We determined the in vitro activity of SMT19969 and 11 comparators, including metronidazole, vancomycin and fidaxomicin, against 107 C. difficile isolates of different antimicrobial resistance phenotypes. Fidaxomicin and SMT19969 were most active. Geometric mean fidaxomicin and SMT19969 MICs were highest in ribotypes known to show multiple resistance. Co-resistance to linezolid and moxifloxacin was evident in ribotypes 001, 017, 027 and 356. High-level ceftriaxone resistance in ribotypes 356 and 018 was location-linked

Freeman, J

Vernon, J

Vickers, R

Wilcox, MH

PubMed

White Rose Research Online

Susceptibility of Clostridium difficile Isolates of Varying AntimicrobialResistance Phenotypes to SMT19969 and 11 ComparatorsJane Freeman,a,b Jonathan Vernon,b Richard Vickers,c Mark H. Wilcoxa,bMicrobiology, Leeds Teaching Hospitals Trust,a and Healthcare Associated Infections Research Group, Leeds Institute for Biomedical and Clinical Sciences,b University ofLeeds, Leeds, United Kingdom; Summit Therapeutics, Abingdon, Oxfordshire, United KingdomcWe determined the in vitro activity of SMT19969 and 11 comparators, including metronidazole, vancomycin, and fidaxomicin,against 107 C. difficile isolates of different antimicrobial resistance phenotypes. Fidaxomicin and SMT19969 were the most ac-tive. The fidaxomicin and SMT19969 geometric mean MICs were highest in ribotypes known to show multiple resistance. Core-sistance to linezolid and moxifloxacin was evident in ribotypes 001, 017, 027, and 356. The high-level ceftriaxone resistance inribotypes 356 and 018 was location linked.Clostridium difficile infection (CDI) is a significant cause of nos-ocomial diarrhea and a continuing burden on health care re-sources (1). Most antimicrobials have been associated with CDIcases at some point, including the treatment agents vancomycinand metronidazole (Clostridium difficile Ribotyping Network[CDRN] for England and Northern Ireland 2011-2013; https://www.gov.uk/government/publications/clostridium-difficile-ribotyping-network-cdrn-report). CDI recurrence is commonfollowing conventional antimicrobial treatment and is associatedwith reduced gut bacterial diversity (2). Treatment options re-main limited, despite the introduction of fidaxomicin for CDI;therefore, development of novel antimicrobial agents, particularlyones with a lower potential for gut microbiota depletion, isneeded. SMT19969 is a novel antimicrobial with potent in vitroactivity against C. difficile (3) but limited activity against gut mi-croflora (4). We investigated the activity of SMT19969 and 11comparators, including predisposing and treatment agents,against C. difficile isolates of different antimicrobial resistancephenotypes.A panel of 107 C. difficile isolates was selected from a collectionassembled during the ClosER study (5) by permission of AstellasPharma Europe. Clinical isolates were collected between July 2011and April 2013 and were selected to maximize the diversity ofantimicrobial resistance phenotypes. The susceptibilities of C. dif-ficile isolates to metronidazole, vancomycin, fidaxomicin, rifam-pin, moxifloxacin, clindamycin, imipenem, chloramphenicol,tigecycline, SMT19969, linezolid, and ceftriaxone were deter-mined using a Wilkins-Chalgren agar incorporation method (5,6). The MIC was defined as the lowest dilution at which growthwas completely inhibited or at which only single colonies re-mained.The MIC results for each isolate were designated susceptible(S), intermediately resistant (I), fully resistant (R), or reducedsusceptibility (RS) according to the breakpoints defined in Table1. The breakpoints were established according to the Clinical Lab-oratory Standards Institute (CLSI), the European Committee onAntimicrobial Susceptibility Testing (EUCAST), or publisheddata. Each result was assigned a score (S  0, I  1, and R  2). Acumulative resistance score (CRS), based on susceptibility to eachof the 11 antimicrobials tested, was generated for each isolate.Thus, an isolate that was fully susceptible to 6, intermediatelyresistant to 2, and resistant to 3 antimicrobials would generate ascore of 8 (0  0  0  0 0  0  1  1  2  2  2).Fidaxomicin was the most active agent, followed bySMT19969, with similar geometric mean (GM) MICs (0.04 mg/liter versus 0.07 mg/liter, respectively) (Table 1) and with no evi-dence of resistance to either agent (Table 1). Fidaxomicin (GMMIC of 0.04 mg/liter) was 10- and 20-fold more active than met-ronidazole (GM MIC of 0.41 mg/liter) and vancomycin (GM MICof 0.80 mg/liter), while SMT19969 (GM MIC of 0.07 mg/liter) was6- and 11-fold more active, respectively. The MICs of both fidax-omicin and SMT19969 were comparable to those observed previ-ously (3, 5, 7, 8). Although the fidaxomicin MICs were slightlyhigher among the highly related ribotype (RT) 027 (n  22) andRT198 (n  8) isolates (GM MIC of 0.08 mg/liter for both) thanfor all isolates (0.04 mg/liter), this was not statistically significant(Kruskal-Wallis P  0.86 and 1.00, respectively). Conversely, thefidaxomicin MICs were statistically significantly lower amongRT001 isolates (Kruskal-Wallis P  0.0001), with a GM MIC of0.01 mg/liter, reflecting previous results (5, 7, 8). The SMT19969MICs for RT027 (GM  0.11 mg/liter) and RT017 (GM  0.12mg/liter) isolates were slightly elevated above those for all isolates,but this was not statistically significant (Kruskal-Wallis P  0.30and 0.29, respectively). Ribotypes 027, 198, and 017 were associ-ated with multiple antimicrobial resistance in a previous study (5).The slightly elevated fidaxomicin and SMT19969 GM MICs ob-served against selected ribotypes are unlikely to have clinical sig-nificance, given the high intraluminal gastrointestinal (GI) con-centrations of both agents (9, 10). The GM metronidazole MICswere also slightly higher among RT027 and RT198 (1 mg/liter forboth) isolates than those for all isolates (0.4 mg/liter), in line withReceived 19 August 2015 Returned for modification 23 September 2015Accepted 4 November 2015Accepted manuscript posted online 9 November 2015Citation Freeman J, Vernon J, Vickers R, Wilcox MH. 2016. Susceptibility ofClostridium difficile isolates of varying antimicrobial resistance phenotypes toSMT19969 and 11 comparators. Antimicrob Agents Chemother 60:689 – 692.doi:10.1128/AAC.02000-15.Address correspondence to Mark H. Wilcox, mark.wilcox@nhs.net.Copyright © 2015 Freeman et al. This is an open-access article distributed underthe terms of the Creative Commons Attribution 4.0 Unported license.crossmarkJanuary 2016 Volume 60 Number 1 aac.asm.org 689Antimicrobial Agents and Chemotherapy on April 19, 2016 by UNIVERSITY OF LEEDShttp://aac.asm.org/Downloaded from previous observations (5, 8). However, despite low gut concentra-tions, metronidazole treatment failure has not been linked to de-creased susceptibility to this agent (8).There was a significant correlation between increased CRS andincreased SMT19969 MICs (Pearson’s product-moment correla-tion r  0.33; P  0.004), metronidazole MICs (r  0.27; P 0.004), and, to a lesser degree, fidaxomicin MICs (r  0.25; p 0.01), but no such correlation for vancomycin (r  0.12; P  0.21). Acomparison of susceptibilities by ribotype in this study would in-evitably contain bias, given that the selection criteria were basedon the resistance phenotypes; however, it is worth noting that theC. difficile isolates with the highest MICs of SMT19969 belongedto ribotypes noted for resistance to multiple antimicrobials.Isolates were selected to represent a broad range of antimicro-bial resistance phenotypes. The results for metronidazole, vanco-mycin, fidaxomicin, rifampin, moxifloxacin, clindamycin, chlor-amphenicol, and tigecycline largely reflected those previouslydetermined (5), with evidence of high-level resistance to rifampin,moxifloxacin, clindamycin, and chloramphenicol (Table 1). Imi-penem resistance was low (3.7%), and reduced susceptibility totigecycline was very rare (1%). Most isolates were linezolid sus-ceptible (85.3%), but, unexpectedly, 13.8% showed high-level re-sistance (16 mg/liter). These isolates belonged to RT001 (7 of22), RT017 (2 of 7), RT027 (4 of 22), and RT356 (2 of 4). Therewas location clustering in RT001, with 3 linezolid-resistant iso-lates from the same geographical location. These isolates alsoshowed resistance to clindamycin and in some cases chloram-phenicol (Table 2). A recent publication also described linezolidresistance among RT001, RT078, RT126, and RT017 isolates fromSpain (11). The authors demonstrated the presence of the multi-drug resistance gene, cfr, in isolates showing high-level resistanceto chloramphenicol, erythromycin, clindamycin, and linezolidfrom RT017, RT078, and RT126. This was linked to a mobilegenetic element, Tn6218, indicating the possibility of transmis-sion between strains. They were unable to demonstrate the pres-ence of cfr in the remaining RT001 isolates that showed lowerchloramphenicol MICs, suggesting that other resistance mecha-nisms are involved (11). There is likely to be more than one etiol-ogy for the linezolid resistance seen in the isolates tested here,given the phenotypes displayed. It is interesting to note that all ofthe linezolid-resistant isolates also displayed moxifloxacin resis-tance. This combination was associated with higher cadazolidMICs (2- to 4-fold higher than those of susceptible isolates withresistance to either moxifloxacin or linezolid) (12) and may not beunexpected since cadazolid is an oxazolidinone-fluoroquinolonehybrid molecule. However, its clinical significance is unknown,given the high fecal cadazolid concentrations achieved (13).Only 6% of isolates were susceptible to ceftriaxone (GM MICof 58.2 mg/liter) according to the breakpoints used (14). The high-est levels of resistance (128 mg/liter) were seen in RT356 isolates(all) and in 5 of 10 RT018 isolates, which are closely related. Ri-botype 356 is exclusive to Italy, and all 5 RT018 isolates showingMICs of 128 mg/liter were also from this location. This high-level ceftriaxone resistance adds to the previously reported multi-drug resistance in RT018 and RT356 isolates from Italy (5). Two ofthe RT356 isolates also showed intermediate imipenem resistance(Table 2).In summary, SMT19969 was highly active against more than100 isolates displaying different antimicrobial resistance pheno-types. There was no evidence of SMT19969 or fidaxomicin resis-TABLE1Susceptibilityof107C.difficileisolatestoSMT19969and11comparatorsParameterResultsfordrug(breakpoint):SMT19969Fidaxomicin(S1;RS15)Metronidazole(S2;I4;R85)Vancomycin(S2;I4;R85)Rifampin(S0.002;I0.003–16;R325)Moxifloxacin(S2;I4;R85)Clindamycin(S2;I4;R85)Imipenem(S4;I8;R165)Chloramphenicol(S8;I16;R325)Tigecycline(S0.25;RS0.255)Linezolid(S4;R411)Cetriaxone(S16;I32;R6414)Geometricmean0.070.040.410.800.039.8119.004.005.970.045.1958.18MIC500.030.060.510.0021616440.03464MIC900.1250.1252232321288160.0616128Range0.015–0.50.004–0.1250.125–20.5–80.001321–1282–1281–1642560.03–0.252–3216256%S100.00100.00100.0070.6431.197.3473.3984.4099.0785.326.42%I0.000.000.002.750.926.4222.9411.010.930.9233.03%R0.000.000.0026.6167.8986.243.674.590.0013.7660.55Freeman et al.690 aac.asm.org January 2016 Volume 60 Number 1Antimicrobial Agents and Chemotherapy on April 19, 2016 by UNIVERSITY OF LEEDShttp://aac.asm.org/Downloaded from tance, but some evidence of modestly higher SMT19969 andfidaxomicin MICs among ribotypes previously noted for multipleantimicrobial resistance. Linezolid resistance was more prevalentthan expected and was also associated with ribotypes noted formultidrug resistance phenotypes. High-level ceftriaxone resis-tance was found in multiresistant RT018 and RT356 isolates fromItaly.ACKNOWLEDGMENTSWe are grateful to Chris Longshaw and Astellas Pharma Europe for per-mission to use C. difficile isolates collected as part of The ClosER Study.R.V. is an employee of Summit plc and was involved in study designand manuscript preparation. J.F. has received research funding fromSummit plc and Astellas Pharma Europe. M.H.W. has received researchfunding/consultancy/honoraria from Abbott, Actelion, Astellas, Astra-Zeneca, Bayer, Cerexa, Subist, Durata, The European Tissue Symposium,The Medicines Company, MedImmune, Merck, Motif Biosciences,Nabriva, Optimer, Paratek, Pfizer, Roche, Sanofi-Pasteur, Seres, Summit,Synthetics Biologics, bioMérieux, and Da Volterra. J.V. declares no con-flicts of interest.FUNDING INFORMATIONSUMMIT plc provided funding to Jane Freeman and Mark Wilcox undergrant number MB14/11296.Richard Vickers is an employee of Summit plc and was involved in studydesign and manuscript preparation.REFERENCES1. Wiegand PN, Nathwani D, Wilcox MH, Stephens J, Shelbaya A, HaiderS. 2012. Clinical and economic burden of Clostridium difficile infection inEurope: a systematic review of healthcare-facility-acquired infection. JHosp Infect 81:1–14. http://dx.doi.org/10.1016/j.jhin.2012.02.004.2. Chang JY, Antonopoulos DA, Kalra A, Tonelli A, Khalife WT, SchmidtTM, Young VB. 2008. Decreased diversity of the fecal microbiome inrecurrent Clostridium difficile-associated diarrhea. J Infect Dis 197:435–438. http://dx.doi.org/10.1086/525047.3. Corbett D, Wise A, Birchall S, Warn P, Baines SD, Crowther G,Freeman J, Chilton CH, Vernon J, Wilcox MH, Vickers RJ. 2015. Invitro susceptibility of Clostridium difficile to SMT19969 and comparators,as well as the killing kinetics and post-antibiotic effects of SMT19969 andcomparators against C. difficile. J Antimicrob Chemother 70:1751–1756.http://dx.doi.org/10.1093/jac/dkv006.4. Goldstein EJ, Citron DM, Tyrrekk KL, Merriam CV. 2013. Comparativein vitro activities of SMT19969, a new antimicrobial agent, against Clos-tridium difficile and 350 Gram-positive and Gram negative aerobic andanaerobic intestinal flora isolates. Antimicrob Agents Chemother 57:4872– 4876. http://dx.doi.org/10.1128/AAC.01136-13.5. Freeman J, Vernon J, Morris K, Nicholson S, Todhunter S, LongshawC, Wilcox MH. 2015. Pan-European longitudinal surveillance of antimi-crobial resistance among prevalent Clostridium difficile ribotypes. ClinMicrobiol Infect 21:248.e9-248.e16. http://dx.doi.org/10.1016/j.cmi.2014.09.017.6. Baines SD, O’Connor R, Freeman J, Fawley WN, Harmanus C, Mas-trantonio P, Kuijper EJ, Wilcox MH. 2008. Emergence of reduced sus-ceptibility to metronidazole in Clostridium difficile. J Antimicrob Che-mother 62:1046 –1052. http://dx.doi.org/10.1093/jac/dkn313.7. Debast SB, Bauer MP, Sanders IM, Wilcox MH, Kuijper EJ, ECDISStudy Group. 2013. Antimicrobial activity of LFF571 and three treatmentagents against Clostridium difficile isolates collected for a pan-Europeansurvey in 2008: clinical and therapeutic implications. J Antimicrob Che-mother 68:1305–1311. http://dx.doi.org/10.1093/jac/dkt013.8. Goldstein EJC, Citron DM, Sears P, Babakhani F, Sambol SP, GerdingDN. 2011. Comparative susceptibilities to fidaxomicin (OPT-80) of iso-lates collected at baseline, recurrence and failure from patients in twophase III trials of fidaxomicin against Clostridium difficile infection. Anti-microb Agents Chemother 55:51945199. http://dx.doi.org/10.1128/AAC.00625-11.TABLE2High-levelresistancetolinezolidamongPCRribotypes001,017,027,and356RibotypeMIC(mg/liter)SMT19969FidaxomicinMetronidazoleVancomycinRifampinMoxifloxacinClindamycinImipenemChloramphenicolTigecyclineLinezolidCeftriaxone0010.0601510.50.00232a128a2256a0.031664a0010.030.00810.50.00116a128a8b16b0.0332a128a0010.060.015120.00216a64a432a0.0332a128a0010.1250.0080.50.50.00216a128a416b0.0332a128a0010.1250.00810.50.00216a128a416b0.0332a128a0010.1250.00810.50.00216a128a416b0.0332a128a0010.060.00810.50.00216a128a216b0.0332a64a0170.1250.060.250.50.00132a128a416b0.0632a32b0170.250.060.1250.532a32a128a464a0.0632a64a0270.060.061132a16a8a8b40.0316a128a0270.250.061132a32a8a8b40.0316a64a0270.250.0610.532a32a128a464a0.0332a128a0270.250.12510.50.00232a128a464a0.0332a64a3560.030.061132a32a8a8b40.0316a256a3560.060.060.5232a32a16a8b80.0316a256aaIsolatethatisresistant.bIsolatethatisintermediate.C. difficile Susceptibility to SMT19969 and ComparatorsJanuary 2016 Volume 60 Number 1 aac.asm.org 691Antimicrobial Agents and Chemotherapy on April 19, 2016 by UNIVERSITY OF LEEDShttp://aac.asm.org/Downloaded from 9. Vickers R, Robinson N, Best E, Echols R, Tillotson G, Wilcox M.2015. A randomised phase 1 study to investigate safety, pharmacoki-netics and impact on gut microbiota following single and multiple oraldoses in healthy male subjects of SMT19969, a novel agent for Clostrid-ium difficile infections. BMC Infect Dis 15:91. http://dx.doi.org/10.1186/s12879-015-0759-5.10. Sears P, Crook DW, Louie TJ, Miller MA, Weiss K. 2012. Fidaxomicinattains high fecal concentrations with minimal plasma concentrations fol-lowing oral administration in patients with Clostridium difficile infection.Clin Infect Dis 55(Suppl 2):S116 –S120. http://dx.doi.org/10.1093/cid/cis337.11. Marín M, Martin A, Alcala L, Cercenado E, Iglesias C, Reigadas E,Bouza E. 2015. Clostridium difficile isolates with high linezolid MICs har-bour the multiresistance gene, cfr. Antimicrob Agents Chemother 59:586 –589. http://dx.doi.org/10.1128/AAC.04082-14.12. Locher HH, Seiler P, Chen X, Scroeder S, Pfaff P, Enderlin M, Klenk A,Fournier E, Hubschwerlen C, Ritz D, Kelly CP, Keck W. 2014. In vitroand in vivo antibacterial evaluation of cadazolid, a new antibiotic for treat-ment of Clostridium difficile infection. Antimicrob Agents Chemother 58:892–900. http://dx.doi.org/10.1128/AAC.01830-13.13. Mackie AE, Desnica B, Nicolas LB, Louie T, Dingemanse J. 2013.Cadazolid, a novel potent antibiotic: minimal systemic cadazolid exposurein subjects with Clostridium difficile associated diarrhea, abstr. A-008. Ab-str 53rd Intersci Conf Antimicrob Agents Chemother, Denver, CO.14. Büchler AC, Rampini SK, Stelling S, Ledergerber B, Peter S, SchweigerA, Ruef C, Sbinden R, Speck RF. 2014. Antibiotic susceptibility ofClostridium difficile is similar worldwide over two decades despite wide-spread use of brad-spectrum antibiotics: an analysis done at the UniversityHospital of Zurich. BMC Infect Dis 14:607. http://dx.doi.org/10.1186/s12879-014-0607-z.Freeman et al.692 aac.asm.org January 2016 Volume 60 Number 1Antimicrobial Agents and Chemotherapy on April 19, 2016 by UNIVERSITY OF LEEDShttp://aac.asm.org/Downloaded from 

Susceptibility of Clostridium difficile isolates of varying antimicrobial resistance phenotypes to SMT19969 and 11 comparators.

ABSTRACT
          
            We determined the
            in vitro
            activity of SMT19969 and 11 comparators, including metronidazole, vancomycin, and fidaxomicin, against 107
            C. difficile
            isolates of different antimicrobial resistance phenotypes. Fidaxomicin and SMT19969 were the most active. The fidaxomicin and SMT19969 geometric mean MICs were highest in ribotypes known to show multiple resistance. Coresistance to linezolid and moxifloxacin was evident in ribotypes 001, 017, 027, and 356. The high-level ceftriaxone resistance in ribotypes 356 and 018 was location linked.
          </jats:p

Jane Freeman

Jonathan Vernon

Richard Vickers

Mark H. Wilcox

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Antimicrobial Agents and Chemotherapy

Susceptibility of Clostridium difficile Isolates of Varying Antimicrobial Resistance Phenotypes to SMT19969 and 11 Comparators

Susceptibility of Clostridium difficile isolates of varying antimicrobial resistance phenotypes to SMT19969 and 11 comparators.

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