S (15) DOI: doi: /j.diagmicrobio Reference: DMB 13832

Transcription

1 Accepted Manuscript A national survey of the molecular epidemiology of Clostridium difficile in Israel: the dissemination of the ribotype-027 strain with reduced susceptibility to vancomycin and metronidazole Amos Adler, Tamar Miller-Roll, Rita Bradenstein, Colin Block, Bracha Mendelson, Miriam Parizade, Yossi Paitan, David Schwartz, Nehama Peled, Yehuda Carmeli, Mitchell J. Schwaber PII: S (15) DOI: doi: /j.diagmicrobio Reference: DMB To appear in: Diagnostic Microbiology and Infectious Disease Received date: 26 February 2015 Revised date: 25 May 2015 Accepted date: 28 May 2015 Please cite this article as: Adler Amos, Miller-Roll Tamar, Bradenstein Rita, Block Colin, Mendelson Bracha, Parizade Miriam, Paitan Yossi, Schwartz David, Peled Nehama, Carmeli Yehuda, Schwaber Mitchell J., A national survey of the molecular epidemiology of Clostridium difficile in Israel: the dissemination of the ribotype-027 strain with reduced susceptibility to vancomycin and metronidazole, Diagnostic Microbiology and Infectious Disease (2015), doi: /j.diagmicrobio This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 1 A national survey of the molecular epidemiology of Clostridium difficile in Israel: the dissemination of the ribotype-027 strain with reduced susceptibility to vancomycin and metronidazole Amos Adler 1 *, Tamar Miller-Roll 1, Rita Bradenstein 2, Colin Block 3, Bracha Mendelson 4, Miriam Parizade 5, Yossi Paitan 6, David Schwartz 7, Nehama Peled 8, Yehuda Carmeli 1, Mitchell J Schwaber National Center of Infection Control, Ministry of Health, Tel Aviv, Israel 2 -Clinical Microbiology Laboratory, Kaplan Medical Center, Rehovot, Israel 3 -Clinical Microbiology Laboratory, Hadassah Medical Center, Jerusalem, Israel 4 -Clinical Microbiology Laboratory, Bnei-Zion Medical Center, Haifa, Israel 5 -Clinical Microbiology Laboratory, Maccabi Health Services, Rehovot, Israel 6 -Clinical Microbiology Laboratory, Meir Medical Center, Kfar Saba, Israel 7- Clinical Microbiology Laboratory, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel 8 -Clinical Microbiology Laboratory, Soroka Medical Center, Be'er-Sheba, Israel Running title: Dissemination of Ribotype 027 in Israel.

3 2 *Corresponding author: National Center for Infection Control and Tel-Aviv Sourasky Medical Center 6 Weizmann St., Tel Aviv, 64239, Israel amosa@tlvmc.gov.il

4 3 Abstract Our goals were to study the molecular epidemiology and antimicrobial susceptibilities of C. difficile strains in Israel. Microbiology laboratories serving 6 general hospitals (GH) and 10 long-term care facilities (LTCF) were asked to submit all stool samples in January-February 2014 that tested positive for C. difficile. Toxigenic C. difficile isolates were recovered in 208 out of 217 samples (95.8%), of which 50 (23.6%) were from LTCFs. Ribotype-027 was the most common type overall, identified in 65 samples (31.8%) and was the predominant strain in the 3 GHs with the highest incidence of C. difficile infections. Other common strains were slpa types cr-02 (n=45) and hr-02 (n=18). The proportions of vancomycin and metronidazole MIC values>2 mg/l were high in Ribotype-027 (87.7% and 44.6%, respectively) and slpacr-02 strains (88.8% and 17.8%, respectively). This study demonstrates that the Ribotype-027 strain has disseminated across Israel and is now the most common strain.

5 4 1. Introduction Clostridium difficile is one of the leading causes of healthcare-associated infection and may be the most common cause of bacterial gastroenteritis in developed countries (Gerding 2010). Since the beginning of the millennium, there has been an increase in the incidence of Clostridium difficile infection (CDI) and possibly also in the severity of illness caused by it. These changes are attributed in part to the spread of an epidemic strain, BI/NAP1/027, that is characterized by the presence of a binary toxin, deletions in the regulatory gene, tcdc and by resistance to moxifloxacin (McDonald et al. 2005; Gerding 2010). Although sporadic cases and isolated outbreaks have been reported from Europe, East Asia and Australia (Barbut et al. 2007; Gerding 2010), this strain has been associated with nationwide epidemics in only a small number of countries, including the USA, Canada and the UK (Freeman et al. 2010; Wilcox et al. 2012; He et al. 2013). The epidemiology of CDI in Israel is difficult to analyze due to the absence of surveillance data before 2009 and the lack of previous molecular epidemiology data. Furthermore, the introduction of new diagnostic methods has led to a significant increase in the tests' positivity rate and thus might have led to an increase in the reported incidence (Adler et al. 2014). The first case of CDI caused by the ribotype 027 strain was reported in Israel in 2009 (Bishara et al. 2011). Subsequently, the 027 strain was responsible for a hospital-wide outbreak in 2013 in Jerusalem, comprising 79% of the strains isolated (Wiener-well et al. 2014). More ominously, these strains exhibited high minimal inhibitory concentration (MIC) values for the two main therapeutic agents, vancomycin and metronidazole. In light of these findings, we initiated a national survey of the molecular epidemiology of toxigenic C. difficile in

6 5 Israel. Our aims were to determine the prevalence of the ribotype 027 and other epidemiologically-important strains (e.g., ribotype 078) in Israel, their molecular characteristics, and their antimicrobial susceptibilities. 2. Methods 2.1. Study design and setting This study was a national, multicenter survey of the molecular epidemiology and antimicrobial susceptibility testing (AST) results of toxigenic C. difficile isolates in general hospitals (GH) and long-term care facilities (LTCF) in Israel conducted by the National Center of Infection Control (NCIC). The following six GH participated in the study: 1) Bnei-Zion Medical Center (BZMC), a 417-bed hospital in the city of Haifa in northern Israel; 2) Hadassah Medical Organization (HMO), a 969-bed, twocampus [Mount Scopus (MS) and Ein Karem (EK)] tertiary-care center in Jerusalem; 3) Kaplan Medical Center (KMC), a 535-bed hospital in the city of Rehovot in central Israel; 4) Meir Medical Center (MMC), a 726-bed hospital in the city of Kfar Saba in central Israel; 5) Soroka Medical Center (SMC), a 1007-bed tertiary-care center in Be'er-Sheba, in southern Israel, and 6) Tel-Aviv Sourasky Medical Center (TASMC), a 1072-bed tertiary-care center in Tel-Aviv, in central Israel. Altogether, participating centers comprised 4,726 (30%) of Israel's 15,739 acute care hospital beds. In addition, samples collected in 10 LTCF and processed by the central laboratory of Maccabee Health Services (MHS) or the KMC laboratories were included. Participating sites were asked to freeze and submit to the NCIC laboratory all positive, patient-unique C. difficile samples collected from adult patients in January-February Microbiological methods

7 6 Laboratory diagnosis of CDI was done by testing non-formed stool samples using three different methods, depending on the study site. TASMC, KMC, SMC, BZMC, MMC used a two-step algorithm. The initial assay was a combined glutamate dehydrogenase antigen (GDH) and Toxin A/B immunochromatographic rapid test (C. DIFF QUIK CHEK COMPLETE, Techlab, Orlando, USA); only if the results of those two tests were discordant, CDT PCR (Xpert C. difficile, Cepheid, Sunnyvale, USA) was performed; MHS used a three-step algorithm, with the ImmunoCard C. difficile PI as the first step, the ImmunoCard Toxin A&B as the second step, and the illumigene C. difficile molecular assay (all assays- Meridian Bioscience Inc., Cincinnati, USA) for discordant results. HMO utilized an in-house proprietary RT- PCR assay based on the tcdb gene, using the Rotor-Gene 2000 platform (Corbett Robotics, Australia). Assay details are confidential due to potential commercial application of the assay. Positive samples were frozen at -80ºC and transported to the NCIC laboratory for further testing. Thawed stool samples were inoculated on pre-reduced ChromID C. difficile agar plates (biomérieux, Marcy l'etoile, France) and incubated in an anaerobic chamber (BACTRON I-2, Shellab, OR, USA) for 48 hours. Suspicious colonies were subcultured onto sheep blood agar plates and identified based on colony morphology, typical odor and molecular tests as described below. Antimicrobial susceptibility testing (AST) was performed for vancomycin, metronidazole and moxifloxacin using the gradient method (Etest, biomérieux, Marcy l'etoile, France) with the technician blinded to the isolate's type. Pre-reduced, supplemented Brucella blood agar plates (Cat. BBL255509, BD, NJ, USA) were inoculated with an inoculum of 0.5 McFarland, incubated and read after 48 hours according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical and Laboratory Standards

8 7 Institute 2012). Minimal inhibitory concentration (MIC) criteria for susceptibility were interpreted according to both the CLSI and the EUCAST recommendations, respectively (Clinical and Laboratory Standards Institute 2012; European Committee on Antimicrobial Susceptibility Testing 2013): Moxifloxacin 2 and 4 µg/ml, metronidazole 8 and 2 µg/ml and vancomycin 4 and 2 µg/ml Molecular methods Identification of C. difficile was confirmed by detection of the species-specific gene, tpi by PCR (Lemee et al. 2004). The presence of the A and B toxins (tcda & tcdb) as well as the binary toxin (cdtb) was tested by PCR (Persson et al. 2008). The presence of in-frame deletions in the tcdc gene was tested by PCR (Persson et al. 2011). The presence of the epidemic strain, BI/NAP1/027, slpa type gc8, was initially detected by observing the 18-bp deletion in the tcdc gene and the cdtb gene (Persson et al. 2011) and confirmed by PCR ribotyping (Xiao et al. 2012). Non-027 isolates (and a minority of 027 isolates) were further typed by PCR and sequencing of the variable region of the slpa gene (Kato 2005; Kato et al. 2010). The amino acid sequences were deduced from the DNA sequences and type was established if the deduced amino acid sequences differed from existing types by more than 20 amino acid residues. Subtypes consisted of groups differing by up to 20 amino acid residues. Designation of slpa types and determination of the inferred ribotype was done based on the nomenclature used by Kato (when present) (Kato 2005; Killgore et al. 2008; Kato et al. 2010); otherwise, a new name was given. Isolates were also typed by ribotyping whenever the corresponding ribotype was available from reference strains. Reference strains for PCR ribotyping were kindly provided by the Cardiff-European Center for Disease Control (ECDC) collection of C. difficile strains; these were first analyzed by slpa typing followed by comparison of the ribotype patterns with the study isolates.

9 Data collection and analysis Data on the incidence of CDI from in the participating GH centers was retrieved from the NCIC database. At the request of the study sites, results are presented without identifying the site. Instead, sites are labeled A-H. One letter (D) stands for the group of 10 LTCFs; the 2 campuses of Hadassah Medical Organization are presented separately. The proportion of resistance to antimicrobials among the different strains were compared by Fisher's exact test using GraphPad online software (GraphPad Software, Inc. LaJolla, CA). 3. Results 3.1. Clonal structure of C. difficile isolates A total of 243 stool samples were submitted from the participating centers, of which 13 were excluded due to insufficient material (B-6, F-1, H-6) and 13 were excluded as non-toxigenic at initial testing (F-8, H-5). Culture was performed on 217 samples, of which toxigenic C. difficile were cultured in 208 samples (95.8%): in 2 samples from center F no growth was observed (0.9%) and 4 samples (1.8%) grew non-toxigenic C. difficile. The frequency of slpa types and ribotype 027 in all study sites combined and the main types (>2 isolates) at each center are presented in Figure 1 and Table 1, respectively. Ribotype 027 was the most common type overall, identified in 65 samples (31.8%). Ribotype 027 was identified in all centers except for center G and was the predominant strain in centers B, C, E and D. Other common strains were cr- 02 (n=45, 4 centers), hr-02 (n=18, 4 centers), hr-05 (ribotype 014, n=13, 3 centers),

10 9 smz-02 IL1 (n=10, 2 centers) and fr-01 (ribotype 017, n=8, 3 centers). Common international strains such as ribotype 001 (slpa gr-01 and gr-03), ribotye 017 (slpa fr- 01 and fr-01 IL1), slpa/ribotype 078 and ribotype 012 (slpa cr-01) were identified in only 4, 9, 4 and 1 sample, respectively. We identified a new slpa type in one isolate (IL Kp-01) and seven new subtypes (all marked IL1). One isolate was untypeable. Seven LTCFs contributed one isolate each.. The three other LTCFs contributed 5, 9 and 29 isolates. In the latter LTCF, a CDI outbreak had taken place during the time of the survey; 16 of the isolates (55%) were ribotype 027 and 9 (31%) were cr Molecular features and antimicrobial susceptibility patterns of C. difficile isolates All isolates harbored the tcda and tcdb genes. Both cdtb and an 18 bp ΔtcdC were present in the ribotype 027 isolates, in the IL-Kp-01 and in the untypeable isolates. The cdtb and a 39 bp ΔtcdC were present in the ribotype 078 isolates and an 18 bp ΔtcdC alone was present in one yok-01 and one gr-01 isolate. The results of AST according to strain are presented in Table 2. The proportion of vancomycin MIC values> 2 mg/l was very high in the two most common strains, 027 (57/65, 87.7%) and cr-02 (40/45, 88.8%). In contrast, a vancomycin MIC values> 2 mg/l was found in only a single isolate of the other strains (p< for both comparisons) and in none of the control strains tested (ribotypes 001, 002, 014, 017, 027, 053, 056 and 078). Similarly, metronidazole MIC values> 2 mg/l were found in 29/65 (44.6%) in the 027 strains and 8/45 (17.8%) in the cr-02 strains but in only a single isolate of the other strains (p-value < and 0.004, respectively) and in none of the control strains. All 027 and cr-02 strains were resistant to moxifloxacin, compared to15 of the other 98 strains (15.3%) (p< for both comparisons).

11 National and institutional incidence of C. difficile infection The quarterly incidence of CDI per 100,000 patient-days in the participating GH is presented in Figure 2. The three centers with the highest CDI incidence in 2013 (B, C and E) were also characterized by the predominance of the ribotype 027 strain (Table 1). 4. Discussion The present study is the first national survey of the molecular epidemiology of C. difficile in Israel and the most extensive study of CDI prevalence conducted in the Near East. The study establishes the similarity between the clonal structure of C. difficile in hospitals in this area and other parts of the world, showing the predominant role of the ribotype 027. Outside of Israel, the 027 strain has been reported in the Near East only once, in Saudi Arabia (Collins et al. 2013; Alzahrani et al. 2013). The 027 strain was present in all but one center included in our study, and was the predominant strain in 3 GH and in the LTCFs samples. Moreover, this strain was specifically associated with centers that had the highest incidence (figure 2), most prominently center C, where all isolates belonged to this strain. These findings, combined with the recent report of an outbreak caused by the 027 strain in Jerusalem (Wiener-well et al. 2014), present an ominous picture of the dissemination of the 027 strain in Israel, which is similar to the conditions reported from the USA, Canada and the UK (Freeman et al. 2010). In addition to GH, our study included samples obtained from LTCFs. Here as well, ribotype 027 was the predominant strain and was responsible for an outbreak in one of the facilities. Considering the propensity for CDI among elderly patients in general and LTCF residents in particular (Simor et al.; Gaynes et al. 2004), these findings are

12 11 not surprising and provide additional evidence for the national dissemination of this strain. Due to the ongoing bi-directional transfer of patients between GH and LTCFs, institutional control of CDI may be extremely challenging; thus, comprehensive transinstitutional prevention programs are necessary. In addition to the ribotype 027 strain, we identified 3 dominant strains that were present in at least 5 of the centers: cr-02, hr-02 and hr-05 (ribotype 014), the latter known as a prevalent strain internationally (Barbut et al. 2007). In contrast, cr-02 and hr-02 that were characterized and reported from Japan by Kato (Kato et al. 2010) were either different from their presumed corresponding ribotype (hr-02 with ribotype 106) (Killgore et al. 2008) or did not match any common ribotype based on the UK classification (Killgore et al. 2008; Kato et al. 2010). Notably, other common international strains such as ribotypes 001, 012, 017 and 078 were rare in our survey. The fact that the distribution of strain types is not uniform throughout the world is not surprising: variation was found even within European countries (Barbut et al. 2007). The presence of a binary toxin and a tcdc deletion were suggested initially as explanations for the virulence of the ribotype 027 strain (McDonald et al. 2005), although later genomic studies have not supported this hypothesis (He et al. 2013). In our study, these features were also found in two new, non-027 strains out of the 67 isolates that were screened positive for the binary toxin and the tcdc deletion. This finding points out the inadequacy of using the Xpert C. difficile assay as the sole method to identify the 027 strain (Kok et al. 2011). We found the binary toxin and a 39 bp tcdc deletion in all ribotype 078 isolates, as reported by other researchers (Persson et al. 2011), but we found a 18 bp deletion in only 1 of several yok-01 and gr-01 (ribotype 001) isolates, demonstrating the variability in this gene within the same strain.

13 12 An ominous finding in our study was the elevated MIC values of both metronidazole and vancomycin that were as high as 6 and 8 µg/ml, respectively. The MIC values were significantly higher in the two most common strains, ribotype 027 and cr-02, compared with all other study strains and the control strains. All 027 and cr-02 isolates were also resistant to moxifloxacin. This phenomenon of the 027 strain's elevated MIC values of metronidazole and vancomycin was observed both in England (Shah et al. 2010) and, recently, in Israel (Wiener-well et al. 2014). It suggests that the reduced susceptibility of these strains may propagate their dissemination. Also, it raises a concern regarding the expected efficacy of metronidazole treatment (Kuijper and Wilcox 2008). This study has several limitations. First, although we found that the study sites in which the ribotype 027 strain was predominant had the highest CDI incidence in 2013, we cannot definitively establish a causal relationship because of the lack of molecular epidemiology data from 2013 or before. Second, the use the slpa typing method, although robust by itself, suffers from a relative paucity of comparative data outside of Japan. We tried to compensate for this problem by comparing our strains to some of the most common UK ribotype strains, which allowed us to correlate between the two methods. Hopefully, with the increased availability of whole genome data in molecular epidemiology studies, such problems will be easier to overcome in the near future. Third, the use of a gradient method for susceptibility testing is of course less optimal compared with the reference agar dilution method. Still, due to practicality considerations this method is widely used in other studies as well (Barbut et al. 2007). In conclusion, this study demonstrates that the epidemic C. difficile strain, ribotype 027, has disseminated across Israel and is now the most common strain, especially in institutions with a high incidence of CDI. This finding demonstrates the need for a

14 13 national intervention program, as was successfully implemented in the UK (Wilcox et al. 2012). Acknowledgement We would like to thank the researchers who oversee the Cardiff-ECDC collection of C. difficile strains for contributing reference strains.

15 14 Table 1. Major (n>2) C. difficile types at the participating centers. Center No. of samples No. of toxigenic isolates Major (n>2) types 1 A hr-02 (N=5) fr-01 (N=4) smz-02 IL1 (N=3) (N=3) B (N=15) C (N=11) D (N=23) cr-02 (N=13) hr-02 (N=5) smz-02 IL1 (N=4) E (N=7) F cr-02 (N=3) G cr-02 (N=16) hr-02 (N=3) hr-05 (N=3) H cr-02 (N=11) 027 (N=6) hr-05 (N=5) hr-02 (N=3) fr-01 (N=3) smz-02 IL1 (N=3) Total (95.8%) 1 -nomenclature according to slpa typing, with the exception of ribotype 027; 2 -Disolates detected at long-term care facilities.

16 15 Table 2. Antimicrobial susceptibility testing of C. difficile isolates. Vancomycin ( MIC Type N median, range, %NS 1 ) Metronidazole (MIC Moxifloxacin median, range, %NS 1 ) ( %NS 1 ) 027 type 65 4, 1-4, , , cr , 1.5-6, , , hr , 0.5-2, 0 0.5, , hr , , , , 0 0 smz-02 IL1 10 1, , , , 0 0 fr , , , , gc , , 0 1, , 0 0 Others 42 1, , , , NS (non-susceptible)-the breakpoints were determined according to the EUCAST criteria: Vancomycin, metronidazole NS>2 mg/l; moxifloxacin NS>4 mg/l.

17 16 Figure 1. Frequency of C. difficile slpa types and ribotype 027 within all participating centers. The slpa corresponding ribotypes were identified as follows: hr-05 and hr-05 IL1=014; fr-01 and fr-01-il1=017; kr-03=070; =078; og39-01=046; y02-01=056; gr-01 and gr-03=001; cr-01=012. Figure 2. Institutional quarterly incidence of C. difficile infection, The letters corresponds with the medical centers as detailed in table 1.

18 17 References Adler a, Schwartzberg Y, Samra Z, Schwartz O, Carmeli Y, Schwaber MJ. Trends and changes in Clostridium difficile diagnostic policies and their impact on the proportion of positive samples: a national survey. Clin Microbiol Infect 2014; 20: O Barbut F, Mastrantonio P, Delmée M, Brazier J, Kuijper E, Poxton I. Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 2007;13: Bishara J, Goldberg E, Madar-Shapiro L, Behor J, Samra Z. Molecular epidemiology of clostridium difficile in a tertiary medical center in Israel: emergence of the polymerase chain reaction ribotype 027. Isr Med Assoc J 2011;13: Alzahrani N, Johani SA. Emergence of a highly resistant Clostridium difficile strain (NAP/BI/027) in a tertiary care center in Saudi Arabia. Ann Saudi Med 2013;33: Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard Eighth Edition. Wayne, PA.: Clinical and Laboratory Standards Institute; Collins DA, Hawkey PM, Riley T V, Asia E. Epidemiology of Clostridium difficile infection in Asia. Antimicrob Resist Infect Control 2013;2:21. European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.1, Freeman J, Bauer MP, Baines SD, Corver J, Fawley WN, Goorhuis B, et al. The changing epidemiology of Clostridium difficile infections. Clin Microbiol Rev 2010;23: Gaynes R, Rimland D, Killum E, Lowery HK, Ii TMJ, Killgore G, et al. Outbreak of Clostridium difficile Infection in a Long-Term Care Facility: Association with Gatifloxacin Use. Clin Infect Dis 2004;38: Gerding DN. Global epidemiology of Clostridium difficile infection in Infect Control Hosp Epidemiol 2010;31 Suppl 1:S32 4. He M, Miyajima F, Roberts P, Ellison L, Pickard DJ, Martin MJ, et al. Emergence and global spread of epidemic healthcare-associated Clostridium difficile. Nat Genet 2013;45: Kato H. Typing by sequencing the slpa gene of Clostridium difficile strains causing multiple outbreaks in Japan. J Med Microbiol 2005;54:

19 18 Kato H, Kato H, Ito Y, Akahane T, Izumida S, Yokoyama T, et al. Typing of Clostridium difficile isolates endemic in Japan by sequencing of slpa and its application to direct typing. J Med Microbiol 2010;59: Killgore G, Thompson A, Johnson S, Brazier J, Kuijper E, Pepin J, et al. Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCRribotyping, multilocus sequence typing, multilocus variable-number tandemrepeat an. J Clin Microbiol 2008;46: Kok J, Wang Q, Thomas LC, Gilbert GL. Presumptive identification of Clostridium difficile strain 027/NAP1/BI on Cepheid Xpert: interpret with caution. J Clin Microbiol Oct;49(10): Kuijper EJ, Wilcox MH. Decreased Effectiveness of Metronidazole for the Treatment of Clostridium difficile Infection? Clin Infect Dis 2008;47:63 5. Lemee L, Dhalluin A, Testelin S, Mattrat M, Maillard K, Pons J. Multiplex PCR Targeting tpi ( Triose Phosphate Isomerase ), tcda ( Toxin A ), and tcdb ( Toxin B ) Genes for Toxigenic Culture of Clostridium difficile. J Clin Microbiol 2004;42: McDonald LC, Killgore GE, Thompson A, Owens RC, Kazakova S V, Sambol SP, et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 200;353: Persson S, Jensen JN, Olsen KEP. Multiplex PCR method for detection of Clostridium difficile tcda, tcdb, cdta, and cdtb and internal in-frame deletion of tcdc. J Clin Microbiol 2011;49: Persson S, Torpdahl M, Olsen KEP. 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: Shah D, Dang M, Hasbun R, Koo HL, Jiang Z, Dupont HL, et al. Clostridium difficile infection: update on emerging antibiotic treatment options and antibiotic resistance. Expert Rev Anti Infect Ther 2010;8: Simor AE, Bradley SF, Strausbaugh LJ, Nicolle LE, Care T, Simor AE, et al. Clostridium difficile in Long-Term Care Facilities for the Elderly. Infect Control Hosp Epidemiol 2002;11: Wiener-well AY, Ben-chetrit E, Abed-eldaim M, Marc V, Miller-roll T, Adler A. Clinical and Molecular Characteristics of an Outbreak Caused by the Pandemic ( BI / NAP1 / 027 ) Clostridium difficile Clone in a Single Center in Israel. Infect Control Hosp Epidemiol. 2014;35:5 8. Wilcox MH, Shetty N, Fawley WN, Shemko M, Coen P, Birtles A, et al. Changing Epidemiology of Clostridium dif fi cile Infection Following the Introduction of a

20 19 National Ribotyping-Based Surveillance Scheme in England. Clin Infect Dis 2012;55: Xiao M, Kong F, Jin P, Wang Q, Xiao K, Jeoffreys N, et al. Comparison of two capillary gel electrophoresis systems for Clostridium difficile ribotyping, using a panel of ribotype 027 isolates and whole-genome sequences as a reference standard. J Clin Microbiol 2012;50: The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.1, p.

21 20 Figure 1

22 21 Figure 2