Activity of Tigecycline (GAR-936), a Novel Glycylcycline, against Enterococci in the Mouse Peritonitis Model

A novel glycylcycline agent, tigecycline (GAR-936), was evaluatedin vivo in the mouse model of peritonitis against three Enterococcusfaecalis and four Enterococcus faecium isolates with differentsusceptibilities to vancomycin and tetracyclines, all of whichwere inhibited by $<=$ 0.125 µg of tigecycline/ml. Using asingle subcutaneous dose, tigecycline displayed a protectiveeffect (50% protective dose, $<=$ 5.7 mg/kg of body weight) againstall strains tested, including two with Tn925 (from the Tn916family), which contains the Tet(M) tetracycline resistance determinant,as well as VanA and VanB strains. As expected, tetracyclineand minocycline were ineffective against the isolates carryingTn925.

INTRODUCTION

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Introduction

Enterococci have long been known to cause endocarditis, urinarytract infections, intraabdominal and pelvic infections, intravascularcatheter-related bacteremia, bacteremia from unknown origin,soft tissue infections, and neonatal sepsis and meningitis (4,8). The interest in this organism has increased in recent years,particularly because of the rising incidence of vancomycin-resistantenterococci (VRE), with a major impact in nosocomial infections,where enterococci have been ranked as the third most commoncause of bloodstream infections (5). Among enterococcal strainscausing nosocomial infections in intensive care units duringthe year 2000, 26.3% were VRE, representing a 31% increase inthe vancomycin resistance rate compared with the mean rate ofvancomycin resistance observed in the previous 5 years, as reportedby the Centers for Disease Control and Prevention (1). Thissituation has led to a search for antienterococcal agents withnew mechanisms of action or to reformulation of older ones.

The efficacy of tetracyclines has been impaired by the widespreadexistence of genes conferring resistance to them, probably secondaryto the long history of tetracycline use in humans, in fieldcrops and fruit trees, and as food additives for growth promotionin animals (14). In 1988, a new tetracycline with modificationsin the minocycline hydrophobic domain conferring activity againsttetracycline-resistant organisms was developed, and this eventuallyled to the synthesis of novel structures called glycylcyclines.The glycylcycline tigecycline (GAR-936) is a 9-t-butylglycylamidoderivative of minocycline; this new agent inhibits protein synthesiseven in the presence of Tet(M)-protected ribosomes, probablydue to its tight binding (11). Tigecycline has shown in vitroactivity against many gram-positive organisms, including vancomycin-susceptibleand vancomycin-resistant Enterococcus faecalis and Enterococcusfaecium, regardless of the susceptibility pattern to tetracyclines(3). The aim of this study was to evaluate the in vivo activityof this new agent against various E. faecalis and E. faeciumstrains, including tetracycline- and vancomycin-resistant ones,in the mouse peritonitis model.

(This work was presented in part at the 41st Interscience Conferenceon Antimicrobial Agents and Chemotherapy, Chicago, Ill., 16to 19 December 2001.)

MATERIALS AND METHODS

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Materials and Methods

Organisms. Three E. faecalis isolates used in this study were OG1RF (ATCC47077), a well-known plasmid-free isolate (9); V583, a vanBand erm(B) clinical isolate (15); and INY1200 (OG1RF::Tn925)(Tn925 is a conjugative transposon from the Tn916 family oftransposons [13]) which carries the tet(M) gene encoding tetracycline-minocyclineresistance (18). The four E. faecium isolates were TX5034, aVanA type of VRE strain resistant also to erythromycin and streptomycin;TX5037 (TX5034::Tn925), a transconjugant of TX5034 and INY1200,carrying the Tet(M) determinant; TX0016 (DO), a tetracycline-and erythromycin-resistant endocarditis isolate (2) (partialsequence available at http://www.hgsc.bcm.tmc.edu/microbial/Efaecium/index.html);and TX2465, another clinical isolate carrying the vanA genecluster, from an oncologic patient with bacteremia.

Antibiotics. Tigecycline in powder form was obtained from Wyeth Research,Pearl River, N.Y. Other antibiotics used were tetracycline hydrochloride,minocycline, vancomycin hydrochloride, erythromycin, and ampicillinsodium salt and were obtained from Sigma, St. Louis, Mo. Quinupristin-dalfopristinwas obtained from Aventis Pharma, Croix-de-Berny, France.

In vitro susceptibility testing. The MICs of each antimicrobial agent were determined by theagar dilution method using Mueller-Hilton agar II (Becton Dickinsonand Company, Cockeysville, Md.) and following the National Committeefor Clinical Laboratory Standards guidelines (10). E. faecalisATCC 29212 and Staphylococcus aureus ATCC 29213 strains wereused for quality control.

In vivo efficacy model. Female, 4- to 6-week-old, outbred ICR mice (Harlan Sprague Dawley,Houston, Tex.) with a mean weight between 20 and 25 g were usedas previously described (16, 17). Each dosing group was composedof six animals. For the bacterial inoculation, the differentstrains of enterococci were grown on brain heart infusion (BHI)agar (Difco Laboratories, Detroit, Mich.) plates, then inoculatedin BHI broth, and incubated at 37°C overnight. Mice wereinjected intraperitoneally with 1 ml of the solution containingthe infecting enterococcal strain in 12.5% sterile rat fecalextract, resulting in an inoculum of about 10 times the minimumlethal dose (16). Antibiotics were given subcutaneously as asingle dose immediately after the intraperitoneal injection.Based on previous observation of the time course of disease,mice infected with E. faecalis were monitored for 96 h, andthose infected with E. faecium were monitored for 120 h. The50% lethal dose and the 50% protective dose (PD₅₀) were determinedby the method described by Reed and Muench (12). Preapprovedguidelines of the Animal Welfare Committee of the Universityof Texas Health Science Center at Houston were followed throughoutthe animal experiments. Bacteria were recovered from the spleensof dead mice and saved for phenotypic comparison and/or pulsed-fieldgel electrophoresis.

RESULTS AND DISCUSSION

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Results and Discussion

The E. faecalis and E. faecium strains used in this study wereall inhibited by $<=$ 0.125 µg of tigecycline/ml (Table 1);the MIC values obtained were not influenced by the tetracyclineor vancomycin susceptibility pattern of the specific isolate.

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TABLE 1. MICs and PD₅₀s after a single subcutaneous dose of several antibiotics for three E. faecalis and four E. faecium strains

Table 1 displays the MICs and PD₅₀s for the three E. faecalisand four E. faecium isolates tested, and Fig. 1 shows two dose-responsecurves. PD₅₀ values from previous experiments done in our laboratoryunder different conditions were included for ampicillin andvancomycin (single subcutaneous dose) against OG1RF and TX2465and for quinupristin-dalfopristin (single intravenous dose)against TX0016 and TX2465, which were previously reported (17).None of the mice in the drug control groups (where only theantibiotic is given) died, and none of the mice in the infectioncontrol groups (where mice are infected and no antimicrobialagent is given) survived.

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FIG. 1. Survival and dose-response curves for mice intraperitoneally infected with wild-type OG1RF and a tetracycline-resistant derivative. Therapy was given subcutaneously immediately after intraperitoneal injection of bacteria. (A) OG1RF, E. faecalis strain sensitive to tetracycline; (B) INY1200 (OG1RF::Tn925), an OG1RF derivative carrying the Tet(M) tetracycline resistance determinant.

Both tetracycline and tigecycline had protective effects (PD₅₀ $<=$ 6 mg/kg) against the tetracycline-susceptible E. faecalis strainOG1RF (ATCC 47077) (Fig. 1A), although the PD₅₀ value for tigecyclinewas approximately 10 times lower than the PD₅₀ obtained withtetracycline. Against the E. faecalis strain INY1200 (OG1RF::Tn925)(Fig. 1B), tetracycline and minocycline did not decrease mouselethality, even when doses up to 200 mg/kg of body weight wereadministered; tigecycline, on the other hand, had a low PD₅₀value (1.9 mg/kg), similar to that obtained against the parentaltetracycline-susceptible strain OG1RF. For E. faecalis V583,tigecycline and minocycline displayed protection against thisstrain. Among the E. faecium isolates tested, tigecycline andminocycline were effective against the VanA-type strain TX5034.Mice infected with the derivative TX5037 (TX5034::Tn925), carryingthe Tet(M) resistance determinant, were effectively protectedby tigecycline (PD₅₀, 3.6 mg/kg), while tetracycline was inactive(PD₅₀, >200 mg/kg). When the tetracycline-resistant E. faeciumclinical isolate TX0016 was tested, minocycline showed no protectiveeffect, vancomycin was moderately effective (PD₅₀ = 16 mg/kg),and tigecycline displayed the lowest PD₅₀ value (5.7 mg/kg).Against the E. faecium strain TX2465, another VanA type of isolate,tigecycline displayed a slightly lower PD₅₀ value (5.5 mg/kg)than that obtained with minocycline (9.3 mg/kg).

In a recent study (6), tigecycline MICs were determined against1,203 clinical isolates by broth microdilution methods. Thetigecycline MICs at which 90% of isolates were inhibited were $<=$ 0.25 µg/ml and 0.12 µg/ml for 100 vancomycin-susceptibleand vancomycin-resistant E. faecalis and E. faecium strainsand for 10 Enterococcus casseliflavus and Entercoccus gallinarum(genotype vanC) strains, respectively. In the same study, allminocycline-resistant enterococci (4 E. faecalis, 13 E. faecium,and 1 E. hirae isolate) had tigecycline MICs of $<=$ 0.25 µg/ml.Another study (3) compared the in vitro activities of tigecyclinewith those of doxycycline, minocycline, and tetracycline against527 gram-positive clinical isolates. One third of the enterococci(including several strains of E. faecalis and E. faecium withVanA and VanB types of glycopeptide resistance) were resistantto minocycline and doxycycline, and 44% were resistant to tetracycline;however, all were inhibited by tigecycline at $<=$ 2 µg/mldemonstrating, again, that tigecycline retains its activityagainst enterococci resistant to tetracyclines as a class. Inother studies in animals, tigecycline produced a statisticallysignificant bacterial count reduction compared to untreatedanimals against vancomycin-susceptible and vancomycin-resistantE. faecalis isolates and a VanA-type E. faecium strain in arat and rabbit model of endocarditis, respectively (7) (A. Lefort,M. Lafaurie, A. Saleh-Mghir, L. Garry, M. C. Peker, D. Le Guludec,C. Carbon, and B. Fantin, Abstr. 39th Intersci. Conf. Antimicrob.Agents Chemother., abstr. 415, 1999).

In summary, the new glycylcycline tigecycline showed good potencyin vitro and in vivo in a mouse peritonitis model against severalE. faecalis and E. faecium strains, including tetracycline-resistantstrains, suggesting that this may be an attractive agent forthe treatment of enterococcal infections and that further studiesin humans may be warranted.

ACKNOWLEDGMENTS

This study was supported by a grant from Wyeth Research.

FOOTNOTES

* Corresponding author. Mailing address: Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School —Houston, 6431 Fannin, 1.728 JFB, Houston, TX 77030. Phone: (713) 500-6745. Fax: (713) 500-5495. E-mail: bem.asst{at}uth.tmc.edu.

REFERENCES

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