandem repeat analysis (n = 509). Heterologous expression and functional analysis of the newly identified methyltransferase were performed. RESULTS: Core genome multilocus sequence typing found 10-37 allele differences. All isolates were resistant to fluoroquinolones (gyrA_p. T82I), aminoglycosides with aac(6')-Ie-aph(2'')-Ia in six isolates. Erythromycin resistance was detected in 21/22 isolates and 15 were also resistant to clindamycin with ermB gene. Fourteen isolates were resistant to rifampicin with rpoB_p. R505K or p. R505K/H502N, and five to imipenem with pbp1_p. P491L and pbp3_p. N537K. PnimBG together with nimB_p. L155I were detected in all isolates but only five were resistant to metronidazole on chocolate agar. The cfrE, vanZ1 and cat-like genes were not associated with linezolid, teicoplanin and chloramphenicol resistance, respectively. The genome comparison identified six transposons carrying antimicrobial resistance genes. The ermB gene was carried by new Tn7808, Tn6189 and Tn6218-like. The aac(6')-Ie-aph(2'')-Ia were carried by Tn6218-like and new Tn7806 together with cfrE gene. New Tn7807 carried a cat-like gene. Tn6110 and new Tn7806 contained an RlmN-type 23S rRNA methyltransferase, designated MrmA, associated with high-level macrolide resistance in isolates without ermB gene. CONCLUSIONS: Multidrug-resistant C. difficile PCR ribotype 176 isolates carry already described and unique transposons. A novel mechanism for erythromycin resistance in C. difficile was identified.

• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Drug Resistance, Bacterial * MeSH
• Bacterial Proteins genetics   MeSH
• Clostridioides difficile * genetics   drug effects   isolation & purification   classification   MeSH
• Genomic Islands * MeSH
• Clostridium Infections * microbiology   epidemiology   MeSH
• Humans MeSH
• Methyltransferases genetics   MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial * genetics   MeSH
• Multilocus Sequence Typing MeSH
• Ribotyping MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH
• Poland MeSH

OBJECTIVES: To assess the effectiveness of shortened regimens of vancomycin or fidaxomicin in the treatment of Clostridioides difficile infection (CDI). METHODS: Adult patients with CDI hospitalized from January 2022 to May 2023 were included in this observational study. In patients with CDI treated with vancomycin or fidaxomicin, antibiotic treatment was discontinued after either 5 or 7 days of vancomycin or 5 days of fidaxomicin if there was a clinical response and improvement in laboratory parameters. The control cohort was treated with the standard 10 day regimen of either vancomycin or fidaxomicin. The follow-up was 60 days. Causative C. difficile strains were characterized by ribotyping and toxin gene detection when available. RESULTS: Twenty-five patients (median age 76 years) received shortened treatment with vancomycin (n = 21), or fidaxomicin (n = 4). Five cases fulfilled the criteria for severe CDI. Twenty-three patients completed follow-up; two died from causes other than CDI, and two developed recurrent CDI (8.0%). Ribotypes (RTs) 001 and 014 were the most prevalent with 20% each. In two C. difficile isolates, binary toxin genes were detected (RTs 078 and 023). In the control group of 22 patients recurrent CDI developed in 5 patients (22.7%). No statistically significant differences were found between the groups. CONCLUSIONS: Shortened treatment regimens for CDI with vancomycin and fidaxomicin were shown to be effective in our cohort of patients compared with 10 days of treatment. The recurrence rate was lower in the study group. A larger, prospective, double-blind, randomized, multicentre study is needed to support our findings.

• MeSH
• Anti-Bacterial Agents * therapeutic use   administration & dosage   MeSH
• Clostridioides difficile * genetics   drug effects   classification   MeSH
• Fidaxomicin * therapeutic use   administration & dosage   MeSH
• Clostridium Infections * drug therapy   microbiology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Ribotyping * MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Vancomycin * therapeutic use   administration & dosage   MeSH
• Treatment Outcome MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Observational Study 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.

• MeSH
• Bacterial Proteins genetics   MeSH
• Clostridioides difficile classification   genetics   isolation & purification   MeSH
• Child MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Phylogeny * MeSH
• Genetic Variation MeSH
• Clostridium Infections microbiology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Bacterial Typing Techniques MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Iran MeSH

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.

• MeSH
• Bacterial Proteins genetics   MeSH
• Bacterial Toxins genetics   MeSH
• Clostridioides difficile classification   genetics   pathogenicity   MeSH
• Child MeSH
• DNA, Bacterial MeSH
• Adult MeSH
• Feces microbiology   MeSH
• Genetic Variation * MeSH
• Genotype MeSH
• Clostridium Infections epidemiology   microbiology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Molecular Typing MeSH
• Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Repressor Proteins genetics   MeSH
• Ribotyping MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Virulence genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Iran MeSH

AIM: To obtain standardized epidemiological data for Clostridium difficile infection (CDI) in Slovakia. METHODS: Between October and December 2016, 36 hospitals in Slovakia used the European Centre for Disease Prevention and Control (ECDC) Clostridium difficile infection (CDI) surveillance protocol. RESULTS: The overall mean CDI incidence density was 2.8 (95% confidence interval 1.9-3.9) cases per 10 000 patient-days. Of 332 CDI cases, 273 (84.9%) were healthcare-associated, 45 (15.1%) were community-associated, and 14 (4.2%) were cases of recurrent CDI. A complicated course of CDI was reported in 14.8% of cases (n=51). CDI outcome data were available for 95.5% of cases (n=317). Of the 35 patients (11.1%) who died, 34 did so within 30 days after their CDI diagnosis. Of the 78 isolates obtained from 12 hospitals, 46 belonged to PCR ribotype 001 (59.0%; 11 hospitals) and 23 belonged to ribotype 176 (29.5%; six hospitals). A total of 73 isolates (93.6%) showed reduced susceptibility to moxifloxacin (ribotypes 001 and 176; p< 0.01). A reduced susceptibility to metronidazole was observed in 13 isolates that subsequently proved to be metronidazole-susceptible when, after thawing, they were retested using the agar dilution method. No reduced susceptibility to vancomycin was found. CONCLUSIONS: These results show the emergence of C. difficile ribotypes 027 and 176 with a predominance of ribotype 001 in Slovakia in 2016. Given that an almost homogeneous reduced susceptibility to moxifloxacin was detected in C. difficile isolates, this stresses the importance of reducing fluoroquinolone prescriptions in Slovak healthcare settings.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Clostridioides difficile classification   drug effects   genetics   isolation & purification   MeSH
• Incidence MeSH
• Clostridium Infections epidemiology   microbiology   MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Moxifloxacin pharmacology   MeSH
• Ribotyping MeSH
• Aged MeSH
• Check Tag
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Slovakia MeSH

Clostridioides difficile infection (CDI) remains a major healthcare problem worldwide, however, little is known about CDI epidemiology in Iran. Between December 2004 and November 2018, 3649 stool samples were collected from patients in 69 hospitals and medical centres in Tehran and were cultured for the presence of C. difficile; isolates were characterized by PCR ribotyping and toxin genes detection. A total of 582 C. difficile isolates were obtained and the overall CDI prevalence was 15.9%; 290 (49.8%) cases were healthcare-associated (HA) and 292 (50.2%) cases were community-associated (CA). Of these, DNA of 513 isolates submitted for ribotyping. The ribotype and/or WEBRIBO type could be assessed in 366 (62.9%) isolates. The most frequent RTs were 001 (n = 75, 12.9%), 126 (n = 65, 11.2%) and 084 (n = 19, 3.3%); the toxin gene profile tcdA+B+/cdtA+B+ (n = 112, 19.2%) was the most common. Fifteen C. difficile isolates (2.6%) did not carry any toxin genes. There was no difference between frequently found RTs in HA-CDI and CA-CDI, except for RT 029 which was more likely to be associated with healthcare origin (12/15, p-value = 0.02). No isolate of RTs 027 or 078 was identified. Importantly, RTs 031, 038, 039, 084, 085 reported previously as RTs with an absence of toxin genes, revealed the presence of toxin genes in our study. Using Simpson's reciprocal index of diversity, we found that RT diversity decreased as the prevalence of the RT 084 increased (R = -0.78, p-value = 0.041). Different patterns in CDI epidemiology underscore the importance of local surveillance and infection control measures in Tehran healthcare settings.

• MeSH
• Clostridioides difficile classification   genetics   isolation & purification   MeSH
• History, 21st Century MeSH
• Child MeSH
• Adult MeSH
• Phylogeny MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Child, Preschool MeSH
• Cross-Sectional Studies MeSH
• Enterocolitis, Pseudomembranous history   microbiology   MeSH
• Ribotyping MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• History, 21st Century MeSH
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Geographicals
• Iran MeSH

OBJECTIVES: This study examined the antimicrobial susceptibility and resistance mechanisms of Clostridium difficile recovered in Greek hospitals during 2012-2015. METHODS: C. difficile isolates (n=88) were collected from clinically-confirmed C. difficile infection from symptomatic patients in 10 Greek hospitals. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by Etest. Isolates were typed by multilocus sequence typing (MLST). Toxin and resistance genes were detected by PCR. Chromosomal mutations in gyrA, gyrB and rpoB were identified by PCR and sequencing. The genetic environment of resistance genes was characterised by Illumina sequencing. RESULTS: The 88 C. difficile isolates comprised 27 sequence types (STs), with ST37 (n=26) and ST11 (n=21) being the most prevalent. All isolates were susceptible to vancomycin and metronidazole, with variable resistance rates to other antimicrobials. Of the 88 isolates, 45.5% were multidrug-resistant and the majority belonged to ST11 and ST37. The presence of chromosomal mutations in gyrA, gyrB and rpoB was mainly observed in high-risk clones such as ST11 and ST37. The antimicrobial resistance genes ermB, mefA, msrA and tetM were identified at different prevalences and combinations. Additionally, cfrB and cfrC were identified for the first time in Greece and were carried by a Tn6218 transposon and a novel plasmid, respectively. CONCLUSIONS: To our knowledge, this is the first study examining the resistance profiles and respective mechanisms of C. difficile recovered in Greek hospitals. Gut commensals such as C. difficile may serve as hubs for further transfer of antimicrobial resistance genes.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• Clostridioides difficile classification   drug effects   genetics   MeSH
• Clostridium Infections microbiology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Multilocus Sequence Typing MeSH
• Mutation MeSH
• Hospitals MeSH
• Bacterial Typing Techniques MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Greece MeSH

• MeSH
• Clostridioides difficile classification   genetics   MeSH
• Disease Outbreaks * MeSH
• Cross Infection diagnosis   epidemiology   MeSH
• Clostridium Infections diagnosis   epidemiology   MeSH
• Humans MeSH
• Minisatellite Repeats genetics   MeSH
• Molecular Epidemiology MeSH
• Multilocus Sequence Typing MeSH
• Ribotyping MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

This study aimed to implement a toxigenic culture as an optional third diagnostic step for glutamate dehydrogenase (GDH)-positive and toxin A/B-negative diarrheal stool samples into a diagnostic algorithm for Clostridioides (Clostridium) difficile infection (CDI), and to characterise C. difficile isolates for epidemiological purposes. During the 5-month study, 481 diarrhoeal stool samples from three Slovak hospitals were investigated and 66 non-duplicated GDH-positive stool samples were found. Of them, 36 were also toxin A/B-positive. Twenty-three GDH-positive and toxin A/B-negative stool samples were shown subsequently to be positive following toxigenic culture (TC). Molecular characterisation of C. difficile isolates showed the predominance of PCR ribotype (RT) 001 (n = 37, 56.1%) and the occurrence of RT 176 (n = 3, 4.5%). C. difficile RT 001 isolates clustered to eight clonal complexes (CCs) using multiple-locus variable-number tandem repeats analysis (MLVA). Interestingly, one third of RT 001 isolates clustering in these CCs were cultured from toxin A/B-negative stool samples. Our observations highlight the need of use multiple step diagnostic algorithm in CDI diagnosis in order to detect all CDI cases and to avoid the spread of C. difficile in healthcare settings.

• MeSH
• Algorithms MeSH
• Bacterial Proteins analysis   MeSH
• Clostridioides difficile classification   isolation & purification   MeSH
• Molecular Diagnostic Techniques MeSH
• Enterotoxins analysis   deficiency   MeSH
• Feces microbiology   MeSH
• Glutamate Dehydrogenase analysis   MeSH
• Clostridium Infections diagnosis   epidemiology   microbiology   MeSH
• Humans MeSH
• Minisatellite Repeats genetics   MeSH
• Molecular Typing * MeSH
• Multilocus Sequence Typing MeSH
• Hospitals MeSH
• Polymerase Chain Reaction * MeSH
• Diarrhea diagnosis   epidemiology   microbiology   MeSH
• Ribotyping methods   MeSH
• Cluster Analysis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Geographicals
• Slovakia MeSH