Antibiotic susceptibility and resistance profiles of Romanian Clostridioides difficile isolates


This study investigated the antibiotic susceptibility patterns and genetic resistance markers of 35 C. difficile strains isolated from patients with C. difficile infection. Vancomycin, metronidazole, tigecycline, teicoplanin, rifampicin, moxifloxacin, cefotaxime, tetracycline, erythromycin, clindamycin, chloramphenicol, linezolid and imipenem MICs were determined for toxigenic strains belonging to PCR ribotypes (PR) 012 (2), 014 (4), 017 (3), 018 (2), 027 (17), 046 (2), 087 (3) and 115 (2). Results showed vancomycin, metronidazole, tigecycline and teicoplanin to be active against all isolates. High resistance rates were noticed against cefotaxime (n = 35), clindamycin (n = 33), imipenem (n = 31), moxifloxacin (n = 25), erythromycin (n = 25) and rifampicin (n = 22). Linezolid-resistance was found in three isolates (PR 017/2, PR 012/1), showing complex resistance (7-9 antibiotics). PR 012, 017, 018, 027 and 046 isolates (n = 26) were resistant to 5-9 antibiotics. Twelve resistance profiles (2-9 antibiotics) were detected. Rifampicin-moxifloxacin-cefotaxime-erythromycin-clindamycin-imipenem-resistance was predominant, being expressed by 18 strains (PR 027/17, PR 018/1). PCR results suggested tetracycline-resistance to be induced by the gene tetM. Three tetM-positive isolates (PRs 012, 046), were also tndX-positive, suggesting the presence of a Tn5397-like element. Only two MLSB-resistant strains (PR 012) had the ermB gene and chloramphenicol-resistance determinant catD was not detected, leaving room for further investigating resistance mechanisms. Multidrug resistance could be attributed to most analysed strains, underlining, once more, the impact of wide-spectrum antimicrobial over prescription, still a tendency in our country, on transmission of antimicrobial resistance and emergence of epidemic C. difficile strains generating outbreaks.

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  • 1. Bartlett JG, Gerding DN. Clinical recognition and diagnosis of Clostridium difficile infection. Clin Infect Dis. 2008 Jan;46(Suppl 1):S12-S18. DOI: 10.1086/521863

  • 2. Bloomfield LE, Riley TV. Epidemiology and risk factors for community-associated Clostridium difficile infection: a narrative review. Infect Dis Ther. 2016 Sep;5(3):231-51. DOI: 10.1007/s40121-016-0117-y

  • 3. Rupnik M. Is Clostridium difficile-associated infection a potentially zoonotic and foodborne disease? Clin Microbiol Infect. 2007 May;13(5):457-9. DOI: 10.1111/j.1469-0691.2007.01687.x

  • 4. Hall IC, O’Toole E. Intestinal flora in new-born infants with a description of a new pathogenic anaerobe, Bacillus difficilis. Am J Dis Child. 1935;49(2):390-402. DOI: 10.1001/archpedi.1935.01970020105010

  • 5. Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev. 2005 Apr;18(2):247-263. DOI: 10.1128/ CMR.18.2.247-263.2005

  • 6. Wilcox MH, Chalmers JD, Nord CE, Freeman J, Bouza E. Role of cephalosporins in the era of Clostridium difficile infection. J Antimicrob Chemother. 2017 Jan;72(1):1-18. DOI: 10.1093/jac/dkw385

  • 7. Bartlett JG, Onderdonk AB, Cisneros RL, Kasper DL. Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. J Infect Dis. 1977 Nov;136(5):701-5. DOI: 10.1093/infdis/136.5.701

  • 8. Owens Jr RC, Donskey CJ, Gaynes RP, Loo VG, Muto CA. Antimicrobial-associated risk factors for Clostridium difficile infection. Clin Infect Dis. 2008 Jan;46(- Suppl 1):S19-S31. DOI: 10.1086/521859

  • 9. Baines SD, Wilcox MH. Antimicrobial resistance and reduced susceptibility in Clostridium difficile: potential consequences for induction, treatment, and recurrence of C. difficile infection. Antibiotics (Basel). 2015 Sep;4(3):267-8. DOI: 10.3390/antibiotics4030267

  • 10. Spigaglia P. Recent advances in the understanding of antibiotic resistance in Clostridium difficile infection. Ther Adv Infect Dis. 2016 Feb;3(1):23-42. DOI: 10.1177/2049936115622891

  • 11. McFarland LV, Ozen M, Dinleyici EC, Goh S. Comparison of pediatric and adult antibiotic-associated diarrhea and Clostridium difficile infections. World J Gastroenterol. 2016 Mar;22(11):3078-104. DOI: 10.3748/wjg.v22.i11.3078

  • 12. Freeman J, Vernon J, Vickers R, Wilcox MH. Susceptibility of Clostridium difficile isolates of varying antimicrobial resistance phenotypes to SMT19969 and 11 comparators. Antimicrob Agents Chemother. 2016 Jan;60(1):689-692. DOI: 10.1128/AAC.02000-15

  • 13. Spigaglia P, Barbanti F, Mastrantonio P, on behalf of the European Study Group on Clostridium difficile (ESGCD). Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother. 2011 Oct;66(10):2227-34. DOI: 10.1093/jac/dkr292

  • 14. Vardakas KZ, Konstantelias AA, Loizidis G, Rafailidis PI, Falagas ME. Risk factors for development of Clostridium difficile infection due to BI/ NAP1/027 strain: a meta-analysis. Int J Infect Dis. 2012 Nov;16(11):e768-e773/. DOI: 10.1016/j.ijid.2012.07.010

  • 15. Freeman J, Vernon J, Morris K, Nicholson S, Todhunter S, Longshaw C, et al. Pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes. Clin Microbiol Infect. 2015 Mar; 21(3):248.e9-248.e16. DOI: 10.1016/j.cmi.2014.09.017

  • 16. Farrow KA, Lyras D, Rood JI. Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile. Microbiology. 2001 Oct;147(10):2717-28. DOI: 10.1099/00221287-147-10-2717

  • 17. Spigaglia P, Mastrantonio P. Comparative analysis of Clostridium difficile clinical isolates belonging to different genetic lineages and time periods. J Med Microbiol. 2004 Nov;53(11):1129-36. DOI: 10.1099/jmm.0.45682-0

  • 18. Dönhöfer A, Franckenberga S, Wicklesa S, Berninghausena O, Beckmann R, Wilson DN. Structural basis for TetM-mediated tetracycline resistance. Proc Natl Acad Sci U S A. 2012 Oct;109(42):16900-5. DOI: 10.1073/pnas.1208037109

  • 19. Mullany P, Wilks M, Lamb I, Clayton C, Wren B, Tabaqchali S. Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis. J Gen Microbiol. 1990 Jul;136(7):1343-9. DOI: 10.1099/00221287-136-7-1343

  • 20. Wang H, Mullany P. The large resolvase TndX is required and sufficient for integration and excision of derivatives of the novel conjugative transposon Tn5397. J Bacteriol. 2000 Dec;182(23):6577-83. DOI: 10.1128/JB.182.23.6577-6583.2000

  • 21. Spigaglia P, Carucci V, Barbanti F, Mastrantonio P. ErmB determinants and Tn916-like elements in clinical isolates of Clostridium difficile. Antimicrob Agents Chemother. 2005 Jun;49(6):2550-3. DOI: 10.1128/AAC.49.6.2550-2553.2005

  • 22. Fry PR, Thakur S, Abley M, Gebreyesa WA. Antimicrobial resistance, toxinotype, and genotypic profiling of Clostridium difficile isolates of swine origin. J Clin Microbiol. 2012 Jul;50(7):2366-72. DOI: 10.1128/JCM.06581-11

  • 23. Spigaglia P, Barbanti F, Mastrantonio P. Tetracycline resistance gene tet(W) in the pathogenic bacterium Clostridium difficile. Antimicrob Agents Chemother.2008 Feb;52(2):770-3. DOI: 10.1128/AAC.00957-07

  • 24. Kuijper EJ, Coignard B, Tüll P, on behalf of the ESCMID Study Group for Clostridium difficile (ESGCD), EU Member States and the European Centre for Disease Prevention and Control (ECDC). Emergence of Clostridium difficile-associated disease in North Americaand Europe. Clin Microbiol Infect. 2006 Oct;12(Suppl. 6):2-18. DOI: 10.1111/j.1469-0691.2006.01580.x

  • 25. Popescu GA, Șerban R, Pistol A, Niculcea A, Preda A, Lemeni D, et al. Clinical and microbiological characterization of Clostridium difficile infection in Romania (2013-2014); a hospital based study. BMC Infect Dis. 2014a;14(Suppl 7):o24. DOI: 10.1186/1471-2334-14-S7-O24

  • 26. Popescu GA, Florea D, Rafila A. Clostridium difficile is emerging in Romania: a story of 027 ribotype and excessive antibiotic consumption. J Gastrointestin Liver Dis. 2014b;23(3):342-3.

  • 27. Florea D, Huhulescu S, Indra A, Badicut I, Rafila A, Otelea D, et al. PCR coupled with mass-spectrometry for detection of Clostridium difficile virulence markers during the emergence of ribotype 027 in Bucharest area. Rev Romana Med Lab. 2015;23(4):449-55. DOI:10.1515/rrlm-2015-0044

  • 28. Macovei IS, Lemeni D, Usein CR, Șerban R, Niculcea A, Popescu GA, et al. The use of PCR Ribotyping for molecular typing of clinically significant Clostridium difficile Romanian isolates. Rom Biotechnol Lett.2017;22(5).

  • 29. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters, version 7.1. 2017;

  • 30. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. CLSI document M100-27th ed. 2017:96-100.

  • 31. Marin M, Martín A, Alcalá LM, Cercenado E, Iglesias C, Reigadas E, et al. Clostridium difficile isolates with high linezolid MICs harbor the multiresistance gene cfr. Antimicrob Agents Chemother. 2015 Jan;59(1):586-9. DOI: 10.1128/AAC.04082-14

  • 32. Marchese A, Ramirez M, Schito GC, Tomasz A. Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996. J Clin Microbiol. 1998 Oct;36(10):2944-9.

  • 33. Baines SD, O’Connor R, Freeman J, Fawley WN, Harmanus C, Mastrantonio P, et al. Emergence of reduced susceptibility to metronidazole in Clostridium difficile. J Antimicrob Chemother. 2008 Nov;62(5):1046-52. DOI: 10.1093/jac/dkn313

  • 34. Marsh JW, Arora R, Schlackman JL, Shutt KA, Curry SR, Harrison LH. Association of relapse of Clostridium difficile disease with BI/NAP1/027. J Clin Microbiol. 2012 Dec;50(12):4078-82. DOI: 10.1128/JCM.02291-12

  • 35. Cherian PT, Wu X, Yang L, Scarborough JS, Singh AP, Alam ZA, et al. Gastrointestinal localization of metronidazole by a lactobacilli-inspired tetramic acid motif improves treatment outcomes in the hamster model of Clostridium difficile infection. J Antimicrob Chemother. 2015 Nov;70(11):3061-9. DOI: 10.1093/jac/dkv231

  • 36. Robinson CD, Auchtung JM, Collins J, Britton RA. Epidemic Clostridium difficile strains demonstrate increased competitive fitness compared to nonepidemic isolates. Infect Immun. 2014 Jul;82(7):2815-25. DOI: 10.1128/IAI.01524-14

  • 37. Lachowicz D, Pituch H, Obuch-Woszczatyński P. Antimicrobial susceptibility patterns of Clostridium difficile strains belonging to different polymerase chain reaction ribotypes isolated in Poland in 2012. Anaerobe. 2015 Feb;31:37-41. DOI: 10.1016/j.anaerobe.2014.09.004

  • 38. Spigaglia P, Barbanti F, Mastrantonio P. Detection of a genetic linkage between genes coding for resistance to tetracycline and erythromycin in Clostridium difficile. Microb Drug Resist. 2007;13(2):90-5. DOI: 10.1089/mdr.2007.723

  • 39. Knight DR, Elliott B, Chang BJ, Perkins TT, Riley TV. Diversity and evolution in the genome of Clostridium difficile. Clin Microbiol Rev. 2015 Jul;28(3):721-41. DOI: 10.1128/CMR.00127-14

  • 40. Büchler AC, Rampini SK, Stelling S, Ledergerber B, Peter S, Schweiger A, et al. Antibiotic susceptibility of Clostridium difficile is similar worldwide over two decades despite widespread use of broad-spectrum antibiotics: an analysis done at the University Hospital of Zurich. BMC Infect Dis. 2014 Nov;14:607. DOI: 10.1186/s12879-014-0607-z

  • 41. Curry SR, Marsh JW, Shutt KA, Muto CA, O’Leary MM, Saul MI, et al. High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis. 2009 Feb;48(4):425-9. DOI: 10.1086/596315


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