In vitro activity of surotomycin against contemporary clinical isolates of toxigenic Clostridium difficile strains obtained in Spain

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. Reigadas 1 3 *, L. Alcalá 1,3,4, M. Marín 1 4, T. Pelaéz 1 4, A. Martin 1,3, C. Iglesias 1,4 and E. Bouza 1 4 1 Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 2 Medicine Department, School of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain; 3 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; 4 CIBER de Enfermedades Respiratorias (CIBERES CB06/06/0058), Madrid, Spain *Corresponding author. Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, C/Dr. Esquerdo, 46, 28007 Madrid, Spain. Tel: +34-91-586-84-53; Fax: +34-91-504-49-06; E-mail: helenrei@hotmail.com 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 MIC 50/90 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 (GM¼0.25 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 (GM¼0.22 mg/l); the highest MICs were for ribotype 078/126 (GM¼0.72 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 patients 1 ) 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: journals.permissions@oup.com 2311

Reigadas et al. which encompasses 715000 people. During 2013, the incidence of CDI episodes at Hospital Gregorio Marañón was 12.2/10000 days of stay. Bacterial isolates We collected 100 C. difficile isolates from the clinical samples of CDI patients received at the microbiology laboratory of Hospital General Universitario Gregorio Marañón 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 (n¼4) and metronidazole (n¼6) were recovered from samples (2007 13) received at the microbiology laboratory of Hospital General Universitario Gregorio Marañón. For this study, the strains were retested using the agar dilution method. Antimicrobial susceptibility testing 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 SigmaChemicalCompany(StLouis,MO,USA).Qualitycontrolstrains 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 Ca 2+. 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. 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 Marañón. Results MIC ranges, geometric means (GMs) and minimum concentrations inhibiting 50% (MIC 50 ) and 90% (MIC 90 ) 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 Table 1. In vitro activity of the antimicrobials tested against 100 clinical isolates of C. difficile Antimicrobial agent Percentages of isolates tested 35 30 25 20 15 10 5 0 MIC range (mg/l) 001 014/020 078/126 106 046 207 070 050 018 002 Other Ribotypes MIC 50/90 (mg/l) Geometric mean (mg/l) Surotomycin 0.06 2 0.25/0.5 0.31 Fidaxomicin 0.015 0.25 0.06/0.125 0.06 Metronidazole 0.06 1 0.25/0.5 0.24 Vancomycin 0.125 2 0.5/1 0.48 Clindamycin 0.125 to.256 4/256 11.71 Rifaximin 0.0019 to.256 0.015/.256 0.20 Moxifloxacin 0.125 32 4/32 4.50 Tigecycline 0.03 0.50 0.125/0.25 0.09 Figure 1. Distribution of ribotypes of 100 clinical isolates of C. difficile isolated in Madrid, Spain in 2013. that presented a lower MIC 90 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 oftheribotypesisshownin Figure 1. The analysis of antimicrobial susceptibility according tothemainribotypesisshownintable2. Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM¼0.22 mg/l) and ribotype 078/126 exhibited the highest surotomycin MICs (GM¼0.72 mg/l). Ribotype 078/126 strains also exhibited higher MICs of fidaxomicin and tigecycline. The selected strains with known high MICs of vancomycin (n¼4) and metronidazole (n¼6) had been originally tested by Etest, with an MIC 50 /MIC 90 of metronidazole of 1.5/8 mg/l and an MIC 50 /MIC 90 of vancomycin of 3/4 mg/l. When retesting these strains by agar dilution for this study, the MIC 50 /MIC 90 of metronidazole was 4/4 mg/l and the MIC 50 /MIC 90 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. 2312

In vitro activity of surotomycin against CDI clinical isolates JAC 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 Antimicrobial agent Ribotype 001 (n¼31) Ribotype 014/020 (n¼17) Ribotype 078/126 (n¼17) MIC range (mg/l) surotomycin 0.06 0.5 0.03 0.5 0.25 2 metronidazole 0.125 0.5 0.06 0.5 0.125 0.5 vancomycin 0.125 2 0.25 1 0.125 1 moxifloxacin 0.5 32 1 8 1 32 tigecycline 0.03 0.25 0.03 0.25 0.06 0.5 fidaxomicin 0.015 0.125 0.015 0.125 0.015 0.25 clindamycin 0.5 to.256 1 16 2 to.256 rifaximin 0.0039 to.256 0.0019 0.03 0.0039 to.256 MIC 50/90 (mg/l) surotomycin 0.25/0.5 0.25/0.5 1/1 metronidazole 0.25/0.5 0.25/0.5 0.25/0.5 vancomycin 0.5/1 0.5/1 0.5/1 moxifloxacin 16/32 2/8 8/32 tigecycline 0.06/0.125 0.125/0.25 0.125/0.5 fidaxomicin 0.03/0.06 0.06/0.125 0.125/0.25 clindamycin 128/256 4/8 8/.256 rifaximin.256/.256 0.0078/0.015 0.015/0.03 Geometric mean MIC (mg/l) surotomycin 0.25 0.22 0.72 metronidazole 0.26 0.25 0.25 vancomycin 0.39 0.54 0.52 moxifloxacin 11.44 2.26 5.11 tigecycline 0.07 0.12 0.15 fidaxomicin 0.03 0.06 0.11 clindamycin 62.58 4.52 26.10 rifaximin 119.52 0.01 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, MIC 50 and MIC 90 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 MIC 50 and MIC 90 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 2313

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) Isolates with high MICs of vancomycin surotomycin 0.25 0.125 0.5 fidaxomicin 0.04 0.015 0.125 metronidazole 0.71 0.25 4 vancomycin 5.65 4 8 clindamycin 64 2 256 rifaximin 44.80 0.03 to.256 moxifloxacin 32 32 tigecycline 0.09 0.06 0.125 Isolates with high MICs of metronidazole surotomycin 0.45 0.25 1 fidaxomicin 0.08 0.03 0.25 metronidazole 4 4 vancomycin 0.79 0.5 8 clindamycin 25.40 2 256 rifaximin 1.25 0.0019 to.256 moxifloxacin 20.16 8 32 tigecycline 0.10 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 MIC 50 /MIC 90 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 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. 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 Río Hortega programme of the Carlos III Health Institute, Spanish Government. Transparency declarations None to declare. References 1 AslamS,HamillRJ,MusherDM.TreatmentofClostridium 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. 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