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Antimicrobial Agents and Chemotherapy, May 2006, p. 1890-1892, Vol. 50, No. 5
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.5.1890-1892.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Antimicrobial Susceptibility Pattern of Clostridium difficile and Its Relation to PCR Ribotypes in a Swedish University Hospital

Olle Aspevall,1,2* Annika Lundberg,1 Lars G. Burman,3 Thomas Åkerlund,3 and Bo Svenungsson4,5

Department of Laboratory Medicine, Division of Clinical Bacteriology,1 Department of Medicine, Division of Infectious Diseases, Karolinska University Hospital at Huddinge, Stockholm, Sweden,4 Department of Clinical Microbiology, Akademiska University Hospital, SE-751 85, Uppsala, Sweden,2 Department of Bacteriology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden,3 Department of Communicable Disease Control and Prevention, Karolinska University Hospital, Stockholm, Sweden5

Received 30 June 2005/ Returned for modification 12 October 2005/ Accepted 13 February 2006


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ABSTRACT
 
All 238 Clostridium difficile isolates were susceptible to metronidazole and vancomycin, whereas 84% and 1% were resistant to clindamycin and fusidic acid. Etest MICs for metronidazole were lower than agar dilution MICs (P < 0.01) but without difference in susceptible-intermediate-resistant categorization. No particular PCR ribotype was associated with clindamycin or fusidic acid resistance.


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TEXT
 
Clostridium difficile-associated diarrhea (CDAD) is common in hospitals (2, 3, 11, 14, 21, 22, 27, 29) and usually treated with metronidazole or vancomycin, which have comparable response rates (80% to 90%) and relapse rates (5% to 25%) (13, 23). Metronidazole is currently the first choice agent due to its lower potential for selecting vancomycin-resistant enterococci and for economical reasons, and fusidic acid has been suggested as an alternative drug (17, 30).

As several studies have reported full susceptibility of C. difficile isolates for metronidazole and vancomycin, clinical laboratories do not routinely perform culture and susceptibility testing of the organism (5, 8, 10, 12). However, resistance to metronidazole in up to 9% of isolates (1, 4, 6, 18), intermediate resistance to vancomycin in 3% of isolates (1), and "relatively poor" outcome of metronidazole therapy in CDAD (15, 19, 28) was recently reported. This emphasizes the need for periodic monitoring of any emergence of drug resistance in C. difficile. The aim of the present study was to investigate its susceptibility to metronidazole, vancomycin, fusidic acid, and clindamycin in a Swedish tertiary care hospital by using the Etest.

The first C. difficile isolate from each of 238 consecutive CDAD patients at Huddinge University Hospital, Stockholm, Sweden, from 10 February 2000 through 10 February 2001, were studied. The definition of CDAD, culture method, and epidemiological data for these patients were recently described (25). All C. difficile isolates were also subject to PCR ribotyping according to the method of Stubbs et al. (24) but with an improved method previously correlated with serotypes (25). Isolates were subcultured three times on horse blood agar prior to susceptibility testing to ensure purity. Bacteria were suspended in tryptic soy broth to a McFarland standard of 1 to 1.5 for testing against clindamycin, fusidic acid, metronidazole, and vancomycin by Etest on Brucella blood agar according to the manufacturer's instructions (AB-Biodisk, Solna, Sweden). Plates were scored after anaerobic incubation at 36°C for 48 h.

A random subset of 34 isolates was also tested using agar dilution to determine metronidazole MICs for comparison with Etest data. Agar dilution was performed on Wilkins-Chalgren agar (1) incubated as described above. Eubacterium lentum ATCC 43055 and Clostridium perfringens ATCC 13124 were used for quality control.

MICs for the drugs tested are shown in Table 1. Using the pharmacological breakpoints (Table 1) recommended by the Swedish Reference Group for Antibiotics (SRGA) (http://www.srga.org), all isolates were susceptible to metronidazole and vancomycin, and 99% were susceptible to fusidic acid. In contrast, only 16% of the isolates were susceptible to clindamycin and 11% were highly resistant to this agent. When using CLSI (formerly NCCLS) breakpoints for clindamycin (susceptible, ≤2; intermediate, 4; resistant, ≥8) 56.3%, 31.1%, and 12.6% were classified as susceptible, intermediate, and resistant, respectively. Interpretation for metronidazole was the same when using CLSI breakpoints as when using SRGA breakpoints. CLSI does not recommend any breakpoints for fusidic acid or vancomycin (7). MIC results for metronidazole from comparison of Etest to agar dilution are presented in Table 2. Etest MICs were generally lower than agar dilution data. One isolate differed by 3 dilution steps, one isolate by 2 steps, and 9 isolates by 1 step (41% lower MICs), whereas only 4 isolates (12%) had 1-step-higher MICs by Etest (P < 0.01, chi-square test). No difference in susceptible-intermediate-resistant categorization resulted from the discrepancies.


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  		TABLE 1. Antibiotic susceptibility of C. difficile isolatesa


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  		TABLE 2. Comparison of metronidazole MICs obtained by agar dilution and Etest for 34 isolates of C. difficile

PCR ribotyping identified 70 different types (25). The most frequent PCR ribotypes were SE20, SE21, SE21b, and SE22 (Table 3). The three isolates that were resistant to fusidic acid belonged to PCR ribotypes SE7b, SE26a, and SE44b. All isolates of several major PCR ribotypes were clindamycin-resistant isolates (SE12, SE14, SE16, SE17, and SE30), whereas other PCR ribotypes comprised 43 to 93% resistant isolates (SE3, SE19, SE20, SE21, SE21b, SE22, SE29b, SE25, and SE37). Thus, no particular PCR ribotype was associated with fusidic acid or clindamycin resistance.


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  		TABLE 3. Response to clindamycin of major PCR ribotypes of C. difficile comprising ≥5 isolates eachc

Treatment of CDAD with metronidazole or vancomycin is effective in most patients (13, 23), and resistance to metronidazole or vancomycin has not been reported as a cause of therapeutic failure or recurrence of CDAD. Several studies indicate that the MICs of vancomycin and metronidazole for C. difficile have remained low over the years (5, 8, 10, 12).

Also among the 238 isolates of C. difficile analyzed here, there was no evidence of resistance to metronidazole and vancomycin, and their MIC ranges were 0.032 mg/liter to 1 mg/liter and 0.5 mg/liter to 2 mg/liter, respectively. In a previous study, comprising 57 C. difficile isolates from consecutive patients at our hospital during 1997, all isolates were susceptible to vancomycin, but one isolate was resistant to metronidazole (MIC ≥ 256 mg/liter) (26). Consequently, both metronidazole and vancomycin still seem to be adequate alternatives for empirical treatment of CDAD in our patients. Of these isolates, 86% were resistant to clindamycin, compared to 83% in the present investigation, despite the fact that only 8% had been treated with this agent within the 2 months prior to infection (data not shown). In contrast to some previous reports (9, 16), no predominant clones associated with clindamycin resistance were found in the present study. However, isolates for which MICs were >256 mg/liter were found only in about half of the major PCR ribotypes.

In earlier studies, metronidazole MICs for C. difficile determined by Etest have been lower than those obtained by agar dilution. In one study, 80% of the Etest MICs were within one dilution of the agar dilution MICs of metronidazole (20). These results were confirmed in our study. The difference between the methods, however, did not result in any discrepancy in susceptible-intermediate-resistant categorization. This suggests that Etest can be used to screen for metronidazole resistance in C. difficile.

We conclude that resistance to metronidazole and vancomycin in C. difficile remains rare in our hospital. As susceptibility testing of C. difficile is labor intensive and thus costly, although less so with Etest compared to agar dilution, routine culture for C. difficile and susceptibility testing of isolates still cannot be recommended. In the present situation, periodic assessment of the antimicrobial susceptibility pattern of C. difficile seems sufficient but remains important for detection of changes over time.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Microbiology, Akademiska University Hospital, SE-751 85, Uppsala, Sweden. Phone: 46 18 6110293. Fax: 46 18 559157. E-mail: olov.aspevall{at}akademiska.se. Back


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REFERENCES
 
    1
  1. Barbut, F., D. Decre, B. Burghoffer, D. Lesage, F. Delisle, V. Lalande, M. Delmee, V. Avesani, N. Sano, C. Coudert, and J. C. Petit. 1999. Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolated in France in 1991 and 1997. Antimicrob. Agents Chemother. 43:2607-2611.[Abstract/Free Full Text]
    2. 2
  2. Bignardi, G. E. 1998. Risk factors for Clostridium difficile infection. J. Hosp. Infect. 40:1-15.[Medline]
    3. 3
  3. Brazier, J. S. 1998. The epidemiology and typing of Clostridium difficile. J. Antimicrob. Chemother. 41(Suppl. C):47-57.[Abstract/Free Full Text]
    4. 4
  4. Brazier, J. S., W. Fawley, J. Freeman, and M. H. Wilcox. 2001. Reduced susceptibility of Clostridium difficile to metronidazole. J. Antimicrob. Chemother. 48:741-742.[Free Full Text]
    5. 5
  5. Drummond, L. J., J. McCoubrey, D. G. Smith, J. M. Starr, and I. R. Poxton. 2003. Changes in sensitivity patterns to selected antibiotics in Clostridium difficile in geriatric in-patients over an 18-month period. J. Med. Microbiol. 52:259-263.[Abstract/Free Full Text]
    6. 6
  6. Fernandez, A., G. Anand, and F. Friedenberg. 2004. Factors associated with failure of metronidazole in Clostridium difficile-associated disease. J. Clin. Gastroenterol. 38:414-418.[Medline]
    7. 7
  7. Hecht, D. W., and National Committee for Clinical Laboratory Standards. 2004. Methods for antimicrobial susceptibility testing of anaerobic bacteria: approved standard, sixth edition. NCCLS, Wayne, Pa.
    8. 8
  8. Jamal, W. Y., E. M. Mokaddas, T. L. Verghese, and V. O. Rotimi. 2002. In vitro activity of 15 antimicrobial agents against clinical isolates of Clostridium difficile in Kuwait. Int. J. Antimicrob. Agents 20:270-274.[Medline]
    9. 9
  9. Johnson, S., M. H. Samore, K. A. Farrow, G. E. Killgore, F. C. Tenover, D. Lyras, J. I. Rood, P. DeGirolami, A. L. Baltch, M. E. Rafferty, S. M. Pear, and D. N. Gerding. 1999. Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. N. Engl. J. Med. 341:1645-1651.[Abstract/Free Full Text]
    10. 10
  10. Johnson, S., J. L. Sanchez, and D. N. Gerding. 2000. Metronidazole resistance in Clostridium difficile. Clin. Infect. Dis. 31:625-626.[CrossRef][Medline]
    11. 11
  11. Karlstrom, O., B. Fryklund, K. Tullus, and L. G. Burman. 1998. A prospective nationwide study of Clostridium difficile-associated diarrhea in Sweden. The Swedish C. difficile Study Group. Clin. Infect. Dis. 26:141-145.[Medline]
    12. 12
  12. Leroi, M. J., S. Siarakas, and T. Gottlieb. 2002. E test susceptibility testing of nosocomial Clostridium difficile isolates against metronidazole, vancomycin, fusidic acid and the novel agents moxifloxacin, gatifloxacin, and linezolid. Eur. J. Clin. Microbiol. Infect. Dis. 21:72-74.[CrossRef][Medline]
    13. 13
  13. Manatsathit, S., H. L. Dupont, M. Farthing, C. Kositchaiwat, S. Leelakusolvong, B. S. Ramakrishna, A. Sabra, P. Speelman, and S. Surangsrirat. 2002. Guideline for the management of acute diarrhea in adults. J. Gastroenterol. Hepatol. 17(Suppl):S54-S71.[Medline]
    14. 14
  14. McFarland, L. V., M. E. Mulligan, R. Y. Kwok, and W. E. Stamm. 1989. Nosocomial acquisition of Clostridium difficile infection. N. Engl. J. Med. 320:204-210.[Abstract]
    15. 15
  15. Musher, D. M., S. Aslam, N. Logan, S. Nallacheru, I. Bhaila, F. Borchert, and R. J. Hamill. 2005. Relatively poor outcome after treatment of Clostridium difficile colitis with metronidazole. Clin. Infect. Dis. 40:1586-1590.[CrossRef][Medline]
    16. 16
  16. Noren, T., Y. J. Tang-Feldman, S. H. Cohen, J. Silva, Jr., and P. Olcen. 2002. Clindamycin resistant strains of Clostridium difficile isolated from cases of C. difficile associated diarrhea (CDAD) in a hospital in Sweden. Diagn. Microbiol. Infect. Dis. 42:149-151.[CrossRef][Medline]
    17. 17
  17. Oppenheimer, M., G. Kronvall, I. Karlsson, and E. Holst. 2000. Fusidic acid disk diffusion testing of clostridium difficile can be calibrated using single-strain regression analysis. Scand. J. Infect. Dis. 32:633-636.[CrossRef][Medline]
    18. 18
  18. Pelaez, T., L. Alcala, R. Alonso, A. Martin-Lopez, V. Garcia-Arias, M. Marin, and E. Bouza. 2005. In vitro activity of ramoplanin against Clostridium difficile, including strains with reduced susceptibility to vancomycin or with resistance to metronidazole. Antimicrob. Agents Chemother. 49:1157-1159.[Abstract/Free Full Text]
    19. 19
  19. Pelaez, T., L. Alcala, R. Alonso, M. Rodriguez-Creixems, J. M. Garcia-Lechuz, and E. Bouza. 2002. Reassessment of Clostridium difficile susceptibility to metronidazole and vancomycin. Antimicrob. Agents Chemother. 46:1647-1650.[Abstract/Free Full Text]
    20. 20
  20. Poilane, I., P. Cruaud, J. C. Torlotin, and A. Collignon. 2000. Comparison of the E test to the reference agar dilution method for antibiotic susceptibility testing of Clostridium difficile. Clin. Microbiol. Infect. 6:155-156.[CrossRef][Medline]
    21. 21
  21. Samore, M. H. 1999. Epidemiology of nosocomial clostridium difficile diarrhoea. J. Hosp. Infect. 43(Suppl.):S183-S190.[Medline]
    22. 22
  22. Samore, M. H., P. C. DeGirolami, A. Tlucko, D. A. Lichtenberg, Z. A. Melvin, and A. W. Karchmer. 1994. Clostridium difficile colonization and diarrhea at a tertiary care hospital. Clin. Infect. Dis. 18:181-187.[Medline]
    23. 23
  23. Schroeder, M. S. 2005. Clostridium difficile-associated diarrhea. Am. Fam. Physician 71:921-928.[Medline]
    24. 24
  24. Stubbs, S. L., J. S. Brazier, G. L. O'Neill, and B. I. Duerden. 1999. PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J. Clin. Microbiol. 37:461-463.[Abstract/Free Full Text]
    25. 25
  25. Svenungsson, B., L. G. Burman, K. Jalakas-Pornull, A. Lagergren, J. Struwe, and T. Akerlund. 2003. Epidemiology and molecular characterization of Clostridium difficile strains from patients with diarrhea: low disease incidence and evidence of limited cross-infection in a Swedish teaching hospital. J. Clin. Microbiol. 41:4031-4037.[Abstract/Free Full Text]
    26. 26
  26. Svenungsson, B., A. Lagergren, and A. Lundberg. 2001. Clostridium difficile cytotoxin B in adults with diarrhea: a comparison of patients treated or not treated with antibiotics prior to infection. Clin. Microbiol. Infect. 7:447-450.[CrossRef][Medline]
    27. 27
  27. Tabaqchali, S., and M. Wilks. 1992. Epidemiological aspects of infections caused by Bacteroides fragilis and Clostridium difficile. Eur. J. Clin. Microbiol. Infect. Dis. 11:1049-1057.[CrossRef][Medline]
    28. 28
  28. Warny, M., J. Pepin, A. Fang, G. Killgore, A. Thompson, J. Brazier, E. Frost, and L. C. McDonald. 2005. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366:1079-1084.[CrossRef][Medline]
    29. 29
  29. Wistrom, J., S. R. Norrby, E. B. Myhre, S. Eriksson, G. Granstrom, L. Lagergren, G. Englund, C. E. Nord, and B. Svenungsson. 2001. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study. J. Antimicrob. Chemother. 47:43-50.[Abstract/Free Full Text]
    30. 30
  30. Wullt, M., and I. Odenholt. 2004. A double-blind randomized controlled trial of fusidic acid and metronidazole for treatment of an initial episode of Clostridium difficile-associated diarrhoea. J. Antimicrob. Chemother. 54:211-216.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, May 2006, p. 1890-1892, Vol. 50, No. 5
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.5.1890-1892.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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