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Antimicrobial Agents and Chemotherapy, July 2003, p. 2334-2338, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2334-2338.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vitro Activities of Ramoplanin, Teicoplanin, Vancomycin, Linezolid, Bacitracin, and Four Other Antimicrobials against Intestinal Anaerobic Bacteria

D. M. Citron,* C. V. Merriam, K. L. Tyrrell, Y. A. Warren, H. Fernandez, and E. J. C. Goldstein

R.M. Alden Research Laboratory, Santa Monica, California

Received 6 December 2002/ Returned for modification 30 January 2003/ Accepted 1 April 2003


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ABSTRACT
 
By using an agar dilution method, the in vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, and five other agents were determined against 300 gram-positive and 54 gram-negative strains of intestinal anaerobes. Ramoplanin was active at ≤2 µg/ml against 287 of 300 (95.7%) gram-positive organisms, including 18 strains of Clostridium difficile for which MICs of ramoplanin were 0.25 to 0.5 µg/ml; for 3 of these, linezolid MICs were 8 to 16 µg/ml. Nineteen Clostridium innocuum strains for which the vancomycin MIC at which 90% of strains were inhibited was 16 µg/ml were susceptible to ramoplanin at 0.06 to 0.25 µg/ml and to teicoplanin at 0.125 to 1.0 µg/ml. All strains of Eubacterium, Actinomyces, Propionibacterium, and Peptostreptococcus spp. were inhibited by ≤0.25 µg of ramoplanin per ml and ≤1 µg of vancomycin per ml. Ramoplanin was also active at ≤4 µg/ml against 15 of 22 of the Prevotella and Porphyromonas strains tested, but ramoplanin MICs for all 31 strains of the Bacteroides fragilis group, the Fusobacterium mortiferum-Fusobacterium varium group, and Veillonella spp. were ≥256 µg/ml. Ramoplanin displays excellent activity against C. difficile and other gram-positive enteric anaerobes, including vancomycin-resistant strains; however, it has poor activity against most gram-negative anaerobes and thus potentially has a lesser effect on the ecological balance of normal fecal flora.


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TEXT
 
Ramoplanin, a glycolipodepsipeptide antibiotic that inhibits peptidoglycan synthesis, is currently being developed as an oral, nonabsorbable agent for the prevention of vancomycin-resistant Enterococcus (VRE) infection in patients with VRE gastrointestinal tract colonization (17). It has demonstrated activity against a wide spectrum of gram-positive organisms, including antibiotic-resistant strains of staphylococci and enterococci and less frequently encountered pathogens such as Corynebacterium jeikeium, Listeria monocytogenes, and Bacillus spp. (5, 9, 10, 11, 12, 14, 16); however, limited data are available on the drug's activity against anaerobic bacteria (3, 14).

(This study was presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, 16 to 19 December 2001, Chicago, Ill. [D. M. Citron, Y. A. Warren, K. L. Tyrrell, C. V. Merriam, H. Fernandez, and E. J. C. Goldstein, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-1417, p. 193, 2001].)

Broad-spectrum antimicrobials with activity against anaerobes may disrupt the ecological balance of the intestinal flora and promote colonization with VRE and Clostridium difficile (6, 7, 8, 15, 19), while antimicrobials with minimal antianaerobe activity preserve the normal intestinal anaerobic flora responsible for colonization resistance (18). Since ramoplanin is intended as treatment for intestinal colonization of VRE, we examined its potential effects on colonic flora by determining its in vitro activity against anaerobic organisms of intestinal origin, including both gram-positive and gram-negative species.

Selected for this study were strains from our collection of anaerobic gram-positive bacilli and cocci that had been isolated from bowel flora or clinical intra-abdominal specimens. Smaller numbers of gram-negative anaerobes of intestinal origin were also included. The majority of the test strains were isolated during the past 3 years. The species and numbers of strains tested are listed in Table 1. Clostridium bifermenions-≤0.125-0.125≤128.125≤128.125Porphyromonas asaccharolytica (10) Ramoplanin≤1-4≤14  Teicoplanin≤1≤1≤1  Vancomycin≤1-424  Bacitracin≤1-4≤12  Linezolid2-222  Cefoxitin0.06-0.250.1250.25  Ampicillin≤0.03≤0.03≤0.03  Clindamycin≤0.03≤0.03≤0.03  Metronidazole0.06-0.1250.060.125 Prevotella spp. (12) Ramoplanin4-12832128  Teicoplanin0.25-40.54  Vancomycin2-643264  Bacitracin0.5-32232  Linezolid0.25-20.51  Cefoxitin≤0.03-160.51  Ampicillin≤0.03-320.1254  Clindamycin≤0.03≤0.03≤0.03  Metronidazole0.125-10.25 Control strains included Staphylococcus aureus ATCC 29213, Eubacterium lentum ATCC 43055, and Bacteroides fragilis ATCC 25285.


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  		TABLE 1. In vitro activities of ramoplanin, teicoplanin, vancomycin, bacitracin, linezolid, and four other agents against intestinal strains of anaerobic bacteria

Susceptibility testing was performed according to the reference agar dilution method described by National Committee on Clinical Laboratory Standards document M11-A5 (12). Antimicrobial agents were obtained as follows: ramoplanin, Intrabiotics, Mountain View, Calif. (ramoplanin is now being developed by Genome Therapeutics Corp., Waltham, Mass.); teicoplanin, Aventis, Romainville, France; vancomycin, Eli Lilly & Co., Indianapolis, Ind.; ampicillin and bacitracin, Sigma Chemical Co., St. Louis, Mo.; linezolid and clindamycin, Pharmacia, Kalamazoo, Mich.; cefoxitin, Merck & Co., Rahway, N.J.; and metronidazole, Searle, Skokie, Ill. The antimicrobials were reconstituted according to their manufacturers' instructions, serially diluted, and added to molten supplemented brucella agar for plate preparation. The plates were inoculated on the day of preparation. Bacitracin plates were prepared on the basis of the weight of the drug. For conversion, 1 µg equals 0.066 U, or 1 U equals 15.2 µg. The binding of ramoplanin to plastic that has been reported in broth microdilution tests was not an issue in agar dilution tests (1).

Isolates were taken from frozen stock and subcultured at least twice on supplemented brucella agar (Anaerobe Systems, Morgan Hill, Calif.) to ensure purity and good growth. Colonies were suspended in brucella broth (Becton Dickinson, Sparks, Md.) to a density equal to the 0.5 McFarland standard. The suspensions were applied to the antibiotic plates with a Steers replicator that delivered a final inoculum of approximately 105 CFU/spot. The plates were incubated in the anaerobic chamber incubator at 36°C for 44 h. The MIC was defined as the concentration of drug that completely inhibited growth or caused a marked reduction in the appearance of growth compared to the drug-free growth control.

The results are presented in Table 1. Ramoplanin was active at ≤2 µg/ml against all gram-positive strains with the exception of all 10 strains of Clostridium clostridioforme, 2 of 5 strains of Clostridium symbiosum, and 1 of 10 strains of Clostridium cadaveris. Moreover, teicoplanin MICs for the C. clostridioforme strains were 1 to 8 µg/ml, which were also higher than those for most of the other clostridia. This finding is of interest because C. clostridioforme and C. symbiosum consistently stain gram-negative, suggesting the presence of a thinner peptidoglycan layer in their cell walls. However, these strains were all susceptible to ≤1 µg of vancomycin per ml, indicating a different mechanism of activity. Ramoplanin was active (MIC, ≤0.125 µg/ml) against the 19 vancomycin-resistant (MIC, 8 to 32 µg/ml) strains of Clostridium innocuum and against all 15 strains of Clostridium ramosum (ramoplanin MIC, ≤0.06 µg/ml), for which vancomycin MICs were 2 to 8 µg/ml and teicoplanin MICs were 0.5 to 1 µg/ml. C. difficile strains were susceptible to ramoplanin at 0.25 to 0.5 µg/ml, including 3 of the18 strains tested for which linezolid MICs were 8 to 16 µg/ml and clindamycin MICs were >128 µg/ml. Other clostridia were susceptible to most of the agents tested, except bacitracin. These results are similar to those obtained by Romano et al. (G. Romano, C. Brunati, A. Bulgheroni, D. Jabes, and G. Privitera, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-2260, p. 196, 2001), who tested 121 Clostridium strains representing 17 species and reported only one isolate of Clostridium rectum for which the ramoplanin MIC was >256 µg/ml; ramoplanin MICs for the remaining Clostridium strains were ≤4 µg/ml. Their study also included 76 strains of C. difficile for which ramoplanin MICs were ≤0.007 to 0.125 µg/ml. Our results are also in agreement with those of Biavasco et al. (3), who tested ramoplanin, teicoplanin, and vancomycin against 70 strains of C. difficile and obtained MICs that were virtually identical to those in our present study. Since C. difficile is frequently isolated from the same types of patients who are colonized with VRE, and since ramoplanin is active against both organisms, it may eradicate both of them (8, 15).

Among the other gram-positive strains, all Eubacterium spp., Propionibacterium spp., Peptostreptococcus spp., Actinomyces spp., and Bifidobacterium spp. were inhibited by ≤0.25 µg of ramoplanin per ml and ≤1 µg of vancomycin per ml. While all 37 Lactobacillus strains were susceptible to ramoplanin at ≤0.5 µg/ml, vancomycin MICs for 16 of these strains were >32 µg/ml and teicoplanin MICs were >64 µg/ml.

Among the gram-negative strains, for the B. fragilis group, the Fusobacterium mortiferum-Fusobacterium varium group, and Veillonella strains, ramoplanin MICs were ≥256 µg/ml. The Prevotella and Porphyromonas strains were somewhat more susceptible, with ramoplanin and vancomycin MICs ranging from ≤1 to 128 µg/ml. All 10 of the Porphyromonas and 5 of the 12 Prevotella strains were susceptible to ≤4 µg of ramoplanin per ml. A previous study also found that ramoplanin inhibited Prevotella bivia and Prevotella melaninogenica (formerly Bacteroides melaninogenicus) at concentrations of 0.5 to 4 µg/ml (13).

Ramoplanin exhibited potent activity against most gram-positive anaerobes while having little or no effect on most of the gram-negative strains; therefore, ramoplanin appears to have less impact on the anaerobic bowel flora than some of the other more broad-spectrum agents that we tested. Our vitro data do not necessarily predict in vivo effect, and clinical data on the impact of ramoplanin on normal fecal flora are needed. Our susceptibility study used an inoculum of 105 CFU/spot that resulted in MICs that were ≤2 µg/ml for 95.7% of the gram-positive strains; however, concentrations of anaerobes are typically in the range of 109 to 1012 CFU/g of feces; therefore, ramoplanin may have even less impact on normal anaerobic gram-positive flora than would be suggested by our test results. Ramoplanin dosed at 400 mg twice daily results in a fecal concentration of 1 to 1.5 mg per g of stool (Timothy Leach [Genome Therapeutics Corp.], personal communication). While enzymatic breakdown of the drug by fecal flora does not occur, there is a nonspecific and reversible adsorption of about 80 to 90% of the drug (2). High concentrations of ramoplanin in feces induce high levels of free antibiotic, and the binding with subsequent release can maintain long-lasting levels in the gastrointestinal tract (2) and provide effective therapy for VRE without causing the major perturbation of the gastrointestinal ecosystem that can occur with administration of broad-spectrum antimicrobials, especially the expanded-spectrum cephalosporins, metronidazole, and the fluoroquinolones (4). Significantly, ramoplanin exhibited excellent activity against the 18 strains of C. difficile tested and might provide an alternative therapy for this pathogen in addition to VRE.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from Intrabiotics.


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FOOTNOTES
 
* Corresponding author. Mailing address: Alden Research Laboratory; 2001 Santa Monica Blvd., Suite 685W, Santa Monica, CA 90404. Phone: (310) 453-7820. Fax: (310) 453-7670. E-mail: d.m.citron{at}verizon.net. Back


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Antimicrobial Agents and Chemotherapy, July 2003, p. 2334-2338, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2334-2338.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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