INTRODUCTION

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Approximately 20% of the 5 million people bitten by animals each year in the United States are allergic to penicillin or beta-lactam agents (5, 20, 22). The selection of an alternative antimicrobial can be problematic. In the past, doxycycline and minocycline have shown in vitro activity against common animal and human bite pathogens, including Pasteurella multocida and Eikenella corrodens (6, 7), and have shown clinical utility (5, 22). However, tetracycline resistance among both aerobic and anaerobic bacteria has increased, and consequently, tetracycline and its derivatives have been relegated to second- and third-line therapies by many clinicians.

GAR-936 is a synthetic analogue of minocycline that has activity against tetracycline-resistant strains that possess either ribosomal protection, such as tet(M), or active efflux mechanisms, such as tet(A), tet(B), etc. (1, 17, 18). Preliminary studies have shown GAR-936 to be active against a broad range of aerobic and anaerobic bacteria, including staphylococci, streptococci, Prevotella spp., and peptostreptococci (2, 3, 9, 17). In addition, GAR-936 is undergoing clinical trials for safety and efficacy in the treatment of complicated skin and soft-tissue infections. In order to determine the potential efficacy of GAR-936 in the treatment of skin and soft-tissue infections associated with human and animal bites, we studied its comparative in vitro activity against 416 clinical isolates.

	MATERIALS AND METHODS

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The strains used in this study were recent isolates from infected skin and soft-tissue bite wounds in humans. All isolates were identified by standard criteria (8, 12, 13, 19). The specific sources were dog bites (184), cat bites (191), human bites (18), and other animal bites (23). The numbers and species of isolates tested are given in Table 1.

Standard laboratory powders were supplied as follows: GAR-936 and minocycline, Wyeth-Ayerst Research, Pearl River, N.Y.; azithromycin and doxycycline, Pfizer Inc., New York, N.Y.; erythromycin and vancomycin, Eli Lilly & Co., Indianapolis, Ind.; levofloxacin, Ortho McNiel Pharmaceuticals, Raritan, N.J.; moxifloxacin, Bayer Corp., West Haven, Conn.; and penicillin G and tetracycline, Sigma Chemical Co., St. Louis, Mo.

Antimicrobial agents were reconstituted according to the manufacturers' instructions. Serial twofold dilutions of antimicrobial agents were prepared on the day of the test and added to the media in various concentrations.

Frozen cultures were transferred twice on tryptic soy agar supplemented with 5% sheep blood or chocolate agar (Hardy Diagnostics, Santa Maria, Calif.) for the aerobes and brucella agar supplemented with hemin, vitamin K₁, and 5% sheep blood (Anaerobe Systems, Morgan Hill, Calif.) for the anaerobes to ensure purity and good growth. Susceptibility testing was performed according to NCCLS standards (14, 15). Brucella agar supplemented with hemin, vitamin K₁, and 5% laked sheep blood was the basal medium used for anaerobic species and for E. corrodens, Bergeyella zoohelcum, and Capnocytophaga spp. Mueller-Hinton agar was used for staphylococci, and Mueller-Hinton agar supplemented with 5% sheep blood was used for the remainder of the organisms.

The agar plates were inoculated with a Steers replicator (Craft Machine Inc., Chester, Pa.). The inoculum used for aerobic bacteria was 10⁴ CFU per spot, and the inoculum used for E. corrodens and anaerobic bacteria was 10⁵ CFU per spot. Control plates without antimicrobial agents were inoculated before and after each set of drug-containing plates. Plates with aerobic isolates were incubated at 35°C in an aerobic environment for 18 to 20 h and then examined. E. corrodens, B. zoohelcum, Capnocytophaga spp., and streptococci were incubated in 5% CO₂ for 42 to 44 h and were then examined. Plates with anaerobes were incubated in an anaerobic chamber (Anaerobe Systems) at 35°C for 48 h and then examined.

The control strains tested included Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Bacteroides fragilis ATCC 25285, and Bacteroides thetaiotaomicron ATCC 29741. These strains were tested simultaneously with the appropriate plates and environments. The MIC was defined as the lowest concentration of an agent that yielded no growth or a marked change in the appearance of growth compared to the growth control plate.

	RESULTS

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The results of our study are shown in Table 1. GAR 936 was very active, with an MIC at which 90% of the strains were inhibited (MIC₉₀) of 0.25 µg/ml, against all aerobic gram-positive strains, including tetracycline-resistant strains of Enterococcus, Streptococcus, coagulase-negative staphylococci, and Corynebacterium spp. GAR 936 was also very active against all aerobic gram-negative strains, with an MIC₉₀ of 0.25 µg/ml for all isolates with the exception of E. corrodens (MIC₉₀, 4 µg/ml), of which 3 of 18 strains required 2 to 4 µg/ml for inhibition while the other 15 strains were susceptible to 0.5 µg of GAR-936/ml. Sixty of the 62 Pasteurellaceae isolates tested, including Pasteurella multocida subsp. multocida, Pasteurella multocida subsp. septica, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis, were susceptible to 0.06 µg of GAR-936/ml; two isolates of P. stomatis required 0.125 µg of GAR-936/ml for inhibition. GAR-936 was also very active against all anaerobic species, including tetracycline-, doxycycline-, and minocycline-resistant strains of Prevotella spp. (such as Prevotella heparinolytica, Prevotella melaninogenica, Prevotella bivia, and Prevotella loeschii, Porphyromonas spp. (such as Porphyromonas levii), Bacteroides tectum, and Peptostreptococcus spp. and had an MIC₉₀ of 0.25 µg/ml. Macrolide (erythromycin and azithromycin)- and fluoroquinolone (levofloxacin and moxifloxacin)-resistant Fusobacterium nucleatum and other Fusobacterium spp. were susceptible to GAR-936 (MIC₉₀, 0.06 µg/ml).

	DISCUSSION

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Selection of an inappropriate antimicrobial agent for the therapy of infected bite wounds can lead to therapeutic failure and long-term sequelae (5, 10, 11). While beta-lactams have been the traditional drugs of choice, many patients report a history of penicillin allergy or side effects and require the selection of an alternative agent. This choice has been somewhat problematic in the past, since erythromycin MICs against bite pathogens have been inconsistent (4) and clinical failures of erythromycin therapy have been reported (10, 11, 16). Other agents, such as the fluoroquinolones, were also attractive, but some relatively common bite isolate species, such as the fusobacteria, were often resistant (6, 7). Our prior clinical experience had suggested that tetracyclines were attractive alternative agents, but tetracycline resistance evolved, both because of efflux-based and ribosomal protection mechanisms, and some bite isolates were resistant (1, 5, 17).

GAR-936 is a derivative of minocycline that has activity against tetracycline-resistant strains that possess either ribosomal protection or active efflux mechanisms (17, 18). In vitro data has shown GAR-936 to be active against a broad range of gram-positive and gram-negative pathogens (1, 3, 17, 18, 21). van Ogtrop et al. (21) have shown GAR-936 to be active in an experimental in vivo murine thigh infection model and to have good activity against S. aureus and other gram-positive and gram-negative aerobic bacteria. They stated that GAR-936 would be a "promising drug(s) for the treatment of staphylococcal infections" and suggested that, based on their model, "the theoretical breakpoint MIC" would be about 0.5 µg/ml.

In our study, GAR-936 showed excellent activity against the full spectrum of 268 aerobic and 148 anaerobic clinical bite wound isolates. Many of our isolates were resistant to tetracycline and tetracycline analogues, such as doxycycline and minocycline, yet, of all the aerobic and anaerobic bacteria studied, the GAR-936 MICs for only 3 of 18 E. corrodens isolates were >0.5 µg/ml. GAR-936 was active against typical primary animal bite pathogens, such as P. multocida subspecies (all 30 strains were susceptible to 0.06 µg/ml), and secondary invaders, such as S. aureus (all 15 isolates were susceptible to 0.125 µg/ml). In addition, GAR-936 was active against macrolide-resistant aerobic isolates, such as Corynebacterium aquaticum, Corynebacterium spp., E. corrodens, enterococci, coagulase-negative staphylococci, and levofloxacin-resistant corynebacteria. Gales and Jones (3) studied the activities of GAR-936 against 1,203 recent clinical isolates and noted its improved activity compared to older tetracyclines as well as its broad spectrum of activity. While most of the isolates in our study are not represented in their data, their GAR-936 MIC₉₀ of 0.25 µg/ml against both oxacillin-susceptible and -resistant S. aureus was one dilution higher than that found for our S. aureus isolates. In our study, moxifloxacin exhibited good in vitro activity against all aerobic bite isolates.

Among anaerobic bacteria, GAR-936 also exhibited excellent activity against isolates, including macrolide-, levofloxacin-, and moxifloxacin-resistant F. nucleatum and other Fusobacterium spp. and tetracycline-, minocycline-, and doxycycline-resistant isolates of B. tectum, P. heparinolytica, Prevotella spp., Porphyromonas macaccae, Porphyromonas spp. (P. levii), and peptostreptococci. Of note, the GAR-936-susceptible peptostreptococci showed resistance to erythromycin, azithromycin, levofloxacin, tetracycline, doxycycline, and minocycline. Edlund and Nord (2) studied the activity of GAR-936 against 327 anaerobes, using PDM-ASM media supplemented with 5% horse blood. The species they studied differed from the species of our bite isolates in most instances. In general, our results were similar for peptostreptococci, and both studies showed GAR-936 MIC₉₀ of 0.06 µg/ml against F. nucleatum isolates.

Overall, GAR-936 exhibited the best activity of the agents tested against the full spectrum of aerobic and anaerobic bite isolates, including multidrug-resistant strains. This excellent in vitro activity warrants its further investigation for clinical use in skin and soft-tissue infections, including those due to human and animal bites.