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.

Department of Clinical Microbiology Unilabs Slovakia Inc Roznava Slovakia

Department of Medical Microbiology Charles University 2nd Faculty of Medicine and Motol University Hospital Prague Czech Republic

Department of Medical Microbiology Medical University of Warsaw Warsaw Poland

Department of Microbiology and Immunology Comenius University Jessenius Faculty of Medicine in Martin Martin Slovakia

Dutch National Expertise Centre for Clostridioides difficile infections Leiden University Center for Infectious Diseases Leiden and Centre for Infectious Disease Control Bilthoven Netherlands

European Society of Clinical Microbiology and Infectious Diseases Basel Switzerland

Experimental Bacteriology Leiden University Center for Infectious Diseases Leiden Medical Center Leiden Netherlands

Institute of Microbiology The Czech Academy of Sciences BIOCEV Vestec Czech Republic

Zobrazit více v PubMed

He M, Miyajima F, Roberts P, et al. . Emergence and global spread of epidemic healthcare-associated Clostridium difficile. Nat Genet. 2013;45(1):109–113. doi:10.1038/ng.2478 PubMed DOI PMC

Viprey VF, Davis GL, Benson AD, et al. . A point-prevalence study on community and inpatient Clostridioides difficile infections (CDI): results from combatting bacterial resistance in Europe CDI (COMBACTE-CDI), July to November 2018. Euro Surveill. 2022;27(26):2100704, doi:10.2807/1560-7917.ES.2022.27.26.2100704 PubMed DOI PMC

Krutova M, Wilcox MH, Kuijper EJ.. The pitfalls of laboratory diagnostics of Clostridium difficile infection. Clin Microbiol Infect. 2018;24(7):682–683. doi:10.1016/j.cmi.2018.02.026 PubMed DOI

Baktash A, Corver J, Harmanus C, et al. . Comparison of whole-genome sequence-based methods and PCR ribotyping for subtyping of Clostridioides difficile. J Clin Microbiol. 2022;60(2):e0173721, doi:10.1128/JCM.01737-21 PubMed DOI PMC

European Centre for Disease Prevention and Control . Study protocol for a survey of whole genome sequencing of Clostridioides difficile isolates from tertiary acute care hospitals, EU/EEA, 2022–2023. Stockholm: ECDC; 2024.

Pituch H, Obuch-Woszczatyński P, Lachowicz D, et al. . Hospital-based Clostridium difficile infection surveillance reveals high proportions of PCR ribotypes 027 and 176 in different areas of Poland, 2011 to 2013. Euro Surveill. 2015;20(38), doi:10.2807/1560-7917.ES.2015.20.38.30025 PubMed DOI

Krutova M, Nyc O, Kuijper EJ, et al. . A case of imported Clostridium difficile PCR-ribotype 027 infection within the Czech Republic which has a high prevalence of C. difficile ribotype 176. Anaerobe. 2014;30:153–155. doi:10.1016/j.anaerobe.2014.09.020 PubMed DOI

Krutova M, Capek V, Nycova E, et al. . The association of a reduced susceptibility to moxifloxacin in causative Clostridium (Clostridioides) difficile strain with the clinical outcome of patients. Antimicrob Resist Infect Control. 2020;9(1):98, doi:10.1186/s13756-020-00765-y PubMed DOI PMC

Nyc O, Krutova M, Liskova A, et al. . The emergence of Clostridium difficile PCR-ribotype 001 in Slovakia. Eur J Clin Microbiol Infect Dis. 2015;34(8):1701–1708. doi:10.1007/s10096-015-2407-9 PubMed DOI

Plankaova A, Brajerova M, Capek V, et al. . Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018-2019. Int J Antimicrob Agents. 2023;62(1):106824, doi:10.1016/j.ijantimicag.2023.106824 PubMed DOI

Novakova E, Kotlebova N, Gryndlerova A, et al. . An outbreak of Clostridium (Clostridioides) difficile infections within an acute and long-term care wards due to moxifloxacin-resistant PCR ribotype 176 genotyped as PCR ribotype 027 by a commercial assay. J Clin Med. 2020;9(11):3738, doi:10.3390/jcm9113738 PubMed DOI PMC

Krehelova M, Nyč O, Sinajová E, et al. . The predominance and clustering of Clostridioides (Clostridium) difficile PCR ribotype 001 isolates in three hospitals in eastern Slovakia, 2017. Folia Microbiol (Praha). 2019;64(1):49–54. doi:10.1007/s12223-018-0629-9 PubMed DOI

Krutova M, Matejkova J, Kuijper EJ, et al. . Clostridium difficile PCR ribotypes 001 and 176 - the common denominator of C. difficile infection epidemiology in the Czech Republic, 2014. Euro Surveill. 2016;21(29), doi:10.2807/1560-7917.ES.2016.21.29.30296 PubMed DOI

Karpiński P, Wultańska D, Piotrowski M, et al. . Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes. Anaerobe. 2022;73:102476, doi:10.1016/j.anaerobe.2021.102476 PubMed DOI

Fawley WN, Knetsch CW, MacCannell DR, et al. . Development and validation of an internationally-standardized, high-resolution capillary gel-based electrophoresis PCR-ribotyping protocol for Clostridium difficile. PLoS One. 2015;10(2):e0118150, doi:10.1371/journal.pone.0118150 PubMed DOI PMC

Persson S, Torpdahl M, Olsen KE.. New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection. Clin Microbiol Infect. 2008;14(11):1057–1064. doi:10.1111/j.1469-0691.2008.02092.x. Erratum in: Clin Microbiol Infect. 2009;15(3):296. PubMed DOI

van den Berg RJ, Schaap I, Templeton KE, et al. . Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis. J Clin Microbiol. 2007;45(3):1024–1028. PubMed PMC

Prjibelski A, Antipov D, Meleshko D, et al. . Using SPAdes De novo assembler. Curr Protocols Bioinf. 2020;70(1), doi:10.1002/cpbi.102 PubMed DOI

Kolmogorov M, Yuan J, Lin Y, et al. . Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019;37(5):540–546. doi:10.1038/s41587-019-0072-8 PubMed DOI

Wick RR, Holt KE.. Polypolish: short-read polishing of long-read bacterial genome assemblies. PLoS Comput Biol. 2022;18(1):e1009802, doi:10.1371/journal.pcbi.1009802 PubMed DOI PMC

Kaas RS, Leekitcharoenphon P, Aarestrup FM, et al. . Solving the problem of comparing whole bacterial genomes across different sequencing platforms. PLoS One. 2014;9(8):e104984, doi:10.1371/journal.pone.0104984 PubMed DOI PMC

Kozlov AM, Darriba D, Flouri T, et al. . RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35(21):4453–4455. doi:10.1093/bioinformatics/btz305 PubMed DOI PMC

Letunic I, Bork P.. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res. 2024;52(W1):W78–W82. doi:10.1093/nar/gkae268 PubMed DOI PMC

Torsten S. (2015). “Abricate.” Github. https://github.com/tseemann/abricate.

Isidro J, Santos A, Nunes A, et al. . Imipenem resistance in Clostridium difficile ribotype 017, Portugal. Emerg Infect Dis. 2018;24(4):741–745. doi:10.3201/eid2404 PubMed DOI PMC

Boekhoud IM, Sidorov I, Nooij S, et al. . Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile. J Antimicrob Chemother. 2021;76(7):1731–1740. doi:10.1093/jac/dkab097 PubMed DOI PMC

Olaitan AO, Dureja C, Youngblom MA, et al. . Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile. Nat Commun. 2023;14(1):4130, doi:10.1038/s41467-023-39429-x PubMed DOI PMC

Smits WK, Harmanus C, Sanders IMJG, et al. . Sequence-Based identification of metronidazole-resistant Clostridioides difficile isolates. Emerg Infect Dis. 2022;28(11):2308–2311. doi:10.3201/eid2811.220615 PubMed DOI PMC

Sullivan MJ, Petty NK, Beatson SA.. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27(7):1009–1010. doi:10.1093/bioinformatics/btr039 PubMed DOI PMC

Bertelli C, Laird MR, Williams KP, et al. . Islandviewer 4: expanded prediction of genomic islands for larger-scale datasets. Nucleic Acids Res. 2017;45(W1):W30–W35. doi:10.1093/nar/gkx343 PubMed DOI PMC

Johansson MHK, Bortolaia V, Tansirichaiya S, et al. . Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: MobileElementFinder. J Antimicrob Chemother. 2021;76(1):101–109. doi:10.1093/jac/dkaa390 PubMed DOI PMC

Wang M, Liu G, Liu M, et al. . Iceberg 3.0: functional categorization and analysis of the integrative and conjugative elements in bacteria. Nucleic Acids Res. 2024;52(D1):D732–D737. doi:10.1093/nar/gkad935 PubMed DOI PMC

Roberts AP, Chandler M, Courvalin P, et al. . Revised nomenclature for transposable genetic elements. Plasmid. 2008;60(3):167–173. doi:10.1016/j.plasmid.2008.08.001 PubMed DOI PMC

Eyre DW, Davies KA, Davis G, et al. . Two Distinct Patterns of Clostridium difficile Diversity Across Europe Indicating Contrasting Routes of Spread. Clin Infect Dis. 2018;67(7):1035–1044. doi:10.1093/cid/ciy252 PubMed DOI PMC

Cizek A, Masarikova M, Mares J, et al. . Detection of plasmid-mediated resistance to metronidazole in Clostridioides difficile from river water. Microbiol Spectr. 2022;10(4):e0080622, doi:10.1128/spectrum.00806-22 PubMed DOI PMC

Yan F, LaMarre JM, Röhrich R, et al. . Rlmn and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA. J Am Chem Soc. 2010;132(11):3953–3964. doi:10.1021/ja910850y PubMed DOI PMC

Atkinson GC, Hansen LH, Tenson T, et al. . Distinction between the Cfr methyltransferase conferring antibiotic resistance and the housekeeping RlmN methyltransferase. Antimicrob Agents Chemother. 2013;57(8):4019–4026. doi:10.1128/AAC.00448-13 PubMed DOI PMC

Hansen LH, Vester B.. A cfr-like gene from Clostridium difficile confers multiple antibiotic resistance by the same mechanism as the cfr gene. Antimicrob Agents Chemother. 2015;59(9):5841–5843. doi:10.1128/AAC.01274-15 PubMed DOI PMC

Baba T, Ara T, Hasegawa M, et al. . Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2; doi:10.1038/msb4100050 PubMed DOI PMC

Sebaihia M, Wren BW, Mullany P, et al. . The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet. 2006;38(7):779–786. doi:10.1038/ng1830 PubMed DOI

Brouwer MS, Warburton PJ, Roberts AP, et al. . Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile. PLoS One. 2011;6(8):e23014, doi:10.1371/journal.pone.0023014 PubMed DOI PMC

Singh KV, Weinstock GM, Murray BE.. An Enterococcus faecalis ABC homologue (Lsa) is required for the resistance of this species to clindamycin and quinupristin-dalfopristin. Antimicrob Agents Chemother. 2002;46(6):1845–1850. doi:10.1128/AAC.46.6.1845-1850.2002 PubMed DOI PMC

Rice LB, Carias LL, Marshall S, et al. . Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion. J Bacteriol. 2005;187(19):6668–6677. doi:10.1128/JB.187.19.6668-6677.2005 PubMed DOI PMC

Guédon G, Lao J, Payot S, et al. . FirmiData: a set of 40 genomes of Firmicutes with a curated annotation of ICEs and IMEs. BMC Res Notes. 2022;15(1):157, doi:10.1186/s13104-022-06036-w PubMed DOI PMC

Gebhart D, Williams SR, Bishop-Lilly KA, et al. . Novel high-molecular-weight, R-type bacteriocins of Clostridium difficile. J Bacteriol. 2012;194(22):6240–47. doi:10.1128/jb.01272-12. PubMed DOI PMC

Haraldsen JD, Sonenshein AL.. Efficient sporulation in Clostridium difficile requires disruption of the σK gene. Molecular Microbiology. 2003;48(3):811–21. doi:10.1046/j.1365-2958.2003.03471.x. PubMed DOI

Woods EC, Wetzel D, Mukerjee M, et al. . Examination of the Clostridioides (Clostridium) difficile VanZ ortholog, CD1240. Anaerobe. 2018;53:108–115. doi:10.1016/j.anaerobe.2018.06.013. PubMed DOI PMC

Giessing AM, Jensen SS, Rasmussen A, et al. . Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria. RNA. 2009;15(2):327–336. doi:10.1261/rna.1371409 PubMed DOI PMC

Long KS, Poehlsgaard J, Kehrenberg C, et al. . The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Antimicrob Agents Chemother. 2006;50(7):2500–2505. doi:10.1128/AAC.00131-06 PubMed DOI PMC

Toh SM, Xiong L, Bae T, et al. . The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA. RNA. 2008;14(1):98–106. doi:10.1261/rna.814408 PubMed DOI PMC

Eyre DW, Fawley WN, Best EL, et al. . Comparison of multilocus variable-number tandem-repeat analysis and whole-genome sequencing for investigation of Clostridium difficile transmission. J Clin Microbiol. 2013;51(12):4141–4149. doi:10.1128/JCM.01095-13 PubMed DOI PMC

Khanafer N, Daneman N, Greene T, et al. . Susceptibilities of clinical Clostridium difficile isolates to antimicrobials: a systematic review and meta-analysis of studies since 1970. Clin Microbiol Infect. 2018;24(2):110–117. doi:10.1016/j.cmi.2017.07.012 PubMed DOI

Dingle KE, Freeman J, Didelot X, et al. . Penicillin binding protein substitutions cooccur with fluoroquinolone resistance in epidemic lineages of multidrug-resistant Clostridioides difficile. mBio. 2023;14(2):e0024323, doi:10.1128/mbio.00243-23 PubMed DOI PMC

Krutova M, Matejkova J, Tkadlec J, et al. . Antibiotic profiling of Clostridium difficile ribotype 176–A multidrug resistant relative to C. difficile ribotype 027. Anaerobe. 2015;36:88–90. doi:10.1016/j.anaerobe.2015.07.009 PubMed DOI

Spigaglia P, Barbanti F, Mastrantonio P, et al. . Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother. 2011;66(10):2227–2234. doi:10.1093/jac/dkr292 PubMed DOI

Kaminska KH, Purta E, Hansen LH, et al. . Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria. Nucleic Acids Res. 2010;38(5):1652–1663. PubMed PMC

Li M, Shen X, Yan J, et al. . GI-type T4SS-mediated horizontal transfer of the 89 K pathogenicity island in epidemic Streptococcus suis serotype 2. Mol Microbiol. 2011;79(6):1670–1683. doi:10.1111/j.1365-2958.2011.07553.x PubMed DOI PMC

Zhang W, Rong C, Chen C, et al. . Type-IVC secretion system: a novel subclass of type IV secretion system (T4SS) common existing in gram-positive genus Streptococcus. PLoS One. 2012;7(10):e46390, doi:10.1371/journal.pone.0046390 PubMed DOI PMC

Sorokina J, Sokolova I, Rybolovlev I, et al. . VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile. J Bacteriol. 2021;203(21):e0035921, doi:10.1128/JB.00359-21 PubMed DOI PMC