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Antimicrobial Agents and Chemotherapy, October 2006, p. 3473-3475, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00479-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vitro Susceptibility of Clostridium difficile Clinical Isolates from a Multi-Institutional Outbreak in Southern Québec, Canada

Anne-Marie Bourgault,1 François Lamothe,1* Vivian G. Loo,2 Louise Poirier,3 and the CDAD-CSI Study Group

Centre Hospitalier de l'Université de Montréal, Montréal, QC, Canada,1 McGill University Health Centre, Montréal, QC, Canada,2 Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada3

Received 18 April 2006/ Returned for modification 23 May 2006/ Accepted 18 July 2006


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ABSTRACT
 
Clostridium difficile isolates from a 2004 outbreak in Québec, Canada, were all found to be susceptible to metronidazole, vancomycin, rifampin, and meropenem but resistant to bacitracin, cefotaxime, ciprofloxacin, and levofloxacin, and most (>80%) were resistant to ceftriaxone, clarithromycin, gatifloxacin, and moxifloxacin. The predominant NAP1 isolates were susceptible to clindamycin, while the NAP2 isolates were resistant.


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TEXT
 
Clostridium difficile is the major identified cause of nosocomial infectious diarrhea (1). The most important risk factor for C. difficile-associated diarrhea (CDAD) is prior antibiotic use. Metronidazole and vancomycin are effective in the treatment of CDAD, but there is a high incidence of relapses (15 to 20%). In the last few years, hospitals in the United States (11) and in Québec, Canada (9, 14), have experienced outbreaks of CDAD, with an increase in the proportion of severe cases and fatal complications (15, 16). In 2004, in a prospective review of CDAD in Québec hospitals, we reported an incidence of 22.5 cases per 1,000 admissions and an attributable mortality of 6.9% (10). A toxin gene variant strain of C. difficile that was more virulent was identified. The purpose of the study was to assess the antibiotic susceptibility patterns of 258 isolates of C. difficile collected during this multi-institutional outbreak of CDAD and to compare them to those of 21 available historic isolates collected from 1987 to 2001.

Isolates (one per patient) were obtained from patients with a clinical diagnosis of diarrhea and a positive assay for toxin A, toxin B, or both. Isolates were identified as C. difficile by Gram stain, typical odor, chartreuse fluorescence under UV light, and Microscreen latex agglutination (Microgen Bioproducts, Camberlye, United Kingdom). The antimicrobial susceptibility profiles for all antibiotics but levofloxacin were determined by the Clinical and Laboratory Standards Institute (CLSI) reference agar dilution method for anaerobes using brucella agar supplemented with laked sheep blood, hemin, and vitamin K1 (13). The antibiotics tested were vancomycin, metronidazole, rifampin, fusidic acid, cefotaxime, and ceftriaxone (Sigma Chemicals, Oakville, Ontario, Canada); ciprofloxacin and moxifloxacin (Bayer HealthCare, Toronto, Ontario, Canada); gatifloxacin (Bristol Myers Squibb Canada Inc., Montreal, QC, Canada); piperacillin-tazobactam (Wyeth Ayerst Research, Pearl River, NY); and meropenem (Astra Zeneca Pharmaceuticals, Wilmington, Delaware). Levofloxacin was tested by Etest (AB Biodisk). CLSI interpretive categories for resistance were used. For fluoroquinolones, trovafloxacin breakpoints were used, as that agent was the only fluoroquinolone for which resistance breakpoints were provided for anaerobes. For rifampin, vancomycin, and clarithromycin, the CLSI interpretive categories for Staphylococcus aureus were used. For fusidic acid, the proposed resistance breakpoint of 2 µg/ml was used (3). Pulsed-field gel electrophoresis (PFGE) of C. difficile isolates was performed according to the method described by Fawley and Wilcox (4), and strain relatedness was determined using the criteria of Tenover et al. (17) as previously described (10).

In Table 1, results are expressed as MIC50, MIC90, range, and percentage of strains resistant at the breakpoint. The isolates were uniformly susceptible to vancomycin, metronidazole, rifampin, and meropenem and resistant to bacitracin, ciprofloxacin, levofloxacin, and cefotaxime. Few isolates were resistant to piperacillin-tazobactam and fusidic acid. Resistance to clindamycin was not prevalent (14.7%). Analysis of drug cross-resistance revealed a strong association between resistance to clarithromycin and resistance to gatifloxacin and moxifloxacin.


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  		TABLE 1. In vitro susceptibilities of 258 isolates of C. difficile

Among the 251 isolates genotyped by PFGE, there were two major clonal strains, types NAP1 (69.3% of the isolates) and NAP2 (11.2% of the isolates). The remaining 49 isolates belonged to other PFGE patterns. Table 2 summarizes the comparative results obtained for genotype NAP1 and NAP2 strains and the other, non-NAP1 non-NAP2 strains. The NAP1 genotype strain was resistant to clarithromycin and fluoroquinolones but susceptible to clindamycin, while the NAP2 strain was resistant to clarithromycin, fluoroquinolones, and clindamycin. The remaining isolates with other PFGE patterns were mostly susceptible to gatifloxacin, moxifloxacin, clindamycin, and clarithromycin.


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  		TABLE 2. Susceptibility profiles of current NAP1, NAP2, and non-NAP1 non-NAP2 isolates of C. difficile

Among the 21 historic isolates, there were 2 (9.5%) NAP1 pattern isolates, 7 (33.3%) NAP2 pattern isolates, and 12 isolates with other PFGE patterns. In contrast to the current NAP1 isolates, the two historic NAP1 isolates were resistant to ciprofloxacin (MICs, ≥32 µg/ml) and levofloxacin (MICs, >32 µg/ml) but susceptible to gatifloxacin and moxifloxacin, with MICs of ≤4 µg/ml. Similar to the current NAP2 isolates, isolates with the NAP2 pattern were resistant to all of the fluoroquinolones and to clindamycin and azithromycin. All historic isolates were susceptible to metronidazole and vancomycin, with MIC90s of 0.25 µg/ml and 2.0 µg/ml, respectively.

Management of CDAD includes withdrawal of the predisposing antibiotic and often therapy with either metronidazole or oral vancomycin (2). The treatment alternatives reported in the literature are diverse and include fusidic acid, rifampin, and bacitracin with reported efficacy (8).

In this study, metronidazole and vancomycin remained highly and uniformly active in vitro. Among the current isolates, the MIC90s to metronidazole and vancomycin have not increased compared to those of historic isolates. However, in vitro antimicrobial susceptibility is not necessarily predictive of a successful therapeutic outcome. Recently, metronidazole treatment of CDAD has been associated with poor outcomes (12) and an increasing risk of relapse (15), calling into question its role as the first-line agent for the treatment of CDAD. Prospective randomized trials are needed to confirm these observations (6).

Fusidic acid and rifampin, two other potentially useful agents for the treatment of CDAD, were very active in vitro. However, bacitracin showed no activity against any of the isolates. As previously observed, the majority of strains were resistant to cefotaxime but susceptible to meropenem and piperacillin-tazobactam (5, 7). Isolates were also highly resistant to clarithromycin and the fluoroquinolones, findings similar to those recently reported in the United Kingdom (7).

The two predominant genotypes were highly resistant to clarithromycin and the fluoroquinolones but differed markedly in their susceptibilities to clindamycin. There was no cross-resistance between clindamycin and clarithromycin. The NAP1 strain was susceptible to clindamycin, while the second most commonly identified genotype, NAP2, was highly resistant to this agent. In contrast to results recently reported in the United States, where clindamycin-resistant clones have been associated with multihospital outbreaks of CDAD (11), our major epidemic strain was susceptible to clindamycin, a finding also observed in the United Kingdom strains (7).

Among 21 historical isolates, two NAP1 isolates were identified. These isolates were susceptible to gatifloxacin and moxifloxacin, in contrast to the current NAP1 isolates. These results corroborate those obtained in the United States when NAP1 contemporary isolates were compared to historic isolates predating 2001.


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ACKNOWLEDGMENTS
 
This work was supported in part by a grant from Bristol Myers Squibb Canada, Inc.

We thank Susan Fenn and Huguette Gilbert for their expert technical assistance.

The members of the CDAD-CSI (Clostridium difficile-Associated Diarrhea-Clinical Study Investigators) group are as follows: Charles H. Frenette (Département de Microbiologie, Division des Maladies Infectieuses, Hôpital Charles LeMoyne), Ruth Horn (Department of Microbiology, Division of Infectious Diseases, McGill University Health Centre), Mirabelle Kelly (Département de Microbiologie, Division des Maladies Infectieuses, Hôpital Jean Talon), Pierre J. Laflamme (Département de Microbiologie, Division des Maladies Infectieuses, Hôpital Sacré Coeur de Montréal), Michael D. Libman (Department of Microbiology [McGill University Health Centre and St. Mary's Hospital] and Division of Infectious Diseases [McGill University Health Centre and St. Mary's Hospital]), Sophie Michaud (Département de Microbiologie, Division des Maladies Infectieuses, Centre Hospitalier Universitaire de Sherbrooke), Mark A. Miller (Division of Medical Microbiology, Division of Infectious Diseases, SMBD-Jewish General Hospital), Tuyen Nguyen (Département de Microbiologie, Cité de la Santé de Laval), Pierre René (Department of Microbiology, Division of Infectious Diseases, McGill University Health Centre), and Anne Vibien (Département de Microbiologie, Division des Maladies Infectieuses, Réseau Santé, Richelieu-Yamaska).


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FOOTNOTES
 
* Corresponding author. Mailing address: Dept. de Microbiologie Médicale et Infectiologie, Hôpital St-Luc, Centre Hospitalier de l'Université de Montréal, 1058 St-Denis, Montréal, QC, Canada H2X 3J4. Phone: (514) 890-8000, ext. 36210. Fax: (514) 412-7412. E-mail: francois.lamothe.CHUM{at}ssss.gouv.qc.ca. Back


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Antimicrobial Agents and Chemotherapy, October 2006, p. 3473-3475, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00479-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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