Clindamycin-Resistant Clone of Clostridium difficile PCR Ribotype 027, Europe

To the Editor: Since 2003, outbreaks of Clostridium difficile–associated disease (CDAD) associated with the emergence of a hypervirulent strain have been reported worldwide (1,2; www.eurosurveillance.org/em/v12n06/1206-221.asp). This strain has been associated with increased disease severity and attributable mortality. Patients infected with C. difficile 027 fail to respond to metronidazole therapy (1). Several typing methods have been applied to further characterize C. difficile PCR ribotype-027, including pulsed-field gel electrophoresis (PFGE) (North American pulsed field type 1) and restriction enzyme analysis (REA) (BI). PFGE and REA are widely used in the United States; PCR ribotyping is more commonly used throughout Europe. More recently, 2 multiple-locus variable-number tandem-repeat analysis (MLVA) protocols have been applied to type C. difficile, and these proved more discriminatory compared to other methods (3,4). Furthermore, MLVA can subgroup geographically diverse 027 isolates (G. Killgore et al., unpub data) as well as 027 isolates that are common to 1 institution (5). 
 
We reported a case of C. difficile PCR 027 in Ireland, where the isolate had an identical antibiogram profile compared with those strains reported across Europe (6,7) (i.e., resistant to fluoroquinolones and erythromycin, susceptible to clindamycin). We have subsequently identified C. difficile 027 in 6 more healthcare settings. To date >100 Irish C. difficile 027 isolates have been characterized by analysis of their antibiogram profiles, toxinotyping, and 16S–23S rDNA PCR ribotyping. All C. difficile 027 isolates were resistant to moxifloxaxin, gatifloxacin, ciprofloxacin (MIC >32 mg/L), and erythromycin (MIC >256 mg/L) but susceptible to metronidazole (MIC 0.25 mg/L) and vancomycin (MIC >0.5 mg/L). Clindamycin susceptibility varied between isolates from unrelated institutions. Isolates from 2 healthcare settings were susceptible to clindamycin (n = 11: MIC90 4 mg/L). However, clindamycin-resistant PCR 027 isolates (n = 96: MIC90 >256 mg/L) were identified in the other 5 healthcare institutions. All clindamycin-resistant PCR 027 isolates were positive for the ermB gene, encoding the macrolide-lincosamide-streptogramin-B genotype. 
 
A subset of clindamycin-sensitive and -resistant Irish 027 strains isolated throughout 2006 (n = 22) were further characterized by using a recently described MLVA protocol (3). Six clindamycin-susceptible isolates were selected from 2 healthcare settings. One hospital conducted active routine laboratory surveillance and molecular genotyping (n = 3). The second hospital submitted only random isolates (n = 3) for typing during a C. difficile outbreak. Sixteen clindamycin-resistant PCR 027 isolates were also included in the MLVA. Resistant isolates were selected from 5 healthcare settings. These included isolates from 2 C. difficile outbreaks with ongoing laboratory surveillance (n = 5, n = 6, respectively); a third hospital with ongoing laboratory surveillance (n = 3) and 2 hospitals that each submitted fecal samples from patients with severe cases of C. difficile disease (n = 1). The Stoke-Mandeville control strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 was included for comparison. 
 
MLVA determined that all strains within the clindamycin-resistant cluster were closely related and were single- or double-locus variants with a maximum 5 summed tandem-repeat difference (STRD). In contrast, the closest relationship between the clindamycin-resistant and the clindamycin-sensitive clusters was a triple-locus variant with an STRD of 17. The nonrelated reference strain of the Stoke-Mandeville outbreak ({"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291) differed considerably from all Irish isolates but was more related to the clindamycin-sensitive cluster than to the clindamycin-resistant cluster (Figure). We thus linked a defined genetic marker with the clindamycin-resistant phenotype in C. difficile PCR-027. MLVA could clearly differentiate clindamycin-resistant and -susceptible isolates from the same geographic region and subgrouped them into 2 distinct clusters (Figure). 
 
 
 
Figure 
 
Minimal spanning tree of 23 Clostridium difficile isolates. In the circles, the individual isolates are mentioned. The numbers between the circles represent the summed tandem repeat differences (STRDs) between multiple-locus variable-number tandem-repeat ... 
 
 
 
Although high-level resistance to fluoroquinolone antimicrobial agents has been well documented in PCR 027 (1,6), resistance to clindamycin is rare. Subsequently, clindamycin has been considered as a “protective” antimicrobial agent for the development of CDAD in an epidemiologic survey in the Netherlands (8). Currently, resistance to this agent in NAP 1/PCR 027 has been restricted to the United States. McDonald and colleagues reported that 19 (79%) of 24 NAP 1 isolates were classified as less susceptible (MIC 4 mg/L) or resistant (MIC 8 mg/L) to clindamycin when Clinical and Laboratory Standards Institute criteria were used (2). Unfortunately, MIC values were not reported, and the corresponding resistance genes were not investigated. In contrast, Canadian studies to date have not reported clindamycin resistance in this strain type. The MIC90 of Canadian NAP 1 isolates for clindamycin was 4 mg/L (9,10). Although outbreaks and sporadic cases of PCR 027 have been identified in several European countries, to date no clindamycin-resistant clone has been reported. 
 
Detection of clindamcyin-resistant C. difficile PCR 027 strains is an important and worrying development. Resistance to this antimicrobal agent increases the risk for CDAD in patients, and its use may be an important factor contributing to the persistence and spread of PCR 027. A similar feature has already been observed when fluoroquinolones and cephalosporins are prescribed. Clindamcyin-resistant PCR 027 probably reflects the emergence of a new clone because MLVA clearly differentiates between clindamycin-susceptible and -resistant isolates.

. Several typing methods have been applied to further characterize C. diffi cile PCR ribotype-027, including pulsed-fi eld gel electrophoresis (PFGE) (North American pulsed fi eld type 1) and restriction enzyme analysis (REA) (BI). PFGE and REA are widely used in the United States; PCR ribotyping is more commonly used throughout Europe. More recently, 2 multiple-locus variable-number tandem-repeat analysis (MLVA) protocols have been applied to type C. diffi cile, and these proved more discriminatory compared to other methods (3,4). Furthermore, MLVA can subgroup geographically diverse 027 isolates (G. Killgore et al., unpub data) as well as 027 isolates that are common to 1 institution (5).
We reported a case of C. diffi cile PCR 027 in Ireland, where the isolate had an identical antibiogram profi le compared with those strains reported across Europe (6,7) (i.e., resistant to fl uoroquinolones and erythromycin, susceptible to clindamycin). We have subsequently identifi ed C. diffi cile 027 in 6 more healthcare settings. To date >100 Irish C. diffi cile 027 isolates have been characterized by analysis of their antibiogram profi les, toxinotyping, and 16S-23S rDNA PCR ribotyping. All C. diffi cile 027 isolates were resistant to moxifl oxaxin, gatifl oxacin, The opinions expressed by authors contributing to this journal do not necessarily refl ect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affi liated. ciprofl oxacin (MIC >32 mg/L), and erythromycin (MIC >256 mg/L) but susceptible to metronidazole (MIC 0.25 mg/L) and vancomycin (MIC >0.5 mg/L). Clindamycin susceptibility varied between isolates from unrelated institutions. Isolates from 2 healthcare settings were susceptible to clindamycin (n = 11: MIC 90 4 mg/L). However, clindamycin-resistant PCR 027 isolates (n = 96: MIC 90 >256 mg/L) were identifi ed in the other 5 healthcare institutions. All clindamycin-resistant PCR 027 isolates were positive for the ermB gene, encoding the macrolide-lincosamide-streptogramin-B genotype.
A subset of clindamycin-sensitive and -resistant Irish 027 strains isolated throughout 2006 (n = 22) were further characterized by using a recently described MLVA protocol (3). Six clindamycin-susceptible isolates were selected from 2 healthcare settings. One hospital conducted active routine laboratory surveillance and molecular genotyping (n = 3). The second hospital submitted only random isolates (n = 3) for typing during a C. diffi cile outbreak. Sixteen clindamycinresistant PCR 027 isolates were also included in the MLVA. Resistant isolates were selected from 5 healthcare settings. These included isolates from 2 C. diffi cile outbreaks with ongoing laboratory surveillance (n = 5, n = 6, respectively); a third hospital with ongoing laboratory surveillance (n = 3) and 2 hospitals that each submitted fecal samples from patients with severe cases of C. diffi cile disease (n = 1). The Stoke-Mandeville control strain R20291 was included for comparison.
MLVA determined that all strains within the clindamycin-resistant cluster were closely related and were single-or double-locus variants with a maximum 5 summed tandem-repeat difference (STRD). In contrast, the closest relationship between the clindamycin-resistant and the clindamycin-sensitive clusters was a triple-locus variant with an STRD of 17.
The nonrelated reference strain of the Stoke-Mandeville outbreak (R20291) differed considerably from all Irish isolates but was more related to the clindamycin-sensitive cluster than to the clindamycin-resistant cluster (Figure). We thus linked a defi ned genetic marker with the clindamycin-resistant phenotype in C. diffi cile PCR-027. MLVA could clearly differentiate clindamycin-resistant and -susceptible isolates from the same geographic region and subgrouped them into 2 distinct clusters (Figure).
Although high-level resistance to fl uoroquinolone antimicrobial agents has been well documented in PCR 027 (1,6), resistance to clindamycin is rare. Subsequently, clindamycin has been considered as a "protective" antimicrobial agent for the development of CDAD in an epidemiologic survey in the Netherlands (8). Currently, resistance to this agent in NAP 1/PCR 027 has been restricted to the United States.
McDonald and colleagues reported that 19 (79%) of 24 NAP 1 isolates were classifi ed as less susceptible (MIC 4 mg/L) or resistant (MIC 8 mg/L) to clindamycin when Clinical and Laboratory Standards Institute criteria were used (2). Unfortunately, MIC values were not reported, and the corresponding resistance genes were not investigated. In contrast, Canadian studies to date have not reported clindamycin resistance in this strain type. The MIC 90 of Canadian NAP 1 isolates for clindamycin was 4 mg/L (9,10). Although outbreaks and sporadic cases of PCR 027 have been identifi ed in several European countries, to date no clindamycin-resistant clone has been reported.
Detection of clindamcyin-resistant C. diffi cile PCR 027 strains is an important and worrying development. Resistance to this antimicrobal agent increases the risk for CDAD in patients, and its use may be an important factor contributing to the persistence and spread of PCR 027. A similar feature has already been observed when fl uoroquinolones and cephalosporins are prescribed. Clindamcyin-resistant PCR Figure. Minimal spanning tree of 23 Clostridium diffi cile isolates. In the circles, the individual isolates are mentioned. The numbers between the circles represent the summed tandem repeat differences (STRDs) between multiple-locus variable-number tandemrepeat analysis types. Straight lines represent single-locus variants, dashed lines doublelocus variants. Curved lines represent triple-locus variants. Two related clusters can be discriminated: the light gray cluster (isolates B1, B4, M246, B6, and M216) and the cluster within dotted lines (isolates V6-44, V6-142, V6-81, 1ML, C1, 4108, V6-35, V6-80, L1, 2191cc, C4, C8, 3ML, C44, C37, and 13ML) The isolates in the light gray cluster are sensitive to clindamycin; isolates in the cluster surrounded by dotted lines are resistant. Two isolates (M278 and R20291) did not belong to a cluster but were more related to the sensitive cluster than to the resistant cluster. Genetically related clusters were defi ned by an STRD <10.

Increasing
Incidence of Clostridium diffi cile-associated Disease, Singapore To the Editor: Clostridium diffi cile-associated disease (CDAD) has increased in incidence across North America and Europe (1). Recent reports document the emergence of an epidemic strain of C. diffi cile, NAP1/ BI/027, associated with increased virulence (2,3). However, less information is available regarding CDAD epidemiology in Asia. We examined the incidence of C. diffi cile among hospitalized patients in Singapore from 2001 through 2006 and conducted a case-control study to evaluate risk factors for testing positive for C. diffi cile toxin (CDT) in our population.
Tan Tock Seng Hospital (TTSH) is a 1,200-bed, acute-care general hospital in Singapore that serves an urban population of 4 million. We calculated CDAD incidence using the number of patients testing positive for CDT per 10,000 patient days from 2001 through 2006. We used this calculation because CDT testing would have been ordered for clinical indications. CDT testing was performed by using the same ELISA (Premier Toxins A&B; Meridian Bioscience, Inc., Cincinnati, OH, USA) throughout the entire period of investigation.
Case-patients and controls were selected from patients hospitalized at TTSH from January 1 through December 31, 2004. Microbiology laboratory records were used to defi ne 3 groups. Case-patients were defi ned as CDTpositive inpatients (group 1). Two sets of negative controls were defi ned: the fi rst (group 2) consisted of patients who tested negative for CDT. However, because false-negatives could nullify differences between groups 1 and 2, we defi ned a second set of negative controls (group 3) from among 18,000 inpatients not tested for CDT.

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