Keywords: CB-183,315; Clostridium difficile; susceptibility; intestinal anaerobes

Transcription

1 AAC Accepts, published online ahead of print on 19 December 2011 Antimicrob. Agents Chemother. doi: /aac Copyright 2011, American Society for Microbiology. All Rights Reserved In vitro activity of CB-183,315, vancomycin and metronidazole against 556 strains of Clostridium difficile, 445 other intestinal anaerobes and 56 Enterobacteriaceae species 1 Diane M. Citron*, 1 Kerin L. Tyrrell, 1 C. Vreni Merriam and 1,2 Ellie J.C. Goldstein 1 R.M. Alden Research Lab, Culver City, CA 2 Geffen School of Medicine, UCLA, Los Angeles, CA Keywords: CB-183,315; Clostridium difficile; susceptibility; intestinal anaerobes Running title: CB-183,315 against C. difficile and intestinal organisms Corresponding author: Diane M. Citron R.M. Alden Research Lab 6133 Bristol Parkway Suite 175 Culver City, CA P F d.m.citron@att.net Abstract 1

2 MICs for CB-183,315, a novel lipopeptide antibiotic, vancomycin and metronidazole were determined for intestinal anaerobes and Enterobacteriaceae. The MIC 90 for gram-negative anaerobes was >8192, 8192 and 4 µg/ml for CB-183,315, vancomycin and metronidazole, respectively. Against Enterobacteriaceae, MIC 90s were >8192 µg/ml, 1024 µg/ml and1024 µg/ml, respectively. CB-183,315 MIC 90 for C. difficile was 0.5 µg/ml. Its lack of activity against normal fecal organisms makes it a promising new agent for treating C. difficile. Clostridium difficile infection (CDI) is the most common cause of healthcare related diarrhea, usually associated with prior exposure to antimicrobial agents. The current theory is that the 2

3 antibiotics disrupt the normal fecal microbiota, changing its complexity and diversity as a primary event, with the subsequent acquisition of toxigenic C. difficile as a secondary event in the development of the disease (6,11,14). CDI has increased in prevalence and severity during the last decade, resulting in increased mortality and complications, recurrent disease and prolonged hospital stay (7,17). Non-healthcare related cases of CDI are now being reported (7,16). Treatment options have been limited by unsatisfactory efficacy of current therapeutic agents, high recurrence rates of disease, disruption of normal intestinal microbiota, and colonization by VRE (1,2,15). Although a recently introduced macrocyclic antibiotic, fidaxomicin, shows equivalent primary cure rates with reduced recurrence rates compared to vancomycin (15) it shows equivalence against REA type BI strain associated disease. CB-183,315 is a novel lipopeptide antibiotic with gram-positive activity and is bactericidal against C. difficile. To study the spectrum of its activity, including effect on the normal components of fecal microbiota, we determined MICs of CB-183,315 and vancomycin against C. difficile and other intestinal anaerobes, plus E. coli, Klebsiella spp. and Enterobacter spp. For comparison, metronidazole was tested against the gram-negative anaerobes and the Enterobacteriaceae spp. The C. difficile strains were recent clinical isolates ( ) recovered from stools of patients with CDI. Other isolates were from stool or infections containing organisms presumed to be of intestinal origin, identified by standard methods (13) and occasionally by 16S rdna sequence analysis (20), and stored in 20% skim milk at -70 o C. MIC values were determined by the agar dilution method according to CLSI procedures (4). Vancomycin and metronidazole laboratory standard powders were obtained from Sigma (St. 3

4 Louis, MO) and CB-183,315 was provided by Cubist Pharmaceuticals, Inc. (Lexington, MA). All assay media for testing CB-183,315 were supplemented with a final concentration of 50 mg/l calcium, where Ca ++ concentrations were confirmed by Laboratory Specialists, Inc. (Westlake, Ohio). The Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. strains were tested using Mueller Hinton agar, and incubated at 37 o C in ambient air, except for metronidazole, which was incubated in the anaerobic chamber (4). Quality control strains included Clostridium difficile ATCC , Bacteroides fragilis ATCC 25285, Staphylococcus aureus ATCC and E. coli ATCC and were included each day of testing of the relevant set of test organisms. Time-kill studies were carried out on one restriction endonuclease analysis (REA) BI type (NAP1, ribotype 027) strain and one REA type Y (NAP4, ribotype 014) strain (3). CB-183,315 and vancomycin were prepared at 2, 4, and 8 times their MICs for each strain in supplemented Brucella broth. A drug-free growth control tube was included. Tubes were inoculated with ~ 10 6 CFU/ml, placed on a shaker and assayed at 0, 2, 4, 8, and 24h of incubation at 37 o C. Table 1 shows the range, MIC 50 and MIC 90 for the major groups of organisms. The CB-183,315 MIC range for C. difficile was µg/ml with MIC 90 of 0.5 µg/ml; other gram-positive strains were inhibited by µg/ml. The MIC 90 for CB-183,315 against the Bacteroides fragilis group, Prevotella spp., gram-negative cocci (Veillonella spp. and Acidaminococcus sp.), 4

5 as well as E. coli, Enterobacter spp., and Klebsiella spp. was greater than 8192 µg/ml. Against fusobacteria, and Porphyromonas spp., the MIC 90 was 8192 µg/ml and 2048 µg/ml, respectively. In contrast, vancomycin had equally high MICs of >8192 µg/ml only against the fusobacteria and the gram-negative cocci; the B. fragilis group was inhibited by vancomycin at MIC 90 of 128 µg/ml (range µg/ml), Porphyromonas by 4 µg/ml, and Prevotella spp. by 256 µg/ml of vancomycin. Vancomycin MICs ranged from µg/ml for E. coli and µg/ml for Enterobacter and Klebsiella species. Metronidazole was active against the gram-negative anaerobes at 0.06 to 8 µg/ml, but showed poor activity against the Enterobacteriaceae, with MICs ranging from 32 to1024 µg/ml. The time-kill studies showed that CB-183,315 at 4 and 8 times the MIC resulted in a 3 log 10 reduction in colony count after 24h incubation against both REA types tested. Vancomycin produced a similar result.. CB-183,315 reaches concentrations of μg/g of feces after a 1g bid dose at day 5 of a 14 day course (Cubist Pharmaceuticals data on file) which is lower than the high MICs (>8192 µg/ml) demonstrated for Bacteroides and other groups of organisms of the intestinal anaerobes, thus CB-183,315 is likely to spare many of these important members of the normal microbiota. In contrast, vancomycin at a dose of 250 mg qid results in fecal levels that are generally above 2,000 μg/ml (12). Vancomycin thus has the potential for killing or inhibiting much of the normal aerobic and anaerobic fecal microbiota. Moreover, oral vancomycin was shown to decrease or suppress Bacteroides in volunteers while increasing occurrence of vancomycin-resistant enterococci (8,14). In contrast, fidaxomicin, a narrow spectrum 5

6 macrocycle, displayed less impact on the Bacteroides populations in patients treated for CDI (14), which has been postulated as one reason for the associated lower relapse rate. Using temporal temperature gradient electrophoresis and fluorescent in situ hybridization and flow cytometry, Tannock et al.(19) compared the impact of fidaxomicin and vancomycin on seven phylogenetic groups, including clostridial clusters IV and XIVa, Bacteroides-Prevotella, Bifidobacterium, Atopobium, enterobacteria, and the Enterococcaceae-Lactobacillaceae group. Minimal changes were seen in these groups for the fidaxomicin treated patients while the vancomycin treated patients showed a marked decrease in Clostridium clusters IV and XIVa, Bacteroides and Bifidobacterium, and an increase in the Enterococcus-Lactobacillus and the enterobacteria groups. MICs for CB-183,315 against clostridia were similar to those reported for fidaxomicin (10), showing decreased activity against several species in Clostridium clusters XIVa, XVI, and XVIII, although unlike fidaxomicin, decreased activity of CB-183,315 was also present against several species in cluster I (5,9,18). MIC 90 for the C. clostridioforme group (cluster XIVa), was 16 µg/ml; for C. innocuum (cluster XVI) 4 µg/ml; for C. ramosum (Cluster XVIII) 8 µg/ml; and C. sphenoides, 1 strain, (cluster XIVa) 16 µg/ml. These findings suggest that CB-183,315 would show a similar lack of impact as fidaxomicin on the composition of the major groups of bowel microbiota. With its excellent activity against C. difficile and its narrower spectrum and lack of activity against other colonic aerobic and anaerobic microbiota, with the potential for fewer relapses, CB- 183,315 is a promising new drug treatment for CDI. 6

7 Acknowledgement This study was sponsored by a grant from Cubist Pharmaceuticals, Inc. We thank Eliza Leoncio for excellent technical assistance. Downloaded from on October 1, 2018 by guest 7

8 148 Reference List Al-Nassir, W. N., A. K. Sethi, Y. Li, M. J. Pultz, M. M. Riggs, and C. J. Donskey Both oral metronidazole and oral vancomycin promote persistent overgrowth of vancomycin-resistant enterococci during treatment of Clostridium difficile-associated disease. Antimicrob.Agents Chemother. 52: Bhalla, A., N. J. Pultz, A. J. Ray, C. K. Hoyen, E. C. Eckstein, and C. J. Donskey Antianaerobic antibiotic therapy promotes overgrowth of antibiotic-resistant, gramnegative bacilli and vancomycin-resistant enterococci in the stool of colonized patients. Infect.Control Hosp.Epidemiol. 24: Clabots, C. R., S. Johnson, K. M. Bettin, P. A. Mathie, M. E. Mulligan, D. R. Schaberg, L. R. Peterson, and D. N. Gerding Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems. J.Clin.Microbiol. 31: Clinical and Laboratory Standards Institute Methods for antimicrobial susceptibility testing of anaerobic bacteria, 7th ed. Approved standard M11-A7. Clinical and Laboratory Standards Institute, Wayne, PA. 5. Collins, M. D., P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. Farrow The phylogeny of the genus 8

9 Clostridium: proposal of five new genera and eleven new species combinations. Int.J.Syst.Bacteriol. 44: Dethlefsen, L., S. Huse, M. L. Sogin, and D. A. Relman The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rrna sequencing. PLoS.Biol. 6:e Dubberke, E. R. and A. I. Wertheimer Review of current literature on the economic burden of Clostridium difficile infection. Infect.Control Hosp.Epidemiol. 30: Edlund, C., L. Barkholt, B. Olsson-Liljequist, and C. E. Nord Effect of vancomycin on intestinal flora of patients who previously received antimicrobial therapy. Clin.Infect.Dis. 25: Finegold, S. M., D. Molitoris, M. L. Vaisanen, Y. Song, C. Liu, and M. Bolanos In vitro activities of OPT-80 and comparator drugs against intestinal bacteria. Antimicrob.Agents Chemother. 48: Finegold, S. M., D. Molitoris, M. L. Vaisanen, Y. Song, C. Liu, and M. Bolanos In vitro activities of OPT-80 and comparator drugs against intestinal bacteria. Antimicrob.Agents Chemother. 48:

10 Gerding, D. N. and S. Johnson Management of Clostridium difficile infection: thinking inside and outside the box. Clin.Infect.Dis. 51: Gonzales, M., J. Pepin, E. H. Frost, J. C. Carrier, S. Sirard, L. C. Fortier, and L. Valiquette Faecal pharmacokinetics of orally administered vancomycin in patients with suspected Clostridium difficile infection. BMC.Infect.Dis. 10: Jousimies-Somer, H. R., P. Summanen, D. M. Citron, E. J. Baron, H. M. Wexler, and S. M. Finegold Wadsworth-KTL Anaerobic Bacteriology Manual. Star Publishing, Belmont, CA. 14. Louie, T. J., J. Emery, W. Krulicki, B. Byrne, and M. Mah OPT-80 eliminates Clostridium difficile and is sparing of bacteroides species during treatment of C. difficile infection. Antimicrob.Agents Chemother. 53: Miller, M., L. Bernard, M. Thompson, D. Grima, and J. Pepin Lack of increased colonization with vancomycin-resistant enterococci during preferential use of vancomycin for treatment during an outbreak of healthcare-associated Clostridium difficile infection. Infect.Control Hosp.Epidemiol. 31: Rouphael, N. G., J. A. O'Donnell, J. Bhatnagar, F. Lewis, P. M. Polgreen, S. Beekmann, J. Guarner, G. E. Killgore, B. Coffman, J. Campbell, S. R. Zaki, and L. C. McDonald Clostridium difficile-associated diarrhea: an emerging threat to pregnant women. Am.J.Obstet.Gynecol. 198:

11 Rupnik, M., M. H. Wilcox, and D. N. Gerding Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat.Rev.Microbiol. 7: Stackebrandt, E., I. Kramer, J. Swiderski, and H. Hippe Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol.Med.Microbiol. 24: Tannock, G. W., K. Munro, C. Taylor, B. Lawley, W. Young, B. Byrne, J. Emery, and T. Louie A new macrocyclic antibiotic, fidaxomicin (OPT-80), causes less alteration to the bowel microbiota of Clostridium difficile-infected patients than does vancomycin. Microbiology 156: Warren, Y. A., K. L. Tyrrell, D. M. Citron, and E. J. Goldstein Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. isolated from human clinical infections. J.Clin.Microbiol. 44:

12 Table 1. In vitro activity of CB-183,315, Vancomycin and Metronidazole against Intestinal Organisms (μg/ml) CB-183,315 Vancomycin Metronidazole Group N Range MIC 50 MIC 90 Range MIC 50 MIC 90 Range MIC 50 MIC 90 Actinomyces species a NT Bifidobacterium species b NT Eggerthella lenta NT Eubacterium limosum NT Eubacterium group (other) c NT Lactobacillus species, VAN-R d >32 - >32 >32 >32 NT Lactobacillus species, VAN-S e NT Propionibacterium species f NT Clostridium clostridioforme NT grp g (CL. XIVa) Clostridium difficile (Cl. XI) NT Clostridium innocuum (Cl.XV) NT Clostridium perfringens (Cl.I) NT Clostridium ramosum (Cl. NT XVIII) Clostridium species (other) h NT Anaerobic gram-positive cocci i 49 < NT Bacteroides fragilis group j > > Fusobacterium species k > > > Porphyromonas species l Prevotella species m > > Gram-negative cocci n >8192 >8192 >8192 > > >8192 > E. coli >

13 >8192- >8192 >8192 Enterobacter species o 18 > >8192 >8192 > Klebsiella species p Cl, phylogenetic cluster a Actinomyces israelii (2), A. meyeri (2), A. naeslundii (1), A. neuii ss anitratus (2), A. neuii ss neuii (1), A. odontolyticus (2), A. turicensis (2), and A. viscosus (3). b Bifidobacterium adolescentis (2), B. bifidum (1), B. breve (4), B. catenulatum (2), B. dentium (1), B. longum (2), B. pseudocatenulatum (1), and B. urinalis (1). c Collinsella aerofaciens (6), Eubacterium biforme (1), E. contortum (1), E. cylindroides (1), E. timidum (3), Pseudoramibacter alactolyticum (4) and Eubacterium species (19). d Lactobacillus brevis (1), L. casei (7), L. fermentum (2), L. rhamnosus (6), and Lactobacillus GG (4). e Atopobium parvulum (1), Lactobacillus acidophilus (2), L. catenaformis (3), L. crispatus (1), L. gasseri (3), L. iners (1), L. jensenii (4), and L. leichmannii (2 ) f Propionibacterium acnes (7), P. avidum (4), and P. granulosum (4). g Clostridium aldenense (4), C. bolteae (5), C. citroniae (3), C. clostridioforme (2), and C. hathewayi (6). h Clostridium bifermentans (1), C. butyricum (5), C. cadaveris (3), C. cochlearium (2), C. fallax (1), C. glycolicum (3), C. hastiforme (1), C. hylemonae (1), C. indolis (1), C. leptum (2), C. nexile (1), C. paraputrificum (4), C. sartagoforme (1), C. scindens (1), C. sordelli (2), C. sphenoides (1), C. sporogenes (2), C. subterminale (1), C. symbiosum (4), and C. tertium (2). 13

14 i Anaerococcus lactolyticus (2), A. octavius (1), A. prevotii (6), A. tetradius (5), A. vaginalis (4) ; Finegoldia magna (7) ; Peptoniphilus asaccharolyticus (5), P. harei (2), P. indolicus (2), P. ivorii (3), and P. lacrimalis (2); Peptostreptococcus anaerobius (5) and Parvimonas micra (5). j Bacteroides caccae (1), B. distasonis (1), B. fragilis (11), B. nordii (1), B. ovatus (3), B. thetaiotaomicron (3), B. vulgatus (1). k Fusobacterium gonidiaformans (1), F. mortiferum-varium group (6), F. necrophorum (2), F. nucleatum (10), and F. russii (1). l Porphyromonas asaccharolytica (9), P. gingivalis (4), P. somerae (6), P. uenonis (2). m Prevotella bivia (10), P. disiens (3), P. intermedia/nigrescens (1), P. loeschii (3), and P. melaninogenica (3). n Acidaminococcus fermentans (8), Veillonella species (13). o Enterobacter aerogenes (6), Enterobacter cloacae (11), Enterobacter sp. (1) p Klebsiella oxytoca (3), Klebsiella pneumoniae (17) 14