Treatments for Clostridium difficile infection remain limited, despite the introduction of fidaxomicin, and development of new agents is necessary. We determined the in vitro susceptibilities of 199 prevalent or emerging Clostridium difficile PCR ribotypes to MCB3681, a novel investigational quinolonyl-oxazolidinone, and 8 comparators (metronidazole, vancomycin, fidaxomicin, moxifloxacin, ciprofloxacin, clindamycin, tigecycline and linezolid). MCB3681 showed good activity against C. difficile with no evidence of MCB3681 resistance in isolates showing either or both moxifloxacin and linezolid resistance

Freeman, J

Pilling, S

Vernon, J

Wilcox, MH

Antimicrobial Agents and Chemotherapy

PubMed

White Rose Research Online

This is a repository copy of In vitro activities of MCB3681 and 8 comparators against Clostridium difficile isolates with known ribotypes and diverse geographical spread.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/111852/Version: Accepted VersionArticle:Freeman, J, Pilling, S, Vernon, J et al. (1 more author) (2017) In vitro activities of MCB3681 and 8 comparators against Clostridium difficile isolates with known ribotypes and diverse geographical spread. Antimicrobial Agents and Chemotherapy, 61 (3). e02077-16. ISSN 0066-4804 https://doi.org/10.1128/AAC.02077-16(c) 2016, Freeman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version - refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher’s website. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request.  1 In vitro activities of MCB3681 and 8 comparators against 2 Clostridium difficile isolates with known ribotypes and 3 diverse geographical spread 4  5 *J Freeman,1,2 S Pilling2, J Vernon2, MH Wilcox1,2 6  7  8 Microbiology, Leeds Teaching Hospitals Trust1 & Healthcare Associated Infections 9 Research Group,2 Leeds Institute for Biomedical and Clinical Sciences,University of 10 Leeds, Leeds, UK. 11  12  13 Running title: C. difficile susceptibility to MCB3681 and comparators 14  15  16 *Corresponding author: Dr Jane Freeman Jane.freeman4@nhs.net  17  18  19  20  21  22 Abstract 23 Treatments for Clostridium difficile infection remain limited, despite the introduction 24 of fidaxomicin, and development of new agents is necessary.  25 We determined the in vitro susceptibilities of 199 prevalent or emerging Clostridium 26 difficile PCR ribotypes to MCB3681, a novel investigational quinolonyl-oxazolidinone, 27 and 8 comparators (metronidazole, vancomycin, fidaxomicin, moxifloxacin, 28 ciprofloxacin, clindamycin, tigecycline and linezolid). 29 MCB3681 showed good activity against C. difficile with no evidence of MCB3681 30 resistance in isolates showing either or both moxifloxacin and linezolid resistance.  31  32 C. difficile infection (CDI) is a major burden on healthcare resources. CDI is thought 33 to arise following the depletion of gut microflora by antimicrobial action, allowing the 34 organism to proliferate and cause disease. Antimicrobial treatments for CDI are 35 currently limited to metronidazole, vancomycin and fidaxomicin. Metronidazole has 36 more recently been associated with treatment failures, while promotion of 37 glycopeptide resistance within the host microflora is a risk associated with 38 vancomycin therapy. 1 Symptomatic recurrence is common following treatment with 39 these agents, 2 requiring further episodes of antimicrobial therapy. Further treatment 40 options are highly desirable to broaden the range of therapeutic choice and 41 strengthen antimicrobial stewardship.  42 MCB3681 is a novel small molecule with structural elements of an oxazolidinone and 43 a quinolone showing good activity against C. difficile, including isolates that were 44 resistant to linezolid, ciprofloxacin, moxifloxacin and clindamycin. 3 It achieves high 45 faecal concentrations after intravenous infusions and has shown activity against 46 Gram positive components of the gut microflora in a clinical Phase 1 study 4. The 47 development of an intravenous treatment agent achieving high faecal concentrations 48 would circumvent issues of rapid gut transit, or impaired delivery of orally 49 administered agents due to ileus, particularly in patients with severe or 50 protracted/multiple recurrent diarrhoeal episodes. 51 We determined the in vitro activities of MCB3681 and 8 comparators (metronidazole, 52 vancomycin, moxifloxacin, ciprofloxacin, clindamycin, tigecycline, linezolid, and 53 fidaxomicin) against a panel of 200 Clostridium difficile isolates of known PCR 54 ribotypes (RT) from 21 European countries (selected from the ClosER study - July 55 2011-April 2013, by kind permission of Astellas Pharma Europe). 5  56  57 In vitro susceptibility testing was performed using a Wilkins-Chalgren agar 58 incorporation method, as previously described.5,7 Briefly, C. difficile test isolates and 59 control strains (C. difficile ATCC 750057, C. difficile E4 PCR ribotype 010, 60 Bacteroides fragilis ATCC 25285, Enterococcus faecalis ATCC 29212 and 61 Staphlyococcus aureus ATCC 29213 were cultured anaerobically at 37oC for 24h in 62 Schaedler’s anaerobic broths prior to dilution to 0.5 McFarland standard 63 equivalence) in pre-reduced sterile saline and inoculation onto antibiotic-containing 64 and control Wilkins-Chalgren agar plates. Inoculated plates were incubated 65 anaerobically at 37oC for 48h.  66  67 MCB3681 is a quinolonyl- oxazolidinone antibacterial which has previously 68 demonstrated good activity against C. difficile. 3 All the CDI treatment agents, 69 including MCB3681 showed good activity against the isolates tested (Table 1). 70 Fidaxomicin was the most active treatment agent (Kruskal-Wallis p<0.0001; 71 geometric mean (GM) MIC=0.05 mg/L), followed by MCB3681 (p<0.0001; GM=0.12 72 mg/L), then metronidazole (p<0.0001; GM=0.33 mg/L), with no evidence of 73 resistance to any of these compounds (Table 1). Vancomycin was the least active 74 (p<0.0001; GM=1.02 mg/L), but resistance was very scarce (1.5%; 75 breakpoint>8mg/L). Reduced metronidazole susceptibility (4 mg/L) was observed in 76 only 1% of isolates. GM metronidazole MICs were elevated in RT027 (0.96 mg/L) 77 and RT106 (0.74 mg/L) vs GM metronidazole MICs for all isolates tested (0.33 78 mg/L), in agreement with previous data.4  79  80 All isolates were resistant to ciprofloxacin according to the breakpoints defined 81 (Table 1), and 48% of isolates showed moxifloxacin resistance, including at least one 82 isolate in each RT group tested. Highly elevated MICs to both moxifloxacin (>32 83 mg/L) and ciprofloxacin (>128 mg/L) were prevalent in RT001, RT027 and RT356. 84 Clindamycin MICs were highest in RT001, RT017 and RT126 (GM MICs = 61.11 85 mg/L; 64 mg/L and 38.05 mg/L, respectively), but there was evidence of clindamycin 86 resistance in all RTs tested (Table 1). There was no evidence of tigecycline 87 resistance (range=0.03-0.125mg/L; GM=0.05mg/L), in agreement with previous data 88 (Table 1).4 The majority of isolates (78.9%) were sensitive to linezolid (Table 1), with 89 a GM MIC of 5.16 mg/L. RT001 and RT017 showed the highest GM linezolid MICs 90 (10.08 mg/L and 7.03 mg/L, respectively). This is also in agreement with previous 91 observations.7 Three RT017, and two RT027 isolates showed dual quinolone-92 oxzolidinone resistant phenotype, and showed MCB3681 MICs of 0.5 mg/L. We 93 have previously reported that these isolates showed high level resistance to 94 chloramphenicol (Table 2).5,8 Marin et al. reported linezolid, chloramphenicol, 95 erythromycin, and clindamycin resistance associated with the presence of the 96 multidrug resistance gene, cfr, in C. difficile RT017, RT078 and RT126 isolates.11 97 The MIC50 and MIC90 values reported here for MCB3681 are similar to those recently 98 described for cadazolid, another quinolonyl-oxazolidinone molecule.9 A previous 99 study investigating susceptibility of C. difficile to cadazolid and comparators, reported 100 an association between resistance to either moxifloxacin or linezolid and 101 moxifloxacin/linezolid double-resistant mutants, and 2- or 4-fold higher cadazolid 102 MICs in mono- or double-resistant isolates, respectively. 10 However, the highest 103 MCB3681 MIC was 0.5 mg/L, and we also found isolates with moxifloxacin, 104 ciprofloxacin, linezolid and chloramphenicol resistance that demonstrated very low 105 MCB3681 MICs (0.008 mg/L) (Table 2). We did not investigate the molecular basis 106 of resistance in these isolates, but the results do not suggest a link between this 107 phenotype and MCB3681 MICs. The results shown here, in conjunction with those 108 previously reported8, 11 would also indicate that other modes of resistance to linezolid 109 (23s rRNA alterations, ribosomal protein modifications) may be at play in 110 combination with quinolone resistance mechanisms. 111  112 Rashid et al. reported MICs of MCB3681 for C. difficile ranged from 0.008-0.5 mg/L,3 113 which were similar to our results (range 0.008-0.5 mg/L). However, in the present 114 study, MIC50 and MIC90 values were 0.125 and 0.25 mg/L, respectively, which were 115 marginally higher than those reported previously, but within 2 doubling dilutions (0.03 116 and 0.06 mg/L, respectively). This may be explained by methodological/agar or C. 117 difficile strain distribution differences. The influence of testing media and 118 components therein on MICs has previously been reported and may have been a 119 factor in the differences observed.7,12 We used a Wilkins-Chalgren agar incorporation 120 method to determine MICs, since is superior to CLSI-recommended Brucella blood 121 agar (BBA) in the detection of reduced susceptibility to metronidazole in C. difficile.7  122  123 This study builds on this previous data of Rashid et al by substantially expanding the 124 diversity of ribotypes examined to include, in particular, RT027 and  several RTs 125 already noted for resistance to multiple antimicrobials: RT001, RT017, RT018, 126 RT027 and RT356.,5,8 There was no evidence of MCB3681 resistance among them. 127 MCB3681 achieves faecal concentrations of 99-226mg/kg after intravenous 128 infusions, far in excess of MIC ranges for C. difficile reported here. MCB3681 has 129 been reported to be active against Gram-positive gut microflora bacteria, but sparing 130 of Gram-negative organisms in human volunteer studies with intravenous 131 administration over 5 days. Further data are needed to assess the impact of 132 MCB3681 on C. difficile and the gut microflora over a longer duration.  133  134 In summary, MCB3681 showed good activity against C. difficile isolates from 135 emerging or prevalent European PCR ribotypes with no evidence of resistance. The 136 presence of quinolone and/or linezolid resistance did not influence MCB3681 MICs. 137  138 Funding 139 This research was funded by Morphochem AG, Munich, Germany 140  141 Acknowledgements 142 We are grateful to Dr Chris Longshaw and Astellas Pharma Europe for kind 143 permission to use C. difficile isolates collected during The ClosER study. 144  145 Transparency Declaration 146 JF has grant/research funding outside this work from Astellas and Melinta 147 Therapeutics. 148 MHW has received grant/research funding outside this work from Abbott, Actelion, 149 Alere, Astellas, Biomerieux, Cerexa, Cubist, Da Volterra, European Tissue 150 Symposium, Merck, Sanofi-Pasteur, Summit, The Medicines Company and Qiagen 151 and consultancies and/or lecture honoraria from Actelion, Alere, Astellas, Astra-152 Zeneca, Basilea, Bayer, Cubist, Durata, European Tissue Symposium, J&J, Merck, 153 Nabriva, Novacta, Novartis, Optimer, Pfizer, Roche, Sanofi-Pasteur and Seres and 154 has been a member of a speaker’s bureau for Pfizer. 155 JV and SP have nothing to declare. 156  157  158 References 159  160  161 1. Al-Nassir WN, Sethi AK, Li Y, Peltz MJ, Riggs MM, Sonskey CJ. 2008. Both oral 162 metronidazole and oral vancomycin promote persistent overgrowth of vancomycin-resistant 163 enterococci during treatment of Clostridium difficile-associated disease. Antimicrob Agents 164 Chemother. 52 (7): 2403-6 165 2. Debast SB, Bauer MP, Kuijper EJ. European Society of Clinical Microbiology and 166 Infectious Diseases. 2014. European Society of Clinical Microbiology and Infectious 167 Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin 168 Microbiol Infect.; 20: suppl 2: 1-26 169 3. Rashid M-U, Dalhoff A, Weintraub A, Nord CE. 2014. In vitro activity of MCB3681 against 170 Clostridium difficile strains. Anaerobe;  28: 216-219  171 4. Dalhoff A, Rashid M-U, Kapsner T,Panagiotidis G, Weintraub A, Nord CE. 2015. Analysis 172 of effects of MCB3681, the antibacterially active substance of prodrug MCB3837, on human 173 resident microflora as proof of principle. Clin Microbiol Infect: 21: 767.e1-767.e4 174 5. Freeman J, Vernon J, Morris K, Nicholson S, Todhunter S, Lonshaw C, Wilcox MH. 2015 175 Pan-European longitudinal surveillance of antimicrobial resistance among prevalent 176 Clostridium difficile ribotypes. Clinical Microbiol and Infect; 21(3):248.e9-248.e16 177 6. Clinical Laboratory Standards Institute. 2014 Principles and Procedures for Detection of 178 Anaerobes in Clinical Specimens; Approved Guideline M56-A. Wayne, PA, USA,  179 7. Baines SD, O’Connor R, Freeman J, Fawley WN, Harmanus C, Mastrantonio P, Kuijper 180 EJ, Wilcox MH.2008. Emergence of reduced susceptibility to metronidazole in Clostridium 181 difficile. J Antimicrob Chemother: 62:1046-1052. 182 8. Freeman J, Vernon JJ, Vickers R, Wilcox MH. 2015. Susceptibility of Clostridium difficile 183 isolates of varying antimicrobial resistance phenotypes to SMT19969 and 11 comparators. 184 Antimicrob Agents Chemother: 60 (1): 689-92 185 9. Gerding DN, Hecht DW, Louie T, Nord CE, Talbot GH, Cornely OA, Buitrago M, Best E, 186 Sambol S, Osmolski JR, Kracker H, Locher HH, Charef P, Wilcox M. 2016 Susceptibility 187 of Clostridium difficile isolates from a Phase 2 clinical trial of cadazolid and vancomycin in C. 188 difficile infection. J. Antimicrob Chemother. 71(1): 213-9 189 10. Locher HH. Seiler P, Chen X Schroeder S, Pfaff P, Enderlin M, Lkenk A, Fournier E, 190 Hubschwerlen C, Ritz D, Kelly CP, Keck W. 2014.. In vitro and in vivo antibacterial 191 evaluation of cadazolid, a new antibiotic for treatment of Clostridium difficile infection. 192 Antimicrob Agents Chemother. 58(2):892-900 193 11. Marin M, Martin A, Alcala L,Cercenado E, Iglesias, Reigadas E, Bouza E. 2015. 194 Clostridium difficile isolates with high linezolid MICs harbour the multiresistance gene, cfr. 195 Antimicrob Agents Chemother; 59 (1): 586-9. 196 12. Wu X, Hurdle JG. 2015. Hemin modulates metronidazole susceptibility of Clostridium difficile. 197 Abstr.  C-576.  Presented at the 55th Interscience Conference on Antimicrobial Agents and 198 Chemotherapy. San Diego, CA. 199  200  201  202  203  204   mg/L MCB3681 FDX MTZ VAN MXF CIP CLI TGC LZD Breakpoints S<4;   R>42 S <1;   RS >14 S<2; I=4; R>84 S<2; I=4; R>84 S<2; I=4; R>84 S <8;   RS >82 S<2;  I=4;  R>84 S<4;   RS>44 S<4;   R>47 %S 100 100 99 96 50.5 - 5.5 100 78.9 %I - - 1 2.5 1 - 29.5 - - %R  -  - 0 1.5 48 100 54 - 21.1 MIC50 0.125 0.06 0.25 1 2 64 16 0.06 4 MIC90 0.25 0.125 1 2 32 256 128 0.06 8 range 0.008-0.5 0.004-0.25 <0.125-4 0.5-8 1->64 8->128 1->64 0.03-0.125 2->64 Geometric mean MIC (mg/L)  RT001 (15) 0.07 0.02 0.42 0.79 16.00 111.43 61.11 0.03 10.08 RT002 (14) 0.11 0.06 0.19 0.87 1.82 27.86 12.13 0.04 4.39 RT005 (16) 0.14 0.06 0.29 1.16 2.00 37.12 9.28 0.04 5.66 RT014 (16) 0.11 0.07 0.28 0.88 3.36 39.74 10.37 0.05 4.36 RT015 (15) 0.14 0.06 0.25 0.87 1.91 26.60 7.29 0.04 4.19 RT017 (16) 0.15 0.04 0.26 0.74 12.88 86.67 64.00 0.06 7.03 RT018 (14) 0.12 0.06 0.41 1.49 6.90 110.33 8.83 0.04 4.42 RT020 (15) 0.10 0.06 0.25 0.75 2.59 36.44 11.31 0.05 4.76 RT027 (16) 0.16 0.09 0.96 1.14 21.67 206.14 19.87 0.05 5.19 RT078 (16) 0.11 0.05 0.26 0.92 2.38 34.90 12.34 0.05 5.42 RT106 (14) 0.11 0.09 0.74 1.10 7.61 81.98 10.77 0.04 4.42 RT126 (16) 0.12 0.06 0.32 1.00 8.35 72.88 38.05 0.06 4.56 RT356 (16) 0.08 0.04 0.27 2.28 29.34 245.15 12.88 0.04 4.76 All isolates (199) 0.12 0.05 0.33 1.02 5.87 66.27 16.17 0.05 5.16 Table 1. Susceptibility of 199 C. difficile isolates to MCB3681 and 8 comparators.  FDX= fidaxomicin; MTZ=metronidazole, VAN= vancomcyin; MXF=moxifloxacin;CIP=ciprofloxacin; CLI=clindamycin; TGC=tigecycline; LZD=linezolid S=sensitive; I=intermediate; R=resistant; RS=reduced susceptibility     MIC (mg/L) RT MXF CIP LZD CLI CHL MCB3681 001 16 128 32 >64 32 0.008 001 32 128 32 >64 32 0.015 001 32 128 32 >64 32 0.015 001 16 64 16 >64 8 0.25 001 16 >128 32 >64 8 0.25 001 16 >128 32 >64 32 0.25 001 16 >128 32 >64 32 0.25 014 16 >128 32 16 16 0.06 017 32 128 64 >64 2 0.06 017 16 64 16 >64 32 0.25 017 32 64 16 >64 64 0.5 017 32 64 32 >64 >64 0.5 017 32 128 32 >64 64 0.5 018 16 >128 8 8 4 0.03 018 32 >128 8 8 2 0.06 027 32 >128 32 >64 64 0.5 027 32 >128 32 >64 64 0.5 078 8 128 8 4 4 0.125 078 16 >128 8 4 64 0.125 106 16 128 8 8 4 0.06 126 16 64 16 >64 4 0.125 356 32 >128 8 8 4 0.03 356 32 >128 8 8 8 0.06 356 32 >128 8 8 4 0.25 356 >64 128 8 16 4 0.25 Table 2 MCB3681 MICs (mg/L) in C. difficile isolates with dual quinolone-oxazolidinone resistance (highlighting indicates resistance). Clindamycin and chloramphenicol MICs4 are also shown. MXF=moxifloxacin; CIP=ciprofloxacin; LZD=linezolid; CLI=clindamycin; CHL=chloramphenicol 

In vitro activities of MCB3681 and 8 comparators against Clostridium difficile isolates with known ribotypes and diverse geographical spread

ABSTRACT
          
            Treatments for
            Clostridium difficile
            infection remain limited, despite the introduction of fidaxomicin, and development of new agents is necessary. We determined the
            in vitro
            susceptibilities of 199 prevalent or emerging
            Clostridium difficile
            PCR ribotypes to MCB3681, a novel investigational quinolonyl-oxazolidinone, and 8 comparators (metronidazole, vancomycin, fidaxomicin, moxifloxacin, ciprofloxacin, clindamycin, tigecycline, and linezolid). MCB3681 showed good activity against
            C. difficile
            with no evidence of MCB3681 resistance in isolates showing either moxifloxacin or linezolid resistance or both moxifloxacin and linezolid resistance.
          </jats:p

Jane Freeman

Sally Pilling

Jonathan Vernon

Mark H. Wilcox

Crossref

<i>In Vitro</i>
            Activities of MCB3681 and Eight Comparators against Clostridium difficile Isolates with Known Ribotypes and Diverse Geographical Spread

In vitro activities of MCB3681 and 8 comparators against Clostridium difficile isolates with known ribotypes and diverse geographical spread

Abstract

Similar works

Full text

Available Versions

White Rose Research Online

Crossref