J Antimicrob Chemother 2015; 70: 2311 – 2315 doi:10.1093/jac/dkv092 Advance Access publication 15 April 2015
In vitro activity of surotomycin against contemporary clinical isolates of toxigenic Clostridium difficile strains obtained in Spain E. Reigadas1–3*, L. Alcala´1,3,4, M. Marı´n1–4, T. Pelae´z1–4, A. Martin1,3, C. Iglesias1,4 and E. Bouza1–4 1
Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Maran˜o´n, Madrid, Spain; 2Medicine Department, School of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain; 3Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n, Madrid, Spain; 4CIBER de Enfermedades Respiratorias (CIBERES CB06/06/0058), Madrid, Spain
Received 17 November 2014; returned 16 January 2015; revised 23 February 2015; accepted 18 March 2015 Objectives: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Metronidazole and vancomycin are the mainstay of treatment, although they are associated with treatment failure and recurrence. Novel agents have emerged to address these shortcomings. We investigated the in vitro activity of a novel agent, surotomycin (formerly CB-183,315), and seven other antimicrobial agents against clinical C. difficile isolates. Methods: Antimicrobial susceptibility to surotomycin, fidaxomicin, metronidazole, vancomycin, clindamycin, rifaximin, moxifloxacin and tigecycline was determined for 100 contemporary clinical isolates of C. difficile collected in 2013. MICs were determined by agar dilution according to CLSI procedures. In addition, 10 strains with reduced susceptibility to metronidazole (n¼6) and vancomycin (n¼4) were also tested. Strains were PCR ribotyped. Results: The MICs of surotomycin for the 100 isolates ranged from ≤0.06 to 2 mg/L, with a geometric mean (GM) of 0.31 mg/L and an MIC50/90 of 0.25/0.5 mg/L. The MIC range of surotomycin was 0.25 –1 mg/L (GM¼0.45 mg/L) for isolates with reduced metronidazole susceptibility and 0.125 – 0.5 mg/L (GM ¼ 0.25 mg/L) for isolates with reduced vancomycin susceptibility. The three most common ribotypes were 001 (31.0%), 014/020 (17.0%) and 078/126 (17.0%). Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM¼ 0.22 mg/L); the highest MICs were for ribotype 078/126 (GM¼ 0.72 mg/L). Conclusions: Surotomycin exhibited potent in vitro activity against all the isolates tested, including those with elevated metronidazole and vancomycin MICs. The potential role of this agent in the treatment of CDI requires further clinical evaluation. Keywords: C. difficile, antimicrobial susceptibility, fidaxomicin, ribotype
Introduction Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Metronidazole and vancomycin are the mainstay of CDI treatment, although they are both associated with treatment failure (affecting 3%–18% of patients1) and disease recurrence (20%).2 Novel agents have emerged to address the shortcomings of current therapeutic agents. Fidaxomicin, a narrow-spectrum macrocyclic antibiotic, was recently included in the therapeutic armamentarium for treatment of CDI.3 The efficacy of other promising narrow-spectrum antibiotics is currently being investigated. Surotomycin (formerly CB-183,315), a promising novel agent, is being developed for treatment of CDI. This agent is a lipopeptide
antibiotic that is structurally similar to daptomycin.4 It has a narrow spectrum of activity and thus spares much of the normal flora, a feature that has been associated with a decreased recurrence rate and higher clinical cure rates.5,6 The aim of the present study was to investigate the in vitro activity of surotomycin and seven other antimicrobial agents against recent clinical C. difficile isolates collected in Madrid, Spain.
Methods Setting Our institution is a large teaching hospital with 1550 beds. The clinical microbiology laboratory receives samples from patients hospitalized at our centre and from all outpatient institutions in our catchment area,
# The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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*Corresponding author. Servicio de Microbiologı´a Clı´nica y Enfermedades Infecciosas, Hospital General Universitario ‘Gregorio Maran˜o´n’, C/Dr. Esquerdo, 46, 28007 Madrid, Spain. Tel: +34-91-586-84-53; Fax: +34-91-504-49-06; E-mail: [email protected]
Reigadas et al.
Table 1. In vitro activity of the antimicrobials tested against 100 clinical isolates of C. difficile
Bacterial isolates
Antimicrobial agent
MIC range (mg/L)
MIC50/90 (mg/L)
Geometric mean (mg/L)
Surotomycin Fidaxomicin Metronidazole Vancomycin Clindamycin Rifaximin Moxifloxacin Tigecycline
≤0.06 –2 ≤0.015 –0.25 0.06 –1 0.125 –2 0.125 to .256 0.0019 to .256 0.125 –32 ≤0.03 –0.50
0.25/0.5 0.06/0.125 0.25/0.5 0.5/1 4/256 0.015/.256 4/32 0.125/0.25
0.31 0.06 0.24 0.48 11.71 0.20 4.50 0.09
Ribotyping All isolates were characterized using PCR ribotyping.9 Phylogenetic analysis of ribotyping profiles was conducted applying the unweighted pair group method with arithmetic mean and Dice coefficients using Bionumerics 5.0 software (Applied Maths). Ribotypes were named according to the designation of the ribotype collection from the Cardiff-ECDC.
Ethics This study was approved by the Ethics Committee of Hospital General Universitario Gregorio Maran˜o´n.
Results MIC ranges, geometric means (GMs) and minimum concentrations inhibiting 50% (MIC50) and 90% (MIC90) of the 100 isolates tested are shown in Table 1. Surotomycin had a range of ≤0.06 – 2 mg/L and a GM of 0.31 mg/L. Overall, 90% of the isolates fell within a range of ≤0.06 – 0.5 mg/L. The antimicrobial agents
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2
he Ot
8
00
0
01
0
05
7
07
6
20
6
04
6
10
0
12 8/
07
4/
02
1
0 00
Susceptibility to eight antimicrobial agents was tested using the agar dilution method according to CLSI procedures.7 The antimicrobial agents tested were surotomycin, fidaxomicin, metronidazole, vancomycin, clindamycin, rifaximin, moxifloxacin and tigecycline. Surotomycin and fidaxomicin were obtained from Cubist Pharmaceuticals (Lexington, MA, USA) and the remaining antimicrobial agents tested were obtained from Sigma Chemical Company (St Louis, MO, USA). Quality control strains included C. difficile ATCC 700057, Bacteroides fragilis ATCC 25285, Bacteroides thetaiotamicron ATCC 29741 and Staphylococcus aureus ATCC 29213. The medium used was Brucella agar supplemented with 5 mg/L haemin, 1 mg/L vitamin K1 and 5% (v/v) laked sheep blood. For in vitro testing of surotomycin, the medium was supplemented to a final concentration of 50 mg/L Ca2+. Surotomycin quality control ranges were 0.125 – 1 mg/L for C. difficile ATCC 700057. S. aureus ATCC 29213 tested anaerobically was also used as an additional control organism for surotomycin with ranges of 0.5–2 mg/L. Fidaxomicin quality control ranges were 0.06 – 0.25 mg/L for C. difficile ATCC 700057.8 When available, the breakpoints for antimicrobials were those established by the CLSI; when no breakpoint was available, recommendations for aerobic bacteria from the CLSI and other sources were used.
30
01
Antimicrobial susceptibility testing
35
Ribotypes Figure 1. Distribution of ribotypes of 100 clinical isolates of C. difficile isolated in Madrid, Spain in 2013.
that presented a lower MIC90 were fidaxomicin (0.125 mg/L) and tigecycline (0.25 mg/L). Resistance rates to clindamycin (≥8 mg/L), moxifloxacin (≥8 mg/L) and rifaximin (≥32 mg/L) were 49.0%, 44.0% and 28.0%, respectively. The most common ribotype was 001 (31.0%), followed by 014/020 (17.0%), 078/126 (17.0%), 106 (9.0%) and other ribotypes (26.0%). The distribution of the ribotypes is shown in Figure 1. The analysis of antimicrobial susceptibility according to the main ribotypes is shown in Table 2. Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM ¼ 0.22 mg/L) and ribotype 078/126 exhibited the highest surotomycin MICs (GM¼ 0.72 mg/L). Ribotype 078/126 strains also exhibited higher MICs of fidaxomicin and tigecycline. The selected strains with known high MICs of vancomycin (n ¼ 4) and metronidazole (n ¼ 6) had been originally tested by Etest, with an MIC50/MIC90 of metronidazole of 1.5/8 mg/L and an MIC50/MIC90 of vancomycin of 3/4 mg/L. When retesting these strains by agar dilution for this study, the MIC50/MIC90 of metronidazole was 4/4 mg/L and the MIC50/MIC90 of vancomycin was 4/8 mg/L. The MIC ranges and GM of all the antimicrobials tested, for isolates with reduced susceptibility to metronidazole and vancomycin, are shown in Table 3. The GM MIC of surotomycin for isolates with reduced susceptibility to metronidazole and vancomycin was 0.45 and 0.25 mg/L, respectively.
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We collected 100 C. difficile isolates from the clinical samples of CDI patients received at the microbiology laboratory of Hospital General Universitario Gregorio Maran˜o´n in 2013. All samples included were recovered from patients aged .2 years meeting criteria for CDI, defined as the presence of a positive test result for toxigenic C. difficile and one of the following: presence of diarrhoea (three or more unformed stools in 24 h) or colonoscopic evidence of pseudomembranous colitis. Out of the 100 C. difficile isolates, 19 strains belonged to patients with CDI recurrence. In addition, strains with known high MICs (these strains had been tested at the moment of isolation by Etest) of vancomycin (n ¼ 4) and metronidazole (n ¼ 6) were recovered from samples (2007 – 13) received at the microbiology laboratory of Hospital General Universitario Gregorio Maran˜o´n. For this study, the strains were retested using the agar dilution method.
Percentages of isolates tested
which encompasses 715000 people. During 2013, the incidence of CDI episodes at Hospital Gregorio Maran˜o´n was 12.2/10000 days of stay.
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In vitro activity of surotomycin against CDI clinical isolates
Table 2. In vitro activity of the antimicrobials tested against clinical isolates of C. difficile according to the most frequent ribotypes encountered in our study Ribotype 014/020 (n¼17)
Ribotype 078/126 (n¼17)
MIC range (mg/L) surotomycin metronidazole vancomycin moxifloxacin tigecycline fidaxomicin clindamycin rifaximin
0.06 –0.5 0.125– 0.5 0.125– 2 0.5–32 0.03 –0.25 0.015– 0.125 0.5 to .256 0.0039 to .256
0.03 –0.5 0.06 –0.5 0.25 –1 1 –8 0.03 –0.25 0.015– 0.125 1 –16 0.0019–0.03
0.25 –2 0.125 –0.5 0.125 –1 1 – 32 0.06 –0.5 0.015 –0.25 2 to .256 0.0039 to .256
MIC50/90 (mg/L) surotomycin metronidazole vancomycin moxifloxacin tigecycline fidaxomicin clindamycin rifaximin
0.25/0.5 0.25/0.5 0.5/1 16/32 0.06/0.125 0.03/0.06 128/256 .256/.256
0.25/0.5 0.25/0.5 0.5/1 2/8 0.125/0.25 0.06/0.125 4/8 0.0078/0.015
1/1 0.25/0.5 0.5/1 8/32 0.125/0.5 0.125/0.25 8/.256 0.015/0.03
Geometric mean MIC (mg/L) surotomycin metronidazole vancomycin moxifloxacin tigecycline fidaxomicin clindamycin rifaximin
0.25 0.26 0.39 11.44 0.07 0.03 62.58 119.52
0.22 0.25 0.54 2.26 0.12 0.06 4.52 0.01
0.72 0.25 0.52 5.11 0.15 0.11 26.10 0.02
Discussion In this study, we evaluated the in vitro activity of eight antimicrobial agents, including the novel antimicrobial surotomycin, against clinical C. difficile isolates. Surotomycin exhibited excellent activity, even against isolates with elevated MICs of vancomycin and metronidazole. Metronidazole and vancomycin have been the primary treatment options in the management of CDI for the past 30 years. However, the need for new antimicrobial agents is evident owing to the disadvantages of current antimicrobial therapies, which include therapeutic failure, VRE selection, high percentage of recurrence and cost.3,10 Fidaxomicin was recently approved for treatment of CDI,3 with similar clinical cure rates to vancomycin and lower recurrence rates.11,12 Unlike traditional treatments, fidaxomicin has bactericidal activity against C. difficile and minimal activity against other constituents of the intestinal microbiota.13 Surotomycin has successfully completed a Phase 2 trial, in which it achieved higher sustained cure rates and statistically less recurrence of CDI than vancomycin.14 A recently published study demonstrated that resistance to surotomycin is very unlikely to emerge in C. difficile, Enterococcus faecalis and Enterococcus faecium (VRE and vancomycin-susceptible enterococci).15 Surotomycin is currently in
Phase 3 trials for treatment of CDI, with a dose regimen of 250 mg twice daily for 10 days. In our study, surotomycin exhibited potent in vitro activity, with an MIC range, MIC50 and MIC90 for our 2013 collection of clinical isolates that were identical to those reported previously by Citron et al.5 against 2005 – 08 clinical isolates. Sanders et al.16 performed a study on isolates collected for a pan-European survey in 2008, presented at ECCMID 2014, in which the MIC50 and MIC90 were also the same as ours; however, they observed a much narrower MIC range. Snydman et al.6 observed similar results in a study of 55 C. difficile isolates, which included quinolone-resistant isolates and isolates with elevated MICs of vancomycin and metronidazole. In our study, surotomycin presented a broader MIC range than reported by Sanders et al.16 Fidaxomicin, tigecycline, metronidazole and vancomycin were also active against our 2013 collection of clinical isolates. The other antimicrobial agents tested showed variable activity against C. difficile isolates. As expected, resistance to clindamycin and moxifloxacin were the most frequent phenotypes observed in our study. Similar results have been reported in other studies from the USA and Europe.17,18 In our study, the three most commonly encountered ribotypes were 001 (30.1%), followed by 014/020 (17.0%) and 078/126 (17.0%). Interestingly, ribotype 106 accounted for 9% of all
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Ribotype 001 (n ¼31)
Antimicrobial agent
Reigadas et al.
Table 3. In vitro activity of the antimicrobials tested against clinical isolates of C. difficile with reduced susceptibility to metronidazole (n¼6) and vancomycin (n¼4) Antimicrobial agent
Geometric mean MIC (mg/L)
MIC range (mg/L)
0.125–0.5 0.015–0.125 0.25– 4 4 –8 2 –256 0.03 to .256 32 0.06– 0.125
Isolates with high MICs of metronidazole surotomycin 0.45 fidaxomicin 0.08 metronidazole 4 vancomycin 0.79 clindamycin 25.40 rifaximin 1.25 moxifloxacin 20.16 tigecycline 0.10
0.25– 1 0.03– 0.25 4 0.5–8 2 –256 0.0019 to .256 8 –32 0.06– 0.125
isolates. Ribotype 106, commonly detected in the UK, was rarely found elsewhere;19 however, it has recently been found to be the fourth most common PCR ribotype encountered in the II national study performed in Spain.20 We found that surotomycin exhibited good activity against the different ribotypes. In a European multicentre study, PCR ribotype 001 had one of the highest percentages of resistance to three or more classes of antibiotics.21 In the present study, the most common ribotype was 001 (31.0%). We observed that 80.6% of ribotype 001 isolates were clindamycin resistant, 83.9% were moxifloxacin resistant and 87.1% were rifaximin resistant. Relatively low resistance rates (2.7%) to rifaximin have usually been described.22 The association of rifaximin resistance with ribotype 001 may be specific to our institution and may merely reflect clonal expansion; however, our results are in line with those from a European multicentre study in which 27 out of 32 (84.4%) PCR ribotype 001 strains were resistant to rifampicin, whose resistance correlates well with rifaximin.21,23,24 High resistance rates to rifaximin (83.8% – 95%) have been observed for ribotype 017,25,26 although in these studies ribotype 001 did not exhibit resistance to rifaximin. Also, resistance to rifamycins has been described in ribotype 027 strains.23,27 Fidaxomicin, surotomycin and tigecycline showed excellent activity against ribotype 001 isolates. Fidaxomicin exhibited the lowest MIC50/MIC90 for this ribotype compared with other frequently found PCR ribotypes 014/020 and 078/126, which is in line with the findings of a study performed on isolates collected for a pan-European survey in 2008.28 Also, surotomycin exhibited the highest MICs for ribotype 078/126. Vancomycin and metronidazole were 100% active against ribotype 001 strains [2013 collection (100 isolates)]. Surotomycin has proven to be active against strains with reduced susceptibility to metronidazole and vancomycin. We found that the most effective antimicrobial
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Acknowledgements Some of the results of this study were previously presented in poster form at the Fifty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2014 (Poster F-242). We thank Thomas O’Boyle for his help in the preparation of the manuscript (English Consultancy Service) and Jeanna Fisher for her assistance in the calcium supplementation procedure during susceptibility testing of surotomycin.
Funding This study was financed by Cubist Pharmaceuticals, the Rafael del Pino Foundation and Fondo de Investigaciones Sanitarias (FIS), Research Project number PI13/00687. E. R. holds a grant from the Rı´o Hortega programme of the Carlos III Health Institute, Spanish Government.
Transparency declarations None to declare.
References 1 Aslam S, Hamill RJ, Musher DM. Treatment of Clostridium difficileassociated disease: old therapies and new strategies. Lancet Infect Dis 2005; 5: 549–57. 2 McFarland LV, Surawicz CM, Rubin M et al. Recurrent Clostridium difficile disease: epidemiology and clinical characteristics. Infect Control Hosp Epidemiol 1999; 20: 43– 50. 3 Debast SB, Bauer MP, Kuijper EJ. European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin Microbiol Infect 2014; 20 Suppl 2: 1– 26. 4 Mascio CT, Mortin LI, Howland KT et al. In vitro and in vivo characterization of CB-183,315, a novel lipopeptide antibiotic for treatment of Clostridium difficile. Antimicrob Agents Chemother 2012; 56: 5023 –30. 5 Citron DM, Tyrrell KL, Merriam CV et al. In vitro activities of CB-183,315, vancomycin, and metronidazole against 556 strains of Clostridium difficile, 445 other intestinal anaerobes, and 56 Enterobacteriaceae species. Antimicrob Agents Chemother 2012; 56: 1613– 5. 6 Snydman DR, Jacobus NV, McDermott LA. Activity of a novel cyclic lipopeptide, CB-183,315, against resistant Clostridium difficile and other Gram-positive aerobic and anaerobic intestinal pathogens. Antimicrob Agents Chemother 2012; 56: 3448 –52.
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Isolates with high MICs of vancomycin surotomycin 0.25 fidaxomicin 0.04 metronidazole 0.71 vancomycin 5.65 clindamycin 64 rifaximin 44.80 moxifloxacin 32 tigecycline 0.09
agents against vancomycin-resistant isolates were fidaxomicin, tigecycline and surotomycin, which were also effective against isolates with reduced susceptibility to metronidazole. Tigecycline has previously demonstrated favourable in vitro activity against C. difficile isolates. Although some reports suggest that tigecycline could be a reasonable addition for treatment of CDI episodes refractory to standard therapy,29,30 more clinical data are needed. Our study is limited by the fact that we were not able to draw conclusions on the performance of surotomycin against ribotype 027 strains owing to the limited number of 027 strains in our centre. In conclusion, surotomycin exhibited potent in vitro activity against all the isolates tested, including those with elevated metronidazole and vancomycin MICs. The potential role of this agent in the treatment of CDI requires further clinical evaluation.
In vitro activity of surotomycin against CDI clinical isolates
7 Clinical and Laboratory Standards. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Eighth Edition: Approved Standard M11-A8. CLSI, Wayne, PA, USA, 2012. 8 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-fourth Informational Supplement M100-S24. CLSI, Wayne, PA, USA, 2014. 9 Stubbs SL, Brazier JS, O’Neill GL et al. 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 1999; 37: 461–3. 10 Cohen SH, Gerding DN, Johnson S et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol 2010; 31: 431–55.
12 Louie TJ, Miller MA, Mullane KM et al. Fidaxomicin versus vancomycin for Clostridium difficile infection. New Engl J Med 2011; 364: 422–31. 13 Babakhani F, Gomez A, Robert N et al. Killing kinetics of fidaxomicin and its major metabolite, OP-1118, against Clostridium difficile. J Med Microbiol 2011; 60: 1213– 7. 14 Chesnel L. Treatment of CDAD with oral CB-183 315: time to recurrence, relapse and re-infection rates compared with vancomycin. Clin Microbiol Infect 2012; 18: Suppl 3: 380. 15 Mascio CT, Chesnel L, Thorne G et al. Surotomycin demonstrates low in vitro frequency of resistance and rapid bactericidal activity in Clostridium difficile, Enterococcus faecalis and Enterococcus faecium. Antimicrob Agents Chemother 2014; 58: 3976 –82. 16 Sanders I, Harmanus C, Debast S et al. Antibiotic susceptibility of surotomycin and five other antibiotics against Clostridium difficile isolates, collected at a pan-European survey in 2008 (n ¼ 119). In: Abstracts of the Twenty-fourth European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, 2014, Abstract P0792. European Society of Clinical Microbiology and Infectious Diseases, Basel, Switzerland.
19 Freeman J, Bauer MP, Baines SD et al. The changing epidemiology of Clostridium difficile infections. Clin Microbiol Rev 2010; 23: 529–49. 20 Alcala´ L, Reigadas E, Marı´n M et al. Multicenter study of Clostridium difficile infection in Spain: an update of the underdiagnosis of Clostridium difficile infection in a whole nation. In: Abstracts of the Twenty-fourth European Congress of Clinical Microbiology and Infectious Diseases, 2014. Abstract P0741. European Society of Clinical Microbiology and Infectious Diseases, Barcelona, Spain. 21 Spigaglia P, Barbanti F, Mastrantonio P. Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother 2011; 66: 2227– 34. 22 Hecht DW, Galang MA, Sambol SP et al. In vitro activities of 15 antimicrobial agents against 110 toxigenic Clostridium difficile clinical isolates collected from 1983 to 2004. Antimicrob Agents Chemother 2007; 51: 2716–9. 23 O’Connor JR, Galang MA, Sambol SP et al. Rifampin and rifaximin resistance in clinical isolates of Clostridium difficile. Antimicrob Agents Chemother 2008; 52: 2813– 7. 24 Miller MA, Blanchette R, Spigaglia P et al. Divergent rifamycin susceptibilities of Clostridium difficile strains in Canada and Italy and predictive accuracy of rifampin Etest for rifamycin resistance. J Clin Microbiol 2011; 49: 4319– 21. 25 Huang H, Weintraub A, Fang H et al. Antimicrobial susceptibility and heteroresistance in Chinese Clostridium difficile strains. Anaerobe 2010; 16: 633–5. 26 Kim J, Kang JO, Pai H et al. Association between PCR ribotypes and antimicrobial susceptibility among Clostridium difficile isolates from healthcare-associated infections in South Korea. Int J Antimicrob Agents 2012; 40: 24– 9. 27 Curry SR, Marsh JW, Shutt KA et al. High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis 2009; 48: 425–9. 28 Debast SB, Bauer MP, Sanders IM et al. Antimicrobial activity of LFF571 and three treatment agents against Clostridium difficile isolates collected for a pan-European survey in 2008: clinical and therapeutic implications. J Antimicrob Chemother 2013; 68: 1305 –11.
17 Tickler IA, Goering RV, Whitmore JD et al. Strain types and antimicrobial resistance patterns of Clostridium difficile isolates from the United States: 2011– 2013. Antimicrob Agents Chemother 2014; 58: 4214 –8.
29 Herpers BL, Vlaminckx B, Burkhardt O et al. Intravenous tigecycline as adjunctive or alternative therapy for severe refractory Clostridium difficile infection. Clin Infect Dis 2009; 48: 1732– 5.
18 Alcala L, Martin A, Marin M et al. The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem? Clin Microbiol Infect 2012; 18: E204–13.
30 Lu CL, Liu CY, Liao CH et al. Severe and refractory Clostridium difficile infection successfully treated with tigecycline and metronidazole. Int J Antimicrob Agents 2010; 35: 311–2.
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11 Cornely OA, Crook DW, Esposito R et al. Fidaxomicin versus vancomycin for infection with Clostridium difficile in Europe, Canada, and the USA: a double-blind, non-inferiority, randomised controlled trial. Lancet Infect Dis 2012; 12: 281–9.
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In vitro activity of surotomycin against contemporary clinical isolates of toxigenic Clostridium difficile strains obtained in Spain E. Reigadas1–3*, L. Alcala´1,3,4, M. Marı´n1–4, T. Pelae´z1–4, A. Martin1,3, C. Iglesias1,4 and E. Bouza1–4 1
Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Maran˜o´n, Madrid, Spain; 2Medicine Department, School of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain; 3Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n, Madrid, Spain; 4CIBER de Enfermedades Respiratorias (CIBERES CB06/06/0058), Madrid, Spain
Received 17 November 2014; returned 16 January 2015; revised 23 February 2015; accepted 18 March 2015 Objectives: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Metronidazole and vancomycin are the mainstay of treatment, although they are associated with treatment failure and recurrence. Novel agents have emerged to address these shortcomings. We investigated the in vitro activity of a novel agent, surotomycin (formerly CB-183,315), and seven other antimicrobial agents against clinical C. difficile isolates. Methods: Antimicrobial susceptibility to surotomycin, fidaxomicin, metronidazole, vancomycin, clindamycin, rifaximin, moxifloxacin and tigecycline was determined for 100 contemporary clinical isolates of C. difficile collected in 2013. MICs were determined by agar dilution according to CLSI procedures. In addition, 10 strains with reduced susceptibility to metronidazole (n¼6) and vancomycin (n¼4) were also tested. Strains were PCR ribotyped. Results: The MICs of surotomycin for the 100 isolates ranged from ≤0.06 to 2 mg/L, with a geometric mean (GM) of 0.31 mg/L and an MIC50/90 of 0.25/0.5 mg/L. The MIC range of surotomycin was 0.25 –1 mg/L (GM¼0.45 mg/L) for isolates with reduced metronidazole susceptibility and 0.125 – 0.5 mg/L (GM ¼ 0.25 mg/L) for isolates with reduced vancomycin susceptibility. The three most common ribotypes were 001 (31.0%), 014/020 (17.0%) and 078/126 (17.0%). Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM¼ 0.22 mg/L); the highest MICs were for ribotype 078/126 (GM¼ 0.72 mg/L). Conclusions: Surotomycin exhibited potent in vitro activity against all the isolates tested, including those with elevated metronidazole and vancomycin MICs. The potential role of this agent in the treatment of CDI requires further clinical evaluation. Keywords: C. difficile, antimicrobial susceptibility, fidaxomicin, ribotype
Introduction Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Metronidazole and vancomycin are the mainstay of CDI treatment, although they are both associated with treatment failure (affecting 3%–18% of patients1) and disease recurrence (20%).2 Novel agents have emerged to address the shortcomings of current therapeutic agents. Fidaxomicin, a narrow-spectrum macrocyclic antibiotic, was recently included in the therapeutic armamentarium for treatment of CDI.3 The efficacy of other promising narrow-spectrum antibiotics is currently being investigated. Surotomycin (formerly CB-183,315), a promising novel agent, is being developed for treatment of CDI. This agent is a lipopeptide
antibiotic that is structurally similar to daptomycin.4 It has a narrow spectrum of activity and thus spares much of the normal flora, a feature that has been associated with a decreased recurrence rate and higher clinical cure rates.5,6 The aim of the present study was to investigate the in vitro activity of surotomycin and seven other antimicrobial agents against recent clinical C. difficile isolates collected in Madrid, Spain.
Methods Setting Our institution is a large teaching hospital with 1550 beds. The clinical microbiology laboratory receives samples from patients hospitalized at our centre and from all outpatient institutions in our catchment area,
# The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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*Corresponding author. Servicio de Microbiologı´a Clı´nica y Enfermedades Infecciosas, Hospital General Universitario ‘Gregorio Maran˜o´n’, C/Dr. Esquerdo, 46, 28007 Madrid, Spain. Tel: +34-91-586-84-53; Fax: +34-91-504-49-06; E-mail: [email protected]
Reigadas et al.
Table 1. In vitro activity of the antimicrobials tested against 100 clinical isolates of C. difficile
Bacterial isolates
Antimicrobial agent
MIC range (mg/L)
MIC50/90 (mg/L)
Geometric mean (mg/L)
Surotomycin Fidaxomicin Metronidazole Vancomycin Clindamycin Rifaximin Moxifloxacin Tigecycline
≤0.06 –2 ≤0.015 –0.25 0.06 –1 0.125 –2 0.125 to .256 0.0019 to .256 0.125 –32 ≤0.03 –0.50
0.25/0.5 0.06/0.125 0.25/0.5 0.5/1 4/256 0.015/.256 4/32 0.125/0.25
0.31 0.06 0.24 0.48 11.71 0.20 4.50 0.09
Ribotyping All isolates were characterized using PCR ribotyping.9 Phylogenetic analysis of ribotyping profiles was conducted applying the unweighted pair group method with arithmetic mean and Dice coefficients using Bionumerics 5.0 software (Applied Maths). Ribotypes were named according to the designation of the ribotype collection from the Cardiff-ECDC.
Ethics This study was approved by the Ethics Committee of Hospital General Universitario Gregorio Maran˜o´n.
Results MIC ranges, geometric means (GMs) and minimum concentrations inhibiting 50% (MIC50) and 90% (MIC90) of the 100 isolates tested are shown in Table 1. Surotomycin had a range of ≤0.06 – 2 mg/L and a GM of 0.31 mg/L. Overall, 90% of the isolates fell within a range of ≤0.06 – 0.5 mg/L. The antimicrobial agents
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Susceptibility to eight antimicrobial agents was tested using the agar dilution method according to CLSI procedures.7 The antimicrobial agents tested were surotomycin, fidaxomicin, metronidazole, vancomycin, clindamycin, rifaximin, moxifloxacin and tigecycline. Surotomycin and fidaxomicin were obtained from Cubist Pharmaceuticals (Lexington, MA, USA) and the remaining antimicrobial agents tested were obtained from Sigma Chemical Company (St Louis, MO, USA). Quality control strains included C. difficile ATCC 700057, Bacteroides fragilis ATCC 25285, Bacteroides thetaiotamicron ATCC 29741 and Staphylococcus aureus ATCC 29213. The medium used was Brucella agar supplemented with 5 mg/L haemin, 1 mg/L vitamin K1 and 5% (v/v) laked sheep blood. For in vitro testing of surotomycin, the medium was supplemented to a final concentration of 50 mg/L Ca2+. Surotomycin quality control ranges were 0.125 – 1 mg/L for C. difficile ATCC 700057. S. aureus ATCC 29213 tested anaerobically was also used as an additional control organism for surotomycin with ranges of 0.5–2 mg/L. Fidaxomicin quality control ranges were 0.06 – 0.25 mg/L for C. difficile ATCC 700057.8 When available, the breakpoints for antimicrobials were those established by the CLSI; when no breakpoint was available, recommendations for aerobic bacteria from the CLSI and other sources were used.
30
01
Antimicrobial susceptibility testing
35
Ribotypes Figure 1. Distribution of ribotypes of 100 clinical isolates of C. difficile isolated in Madrid, Spain in 2013.
that presented a lower MIC90 were fidaxomicin (0.125 mg/L) and tigecycline (0.25 mg/L). Resistance rates to clindamycin (≥8 mg/L), moxifloxacin (≥8 mg/L) and rifaximin (≥32 mg/L) were 49.0%, 44.0% and 28.0%, respectively. The most common ribotype was 001 (31.0%), followed by 014/020 (17.0%), 078/126 (17.0%), 106 (9.0%) and other ribotypes (26.0%). The distribution of the ribotypes is shown in Figure 1. The analysis of antimicrobial susceptibility according to the main ribotypes is shown in Table 2. Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM ¼ 0.22 mg/L) and ribotype 078/126 exhibited the highest surotomycin MICs (GM¼ 0.72 mg/L). Ribotype 078/126 strains also exhibited higher MICs of fidaxomicin and tigecycline. The selected strains with known high MICs of vancomycin (n ¼ 4) and metronidazole (n ¼ 6) had been originally tested by Etest, with an MIC50/MIC90 of metronidazole of 1.5/8 mg/L and an MIC50/MIC90 of vancomycin of 3/4 mg/L. When retesting these strains by agar dilution for this study, the MIC50/MIC90 of metronidazole was 4/4 mg/L and the MIC50/MIC90 of vancomycin was 4/8 mg/L. The MIC ranges and GM of all the antimicrobials tested, for isolates with reduced susceptibility to metronidazole and vancomycin, are shown in Table 3. The GM MIC of surotomycin for isolates with reduced susceptibility to metronidazole and vancomycin was 0.45 and 0.25 mg/L, respectively.
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We collected 100 C. difficile isolates from the clinical samples of CDI patients received at the microbiology laboratory of Hospital General Universitario Gregorio Maran˜o´n in 2013. All samples included were recovered from patients aged .2 years meeting criteria for CDI, defined as the presence of a positive test result for toxigenic C. difficile and one of the following: presence of diarrhoea (three or more unformed stools in 24 h) or colonoscopic evidence of pseudomembranous colitis. Out of the 100 C. difficile isolates, 19 strains belonged to patients with CDI recurrence. In addition, strains with known high MICs (these strains had been tested at the moment of isolation by Etest) of vancomycin (n ¼ 4) and metronidazole (n ¼ 6) were recovered from samples (2007 – 13) received at the microbiology laboratory of Hospital General Universitario Gregorio Maran˜o´n. For this study, the strains were retested using the agar dilution method.
Percentages of isolates tested
which encompasses 715000 people. During 2013, the incidence of CDI episodes at Hospital Gregorio Maran˜o´n was 12.2/10000 days of stay.
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In vitro activity of surotomycin against CDI clinical isolates
Table 2. In vitro activity of the antimicrobials tested against clinical isolates of C. difficile according to the most frequent ribotypes encountered in our study Ribotype 014/020 (n¼17)
Ribotype 078/126 (n¼17)
MIC range (mg/L) surotomycin metronidazole vancomycin moxifloxacin tigecycline fidaxomicin clindamycin rifaximin
0.06 –0.5 0.125– 0.5 0.125– 2 0.5–32 0.03 –0.25 0.015– 0.125 0.5 to .256 0.0039 to .256
0.03 –0.5 0.06 –0.5 0.25 –1 1 –8 0.03 –0.25 0.015– 0.125 1 –16 0.0019–0.03
0.25 –2 0.125 –0.5 0.125 –1 1 – 32 0.06 –0.5 0.015 –0.25 2 to .256 0.0039 to .256
MIC50/90 (mg/L) surotomycin metronidazole vancomycin moxifloxacin tigecycline fidaxomicin clindamycin rifaximin
0.25/0.5 0.25/0.5 0.5/1 16/32 0.06/0.125 0.03/0.06 128/256 .256/.256
0.25/0.5 0.25/0.5 0.5/1 2/8 0.125/0.25 0.06/0.125 4/8 0.0078/0.015
1/1 0.25/0.5 0.5/1 8/32 0.125/0.5 0.125/0.25 8/.256 0.015/0.03
Geometric mean MIC (mg/L) surotomycin metronidazole vancomycin moxifloxacin tigecycline fidaxomicin clindamycin rifaximin
0.25 0.26 0.39 11.44 0.07 0.03 62.58 119.52
0.22 0.25 0.54 2.26 0.12 0.06 4.52 0.01
0.72 0.25 0.52 5.11 0.15 0.11 26.10 0.02
Discussion In this study, we evaluated the in vitro activity of eight antimicrobial agents, including the novel antimicrobial surotomycin, against clinical C. difficile isolates. Surotomycin exhibited excellent activity, even against isolates with elevated MICs of vancomycin and metronidazole. Metronidazole and vancomycin have been the primary treatment options in the management of CDI for the past 30 years. However, the need for new antimicrobial agents is evident owing to the disadvantages of current antimicrobial therapies, which include therapeutic failure, VRE selection, high percentage of recurrence and cost.3,10 Fidaxomicin was recently approved for treatment of CDI,3 with similar clinical cure rates to vancomycin and lower recurrence rates.11,12 Unlike traditional treatments, fidaxomicin has bactericidal activity against C. difficile and minimal activity against other constituents of the intestinal microbiota.13 Surotomycin has successfully completed a Phase 2 trial, in which it achieved higher sustained cure rates and statistically less recurrence of CDI than vancomycin.14 A recently published study demonstrated that resistance to surotomycin is very unlikely to emerge in C. difficile, Enterococcus faecalis and Enterococcus faecium (VRE and vancomycin-susceptible enterococci).15 Surotomycin is currently in
Phase 3 trials for treatment of CDI, with a dose regimen of 250 mg twice daily for 10 days. In our study, surotomycin exhibited potent in vitro activity, with an MIC range, MIC50 and MIC90 for our 2013 collection of clinical isolates that were identical to those reported previously by Citron et al.5 against 2005 – 08 clinical isolates. Sanders et al.16 performed a study on isolates collected for a pan-European survey in 2008, presented at ECCMID 2014, in which the MIC50 and MIC90 were also the same as ours; however, they observed a much narrower MIC range. Snydman et al.6 observed similar results in a study of 55 C. difficile isolates, which included quinolone-resistant isolates and isolates with elevated MICs of vancomycin and metronidazole. In our study, surotomycin presented a broader MIC range than reported by Sanders et al.16 Fidaxomicin, tigecycline, metronidazole and vancomycin were also active against our 2013 collection of clinical isolates. The other antimicrobial agents tested showed variable activity against C. difficile isolates. As expected, resistance to clindamycin and moxifloxacin were the most frequent phenotypes observed in our study. Similar results have been reported in other studies from the USA and Europe.17,18 In our study, the three most commonly encountered ribotypes were 001 (30.1%), followed by 014/020 (17.0%) and 078/126 (17.0%). Interestingly, ribotype 106 accounted for 9% of all
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Ribotype 001 (n ¼31)
Antimicrobial agent
Reigadas et al.
Table 3. In vitro activity of the antimicrobials tested against clinical isolates of C. difficile with reduced susceptibility to metronidazole (n¼6) and vancomycin (n¼4) Antimicrobial agent
Geometric mean MIC (mg/L)
MIC range (mg/L)
0.125–0.5 0.015–0.125 0.25– 4 4 –8 2 –256 0.03 to .256 32 0.06– 0.125
Isolates with high MICs of metronidazole surotomycin 0.45 fidaxomicin 0.08 metronidazole 4 vancomycin 0.79 clindamycin 25.40 rifaximin 1.25 moxifloxacin 20.16 tigecycline 0.10
0.25– 1 0.03– 0.25 4 0.5–8 2 –256 0.0019 to .256 8 –32 0.06– 0.125
isolates. Ribotype 106, commonly detected in the UK, was rarely found elsewhere;19 however, it has recently been found to be the fourth most common PCR ribotype encountered in the II national study performed in Spain.20 We found that surotomycin exhibited good activity against the different ribotypes. In a European multicentre study, PCR ribotype 001 had one of the highest percentages of resistance to three or more classes of antibiotics.21 In the present study, the most common ribotype was 001 (31.0%). We observed that 80.6% of ribotype 001 isolates were clindamycin resistant, 83.9% were moxifloxacin resistant and 87.1% were rifaximin resistant. Relatively low resistance rates (2.7%) to rifaximin have usually been described.22 The association of rifaximin resistance with ribotype 001 may be specific to our institution and may merely reflect clonal expansion; however, our results are in line with those from a European multicentre study in which 27 out of 32 (84.4%) PCR ribotype 001 strains were resistant to rifampicin, whose resistance correlates well with rifaximin.21,23,24 High resistance rates to rifaximin (83.8% – 95%) have been observed for ribotype 017,25,26 although in these studies ribotype 001 did not exhibit resistance to rifaximin. Also, resistance to rifamycins has been described in ribotype 027 strains.23,27 Fidaxomicin, surotomycin and tigecycline showed excellent activity against ribotype 001 isolates. Fidaxomicin exhibited the lowest MIC50/MIC90 for this ribotype compared with other frequently found PCR ribotypes 014/020 and 078/126, which is in line with the findings of a study performed on isolates collected for a pan-European survey in 2008.28 Also, surotomycin exhibited the highest MICs for ribotype 078/126. Vancomycin and metronidazole were 100% active against ribotype 001 strains [2013 collection (100 isolates)]. Surotomycin has proven to be active against strains with reduced susceptibility to metronidazole and vancomycin. We found that the most effective antimicrobial
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Acknowledgements Some of the results of this study were previously presented in poster form at the Fifty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2014 (Poster F-242). We thank Thomas O’Boyle for his help in the preparation of the manuscript (English Consultancy Service) and Jeanna Fisher for her assistance in the calcium supplementation procedure during susceptibility testing of surotomycin.
Funding This study was financed by Cubist Pharmaceuticals, the Rafael del Pino Foundation and Fondo de Investigaciones Sanitarias (FIS), Research Project number PI13/00687. E. R. holds a grant from the Rı´o Hortega programme of the Carlos III Health Institute, Spanish Government.
Transparency declarations None to declare.
References 1 Aslam S, Hamill RJ, Musher DM. Treatment of Clostridium difficileassociated disease: old therapies and new strategies. Lancet Infect Dis 2005; 5: 549–57. 2 McFarland LV, Surawicz CM, Rubin M et al. Recurrent Clostridium difficile disease: epidemiology and clinical characteristics. Infect Control Hosp Epidemiol 1999; 20: 43– 50. 3 Debast SB, Bauer MP, Kuijper EJ. European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin Microbiol Infect 2014; 20 Suppl 2: 1– 26. 4 Mascio CT, Mortin LI, Howland KT et al. In vitro and in vivo characterization of CB-183,315, a novel lipopeptide antibiotic for treatment of Clostridium difficile. Antimicrob Agents Chemother 2012; 56: 5023 –30. 5 Citron DM, Tyrrell KL, Merriam CV et al. In vitro activities of CB-183,315, vancomycin, and metronidazole against 556 strains of Clostridium difficile, 445 other intestinal anaerobes, and 56 Enterobacteriaceae species. Antimicrob Agents Chemother 2012; 56: 1613– 5. 6 Snydman DR, Jacobus NV, McDermott LA. Activity of a novel cyclic lipopeptide, CB-183,315, against resistant Clostridium difficile and other Gram-positive aerobic and anaerobic intestinal pathogens. Antimicrob Agents Chemother 2012; 56: 3448 –52.
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Isolates with high MICs of vancomycin surotomycin 0.25 fidaxomicin 0.04 metronidazole 0.71 vancomycin 5.65 clindamycin 64 rifaximin 44.80 moxifloxacin 32 tigecycline 0.09
agents against vancomycin-resistant isolates were fidaxomicin, tigecycline and surotomycin, which were also effective against isolates with reduced susceptibility to metronidazole. Tigecycline has previously demonstrated favourable in vitro activity against C. difficile isolates. Although some reports suggest that tigecycline could be a reasonable addition for treatment of CDI episodes refractory to standard therapy,29,30 more clinical data are needed. Our study is limited by the fact that we were not able to draw conclusions on the performance of surotomycin against ribotype 027 strains owing to the limited number of 027 strains in our centre. In conclusion, surotomycin exhibited potent in vitro activity against all the isolates tested, including those with elevated metronidazole and vancomycin MICs. The potential role of this agent in the treatment of CDI requires further clinical evaluation.
In vitro activity of surotomycin against CDI clinical isolates
7 Clinical and Laboratory Standards. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Eighth Edition: Approved Standard M11-A8. CLSI, Wayne, PA, USA, 2012. 8 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-fourth Informational Supplement M100-S24. CLSI, Wayne, PA, USA, 2014. 9 Stubbs SL, Brazier JS, O’Neill GL et al. 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 1999; 37: 461–3. 10 Cohen SH, Gerding DN, Johnson S et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol 2010; 31: 431–55.
12 Louie TJ, Miller MA, Mullane KM et al. Fidaxomicin versus vancomycin for Clostridium difficile infection. New Engl J Med 2011; 364: 422–31. 13 Babakhani F, Gomez A, Robert N et al. Killing kinetics of fidaxomicin and its major metabolite, OP-1118, against Clostridium difficile. J Med Microbiol 2011; 60: 1213– 7. 14 Chesnel L. Treatment of CDAD with oral CB-183 315: time to recurrence, relapse and re-infection rates compared with vancomycin. Clin Microbiol Infect 2012; 18: Suppl 3: 380. 15 Mascio CT, Chesnel L, Thorne G et al. Surotomycin demonstrates low in vitro frequency of resistance and rapid bactericidal activity in Clostridium difficile, Enterococcus faecalis and Enterococcus faecium. Antimicrob Agents Chemother 2014; 58: 3976 –82. 16 Sanders I, Harmanus C, Debast S et al. Antibiotic susceptibility of surotomycin and five other antibiotics against Clostridium difficile isolates, collected at a pan-European survey in 2008 (n ¼ 119). In: Abstracts of the Twenty-fourth European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, 2014, Abstract P0792. European Society of Clinical Microbiology and Infectious Diseases, Basel, Switzerland.
19 Freeman J, Bauer MP, Baines SD et al. The changing epidemiology of Clostridium difficile infections. Clin Microbiol Rev 2010; 23: 529–49. 20 Alcala´ L, Reigadas E, Marı´n M et al. Multicenter study of Clostridium difficile infection in Spain: an update of the underdiagnosis of Clostridium difficile infection in a whole nation. In: Abstracts of the Twenty-fourth European Congress of Clinical Microbiology and Infectious Diseases, 2014. Abstract P0741. European Society of Clinical Microbiology and Infectious Diseases, Barcelona, Spain. 21 Spigaglia P, Barbanti F, Mastrantonio P. Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother 2011; 66: 2227– 34. 22 Hecht DW, Galang MA, Sambol SP et al. In vitro activities of 15 antimicrobial agents against 110 toxigenic Clostridium difficile clinical isolates collected from 1983 to 2004. Antimicrob Agents Chemother 2007; 51: 2716–9. 23 O’Connor JR, Galang MA, Sambol SP et al. Rifampin and rifaximin resistance in clinical isolates of Clostridium difficile. Antimicrob Agents Chemother 2008; 52: 2813– 7. 24 Miller MA, Blanchette R, Spigaglia P et al. Divergent rifamycin susceptibilities of Clostridium difficile strains in Canada and Italy and predictive accuracy of rifampin Etest for rifamycin resistance. J Clin Microbiol 2011; 49: 4319– 21. 25 Huang H, Weintraub A, Fang H et al. Antimicrobial susceptibility and heteroresistance in Chinese Clostridium difficile strains. Anaerobe 2010; 16: 633–5. 26 Kim J, Kang JO, Pai H et al. Association between PCR ribotypes and antimicrobial susceptibility among Clostridium difficile isolates from healthcare-associated infections in South Korea. Int J Antimicrob Agents 2012; 40: 24– 9. 27 Curry SR, Marsh JW, Shutt KA et al. High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis 2009; 48: 425–9. 28 Debast SB, Bauer MP, Sanders IM et al. Antimicrobial activity of LFF571 and three treatment agents against Clostridium difficile isolates collected for a pan-European survey in 2008: clinical and therapeutic implications. J Antimicrob Chemother 2013; 68: 1305 –11.
17 Tickler IA, Goering RV, Whitmore JD et al. Strain types and antimicrobial resistance patterns of Clostridium difficile isolates from the United States: 2011– 2013. Antimicrob Agents Chemother 2014; 58: 4214 –8.
29 Herpers BL, Vlaminckx B, Burkhardt O et al. Intravenous tigecycline as adjunctive or alternative therapy for severe refractory Clostridium difficile infection. Clin Infect Dis 2009; 48: 1732– 5.
18 Alcala L, Martin A, Marin M et al. The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem? Clin Microbiol Infect 2012; 18: E204–13.
30 Lu CL, Liu CY, Liao CH et al. Severe and refractory Clostridium difficile infection successfully treated with tigecycline and metronidazole. Int J Antimicrob Agents 2010; 35: 311–2.
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11 Cornely OA, Crook DW, Esposito R et al. Fidaxomicin versus vancomycin for infection with Clostridium difficile in Europe, Canada, and the USA: a double-blind, non-inferiority, randomised controlled trial. Lancet Infect Dis 2012; 12: 281–9.
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