In Vitro Activities of Daptomycin, Vancomycin, Quinupristin- Dalfopristin, Linezolid, and Five Other Antimicrobials against 307 Gram-Positive Anaerobic and 31 Corynebacterium Clinical Isolates

The activities of daptomycin, a cyclic lipopeptide, and eightother agents were determined against 338 strains of gram-positiveanaerobic bacteria and corynebacteria by the NCCLS referenceagar dilution method with supplemented brucella agar for theanaerobes and Mueller-Hinton agar for the corynebacteria. Thedaptomycin MICs determined on Ca²⁺-supplemented (50 mg/liter)brucella agar plates were one- to fourfold lower than thosedetermined in unsupplemented media. Daptomycin was highly active(MICs, $<=$ 2 µg/ml) against many strains including 36 of 37peptostreptococci, 37 of 48 isolates of the Eubacterium group,and all strains of Propionibacterium spp., Clostridium perfringens,Clostridium difficile, and other Clostridium spp. It was fourfoldor greater more active than vancomycin against Clostridium innocuumand 16 of 34 strains of vancomycin-resistant lactobacilli. Threestrains of C. difficile for which quinupristin-dalfopristinand linezolid MICs were >8 µg/ml were inhibited by<1 µg of daptomycin per ml. Daptomycin MICs were $>=$ 4µg/ml for most strains of Clostridium clostridioforme,Clostridium paraputrificum, Clostridium tertium, and Clostridiumramosum; the isolates were generally more resistant to otherantimicrobials. Daptomycin was two- to fourfold less activeagainst Actinomyces spp. than vancomycin, quinupristin-dalfopristin,or linezolid. Twenty-nine of 31 strains of Corynebacterium spp.,including Corynebacterium jeikeium, Corynebacterium amycolatum,and Corynebacterium pseudodiphtheriticum, were inhibited by $<=$ 0.25 µg of daptomycin per ml. For two strains of "Corynebacteriumaquaticum," 8 µg of daptomycin per ml was required forinhibition. Daptomycin demonstrated very good activities againsta broad range of gram-positive organisms including vancomycin-resistantC. innocuum and lactobacillus strains and quinupristin-dalfopristin-and linezolid-resistant C. difficile strains.

INTRODUCTION

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Introduction

Daptomycin is a cyclic lipopeptide antibiotic derived from Streptomycesroseoporus with bactericidal activity against resistant gram-positivebacteria including methicillin-resistant Staphylococcus aureus,vancomycin-resistant enterococci, and penicillin-resistant pneumococci.It is undergoing clinical trials at present (24). Daptomycinacts by disrupting the bacterial cytoplasmic membrane and bydissipation of the membrane potential (1). Spontaneous resistanceto daptomycin is rare (22).

A few studies performed in the late 1980s or early 1990s havenoted that daptomycin is active against a very limited numberof anaerobes (4, 6, 9, 13, 21). Since the anaerobic gram-positiveisolates involved in diverse clinical infections comprise alarge variety of species and many clinical laboratories areeither unable to perform or choose not to perform anaerobicsusceptibility studies, clinicians are often reliant on thepublished literature to select both empirical and specific therapies.Because of the unusual resistance patterns recently manifestedby gram-positive anaerobes such as Clostridium and Peptostreptococcusspecies (2, 5, 22), information on recent isolates is desirable.In addition, most of the prior studies of the activity of daptomycinagainst anaerobes used Wilkins-Chalgren agar or brain heartinfusion agar (6, 9, 11, 13, 15), but these have now been replacedby supplemented brucella blood agar as the current NCCLS anaerobicreference susceptibility testing medium (18). Therefore, usingmore contemporary isolates, a larger number of defined species,and supplemented brucella blood agar, we evaluated the potentialutility of daptomycin against a variety of recently isolatedanaerobic gram-positive organisms. A set of pathogenic corynebacterialstrains was tested under aerobic conditions by NCCLS methods(17).

The antibacterial activity of daptomycin against aerobes hasbeen noted to be affected by the free Ca²⁺ concentration, requiringsupplementation of the growth medium to achieve the levels encounteredin normal human serum ( $~$ 50 mg/liter) (3, 14, 24). To determinethe effect of Ca²⁺ supplementation on daptomycin MICs for anaerobes,we compared non-calcium-supplemented with calcium-supplementedbrucella agar.

MATERIALS AND METHODS

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Materials and Methods

Isolates from human clinical specimens were identified by standardcriteria (2, 16, 23). Strains of anaerobic gram-positive anaerobesand corynebacteria were taken from a freezer and subculturedat least twice on brucella agar supplemented with vitamin K₁,hemin, and laked sheep blood or Trypticase soy blood agar, respectively,to ensure purity and good growth. The species and numbers ofisolates tested are listed in Table 1. Control strains includedStaphylococcus aureus ATCC 29213 and Eubacterium lentum ATCC40335. Agar dilution tests were performed according to NCCLSguidelines (17, 18). Antimicrobial agents were reconstitutedaccording to the instructions of the manufacturers, seriallydiluted, and added to molten supplemented brucella agar forthe anaerobes and to Mueller-Hinton agar for the corynebacteria.Daptomycin plates were prepared with and without additionalCa²⁺ to achieve a daptomycin concentration of 50 mg/liter. Thecalcium concentrations in the agar and broth media were measuredwith a Beckman Synchron CX 7 analyzer to determine the amountof supplementation needed to achieve a concentration of 50 mg/liter.Drug-free plates were inoculated before and after inoculationof each antimicrobial plate series and were used as growth controls.

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TABLE 1. In vitro activities of daptomycin and seven other antimicrobial agents against gram-positive anaerobes and corynebacteria

Colonies were suspended in brucella broth to a turbidity equalto that of a 0.5 McFarland standard and applied to the plateswith a Steers replicator that delivered approximately 10⁵ CFU/spotfor anaerobes. The anaerobic plates were incubated in the anaerobicchamber incubator for 44 h at 36°C prior to interpretationof the results. Corynebacteria were tested with an inoculumof 10⁴ CFU/spot, and the plates were incubated at 36°C for24 h under ambient conditions. The MIC was the concentrationthat completely inhibited growth or caused a marked reductionin the appearance of growth compared to that for the drug-freegrowth control (17, 18).

RESULTS

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Results

The results of our in vitro study are presented in Table 1.The modal daptomycin MIC for the S. aureus ATCC 29213 controlorganisms on calcium-supplemented plates was 0.5 µg/ml,while the modal MIC on unsupplemented agar was 2 µg/ml,demonstrating that calcium supplementation is required for agardilution tests. The MICs for the anaerobic isolates tested onbrucella agar plates supplemented with calcium were generallyone- to fourfold lower than those on unsupplemented plates.

Daptomycin was highly active against many strains. The MICsfor 18 strains of Clostridium difficile were $<=$ 1 µg/ml;these included 3 strains for which linezolid MICs were 8 to16 µg/ml, 3 strains for which quinupristin-dalfopristinMICs were 8 to 16 µg/ml, and 3 strains for which imipenemMICs were 16 µg/ml. Daptomycin was also active (MICs,1 to 4 µg/ml) against 19 strains of Clostridium innocuumfor which vancomycin MICs were 8 to 32 µg/ml. All strainsof Clostridium perfringens were inhibited by 0.06 to 0.5 µgof daptomycin per ml. Daptomycin MICs were $>=$ 4 µg/ml formost strains of Clostridium ramosum, Clostridium paraputrificum,and Clostridium tertium; and these strains were generally moreresistant to the other agents tested. The daptomycin MIC forClostridium clostridioforme, one of the clostridia that stainsgram negative, did not demonstrate the shift to a lower valuein the Ca²⁺-supplemented medium.

Daptomycin showed excellent activity (MIC, $<=$ 1 µg/ml) againstall peptostreptococci except one strain of Peptostreptococcusprevotii. Members of the Eubacterium group were generally susceptible,with MICs only occasionally being >4 µg/ml for strainsof E. lentum and Collinsella aerofaciens. All strains of Propionibacteriumspp. were susceptible to $<=$ 2 µg of daptomycin per ml andwere also susceptible to the other agents tested. DaptomycinMICs were $>=$ 16 µg/ml for 5 of 32 strains of Lactobacillusspp., but 16 vancomycin-resistant strains were all susceptibleto daptomycin. Vancomycin was generally two- to fourfold moreactive than daptomycin against Actinomyces spp.

Penicillin and the other beta-lactam agents tested were themost active agents tested against Actinomyces group speciesand many of the clostridia including Clostridium bifermentans,Clostridium sordellii, C. clostridioforme, C. innocuum, andC. perfringens. The daptomycin MICs were within 1 or 2 dilutionshigher than the vancomycin MICs for the clostridia. Daptomycinwas fourfold or greater more active than vancomycin againstC. innocuum. The activities of quinupristin-dalfopristin andlinezolid were more variable than those of daptomycin againstthe species tested.

DISCUSSION

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Discussion

Daptomycin is undergoing clinical evaluation for the treatmentof infections caused by drug-resistant gram-positive organisms(24). While most attention has focused on infections causedby methicillin-resistant S. aureus and drug-resistant enterococcaland pneumococcal strains, daptomycin may potentially be usedagainst anaerobes as well. Jones and Barry (15) and Fuchs etal. (10) proposed tentative interpretive criteria of a daptomycinMIC of $<=$ 2 µg/ml for susceptible strains and a daptomycinMIC of $>=$ 8 µg/ml for resistant strains. Calcium supplementationof brucella-based media resulted in MICs one- to fourfold loweron plates supplemented with Ca²⁺ (50 mg/liter) than on platesnot supplemented with calcium. This is in accord with the findingsof others for anaerobes with Wilkins-Chalgren agar and brainheart infusion agar (6, 9, 11, 13, 15). Similar results werenoted in our study with the corynebacteria tested, as previouslynoted by others (3, 8, 10) for aerobes, also with Mueller-Hintonmedium. Supplementation with calcium to physiologic levels appearsto be required for accurate daptomycin MIC determinations byboth aerobic and anaerobic agar dilution testing, regardlessof the basal medium used.

Clinical failures of metronidazole therapy of C. difficile-associatedcolitis and the in vitro resistance of C. difficile isolatesto metronidazole have been reported (5). While vancomycin hasbeen the alternative agent of choice for such cases, the developmentof vancomycin-resistant enterococci has led to guidelines suggesting"the prudent use of vancomycin" (12). The development of alternativeagents for the treatment of infections caused by such organismsseems desirable. Daptomycin has been noted to be active in vitroagainst C. difficile (4, 9, 13, 21). Faruki et al. (9), usingWilkins-Chalgren broth, studied 62 C. difficile isolates andfound an MIC at which 90% of isolates are inhibited (MIC₉₀)of 0.12 µg/ml with calcium-supplemented Wilkins-Chalgrenagar and an MIC₉₀ of 8 µg/ml with unsupplemented agar.Bartoloni et al. (4) found a daptomycin MIC₉₀ of 0.87 µg/mland suggested that it might be effective as therapy for C. difficile-associatedcolitis. This was supported by the study of Silva et al. (21),who used supplemented brain heart infusion agar and who notedthat daptomycin is more active in vitro than both metronidazoleand vancomycin against C. difficile isolates. In our study daptomycinwas more active than vancomycin, linezolid, or quinupristin-dalfopristinagainst C. difficile isolates. Three strains of C. difficilefor which quinupristin-dalfopristin and linezolid MICs were>8 µg/ml were inhibited by daptomycin at <1 µg/ml.

Dong et al. (7) reported that daptomycin at 0.05 mg/day waseffective in preventing death in a hamster C. difficile colitismodel. Nord et al. (19) studied the in vitro activity of daptomycinagainst C. difficile and subsequently (20) used 5 mg/day andalso found that it was protective in the hamster model. At present,daptomycin therapy involves intravenous dosing; an oral formulationwould be needed to treat C. difficile-associated diarrheal diseases.

Greenwood and Palfreyman (11) reported that daptomycin is activeagainst Peptostreptococcus species, but they noted a dramaticpotentiation of MICs related to calcium supplementation of thegrowth medium. They noted a wide range of MICs ( $<=$ 0.12 to >8µg/ml) but did not identify the species of their isolates.Silva et al. (21) studied the activities of daptomycin against23 peptostreptococcal strains (the species were not identified)and found that a daptomycin concentration of >4 µg/mlwas required for inhibition of 26% of the isolates, but theynoted a broad range of MICs (0.5 to 32 µg/ml). Our studynoted variations in susceptibility to daptomycin by species.All Peptostreptococcus asaccharolyticus isolates were susceptibleto daptomycin at $<=$ 0.125 µg/ml, all Peptostreptococcus microsstrains were susceptible to daptomycin at $<=$ 0.5 µg/ml, andPeptostreptococcus magnus isolates required up to 1 µgof daptomycin per ml for inhibition, while one of three Peptostreptococcusprevotii strains required 16 µg of daptomycin per ml forinhibition.

Barry et al. (3) studied 10 Lactobacillus isolates, many ofwhich were resistant to vancomycin, and found an MIC₉₀ of 4.0µg/ml on calcium-supplemented medium and an MIC₉₀ of 8.0µg/ml on unsupplemented medium. Our study noted a bimodalpattern of susceptibility of lactobacilli to daptomycin. Whilemost strains were susceptible to daptomycin at $<=$ 2 µg/ml,two strains of Lactobacillus plantarum, one strain of Lactobacillusrhamnosus, and one strain of Lactobacillus whose species wasunknown but that was also metronidazole resistant required >16µg of daptomycin per ml for inhibition. Daptomycin wasfourfold or greater more active than vancomycin against 16 of34 strains of vancomycin-resistant lactobacilli.

Our study showed that daptomycin inhibited 29 of 31 strainsof Corynebacterium spp. at $<=$ 0.25 µg/ml, including 10 strainsof Corynebacterium jeikeium that were resistant to penicillin,ampicillin, imipenem, and piperacillin-tazobactam. Two strainsof Corynebacterium aquaticum required treatment with 8 µg/mlfor inhibition. Barry et al. (3) reported the activities ofdaptomycin against 22 Corynebacterium sp. isolates (the specieswere not identified) and 7 C. jeikeium strains and reportedan MIC₉₀ of 0.5 µg/ml on calcium-supplemented medium andan MIC₉₀ of 1.0 µg/ml on unsupplemented medium, but theydid not report any variation in MIC by species. Fuchs et al.(10) noted that the geometric mean MIC of daptomycin for 21isolates of C. jeikeium was 0.25 µg/ml and that it ismore active than vancomycin and teichoplanin. Eliopoulos etal. (8) studied unidentified diphtheroids and noted that thedaptomycin MIC₉₀ for the isolates was 4 µg/ml.

Daptomycin demonstrated very good activities against a broadrange of anaerobic gram-positive organisms including vancomycin-resistantC. innocuum and lactobacillus strains and quinupristin-dalfopristin-resistantstrains of C. difficile. Due to marked differences in the patternsof susceptibility to daptomycin by species, laboratories shouldconsider identification of the species of clinically significantanaerobic isolates, especially when susceptibility studies arenot routinely performed.

ACKNOWLEDGMENTS

This study was funded, in part, by a grant from Cubist Pharmaceuticals,Lexington, Mass.

We thank Judee H. Knight and Alice E. Goldstein for assistance.

FOOTNOTES

* Corresponding author. Mailing address: R. M. Alden Research Laboratory, 2021 Santa Monica Blvd., Suite 740 East, Santa Monica, CA 90404. Phone: (310) 315-1511. Fax: (310) 315-3662. E-mail: ejcgmd{at}aol.com.

REFERENCES

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