INTRODUCTION

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Ertapenem (MK-0826; L749,345; 1--methyl carbapenem (4R,5S,6S,8R,2¹S,4¹S)-3-[2-[[(3-carboxyphenyl)amino]carbonyl]pyrrolidin-4-yl]- 4-methyl-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-en-2-carboxylic acid monosodium salt) is a new parenteral carbapenem that is highly resistant to inactivation by a wide variety of beta-lactamases and has a broad spectrum of antimicrobial activity (4, 5, 8, 9, 14). It binds preferentially to penicillin-binding proteins 1b, 2, and 3. It is more resistant than imipenem to renal dehydropeptidase 1 inactivation and therefore does not require the addition of cilastatin. MK-0826 has a half-life of ~4.5 h, ~30 to 40% of a dose is excreted as intact drug in the urine, and its pharmacokinetic profile allows single daily dosing (5, 14). While the in vitro activity of MK-0826 against many aerobes has been reported (4, 5, 8, 9, 13), few data regarding its activity against anaerobes have been reported (M. D. Appleman, D. M. Citron, P. N. R. Heseltine, H. Belzberg, A. E. Yellin, J. Murray, and T. V. Berne, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-43, p. 244, 1998). In order to evaluate MK-0826's activity against anaerobes and to assess its potential utility in mixed infections, we studied its in vitro activity against 1,001 clinical isolates from human intra-abdominal infections.

	MATERIALS AND METHODS

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The 1,001 anaerobic strains studied were recently isolated (1998 to 1999) from human clinical infections as part of an international, multicenter clinical trial. Aspirated specimens of abdominal pus were obtained intraoperatively and were placed in anaerobic transport tubes (Anaerobe Systems, Morgan Hill, Calif.) and sent via express delivery services from the study site to the R. M. Alden Research Laboratory in Santa Monica, Calif. Twenty-nine study centers located in 17 countries worldwide contributed various numbers of specimens (see Table 1). Upon receipt, the specimens were placed in an anaerobe chamber and plated onto anaerobic media including supplemented brucella, phenylethyl alcohol blood, Bacteroides bile esculin, and kanamycin-vancomycin laked blood agars (Anaerobe Systems) and were incubated anaerobically at 37°C for 5 days. Trypticase soy blood agar, Rose agar, and MacConkey agar (Hardy Diagnostics, Santa Maria, Calif.) were used to recover aerobic organisms. Isolates were identified by standard criteria (7, 13) and were stored in skim milk at 70°C.

For the in vitro susceptibility studies, isolates were taken from frozen stocks and were subcultured twice on brucella agar supplemented with hemin, vitamin K₁, and 5% sheep blood. Susceptibility testing was performed by the reference agar dilution method according to the standards of the National Committee for Clinical Laboratory Standards (11). Brucella agar supplemented with hemin, vitamin K₁, and 5% laked sheep blood was the basal medium. The antimicrobial agents were reconstituted according to the manufacturers' instructions. Serial twofold dilutions of various concentrations of antimicrobial agents were prepared on the day of the test and added to the agar medium. Ampicillin-sulbactam was fixed in a ratio of 2:1; ticarcillin was diluted with clavulanate and was tested at a constant concentration of 2 µg/ml, and piperacillin was diluted with tazobactam and was tested at a constant concentration of 4 µg/ml.

The agar plates were inoculated with a Steers replicator (Craft Machine Inc., Chester, Pa.) with an inoculum of 10⁵ CFU per spot. The plates were incubated in an anaerobic chamber for 44 h at 37°C prior to examination. 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 on a control plate. Control strains Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741 were included for each drug tested. The numbers and species of isolates tested are given in Table 2.

Standard laboratory powders were supplied as follows: MK-0826, imipenem, and cefoxitin, Merck & Co., West Point, Pa.; ticarcillin and clavulanate, SmithKline Beecham, Philadelphia, Pa.; piperacillin and tazobactam, Wyeth-Ayerst, Philadelphia, Pa.; ampicillin and sulbactam, Pfizer Inc., New York, N.Y.; clindamycin, Pharmacia Upjohn Co., Kalamazoo, Mich.; metronidazole, Searle Research & Development, Skokie, Ill.; penicillin G and chloramphenicol, Sigma Chemical Co., St. Louis, Mo.; ceftriaxone, Roche, Nutley, N.J.; and vancomycin, Eli Lilly & Co., Indianapolis, Ind.

	RESULTS AND DISCUSSION

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Table 1 analyzes the demographics of the specimens received. Four hundred twenty-seven individual specimens were received from the 29 study sites in 17 countries. Bacterial growth was obtained for 72.7 to 100% of specimens (mean, 93%; median, 95.2%), usually as mixed growth, and yielded an average of 3 aerobes (range, 0 to 9) and 2.3 anaerobes (range, 0 to 13) per specimen. This particularly attests to the quality of the transport system used, since the median time from collection of specimens to processing was 4 days (range, 2 to 62 days). While specimens from Canada and certain European countries generally arrived within 36 to 72 h, the sometimes erratic policies of other international airlines and couriers meant that specimens from Mexico or South Africa, for example, often sat in transit up to 1 week from the day of collection; in the case of Russia, one specimen was "lost" for 6 weeks, yet it still yielded eight anaerobes.

Baron et al. (2) looked at the effect of long-distance transport on 10 specimens from diverse locations in the United States on isolate survival and found that it "did not compromise recovery of clinically relevant microbes." They also noted no difference due to the weather conditions at the source location on recovery. A study by Bennion et al. (3) on the bacteriology of samples obtained from 30 patients with gangrene and perforated appendicitis noted the recovery of 2.7 aerobes and 7.4 anaerobes per specimen. They used local transportation (mean time, 15.7 h) and had optimal conditions of collection, transport, and culture. A survey of all relevant literature prior to that time noted an overall mean of 1.2 aerobes and 0.9 anaerobes per patient (3).

The comparative activities of MK-0826 and the other agents tested are presented in Table 2. Determination of susceptibility to MK-0826 was based on the published preliminary breakpoints for susceptibility (4 µg/ml), intermediate (8 µg/ml), and resistance (16 µg/ml) (4). In general, MK-0826 was active at 8 µg/ml against all anaerobes tested, including B. fragilis group species, with the exception of 12 of 61 (20%) Bilophila wadsworthia isolates, 3 isolates of Lactobacillus spp. (1 L. acidophilus isolate and 2 L. casei isolates), and 1 isolate of Acidaminococcus fermentans, for which the MICs were 16 µg/ml. MK-0826 was generally 1 to 2 dilutions less active than imipenem except against B. wadsworthia. Appleman et al. (38th ICAAC) also studied the comparative activities of MK-0826 against 88 anaerobic isolates obtained from 60 patients with serious intra-abdominal infections and found that MK-0826 had "excellent activity" against the 41 B. fragilis group strains (MICs at which 90% of isolates are inhibited [MIC₉₀s], 4 µg/ml). Using the same agar dilution method that we used, they found that 99% of their isolates were susceptible to both MK-0826 and imipenem (MICs, 4 µg/ml).

The gene for metalloenzymes, both constitutive and plasmid mediated, has been reported to occur in ~1 to 3% of Bacteroides spp. (1, 12, 15). Expression requires the presence of zinc and a single-step conversion with insertion of an insertion sequence element into a promoter to cause carbapenem resistance. While this has been reported in clinical isolates, especially from Japan (1), only three strains of B. fragilis species showed some degree of carbapenem resistance in our study. For one strain of B. fragilis the MK-0826 MIC was 8 µg/ml (intermediate), for one strain of Bacteroides caccae the imipenem MIC was 32 µg/ml and the MK-0826 MIC was 8 µg/ml, and for one strain of Bacteroides uniformis the imipenem MIC was 16 µg/ml.

As expected with a "static" agent, the chloramphenicol MICs covered a wide range, with the chloramphenicol MICs for many isolates clustered at 4 to 8 µg/ml (the MIC for intermediate susceptibility is 16 µg/ml). While occasional metronidazole resistance has been noted (10, 15), including one report in which it was also associated with imipenem resistance, all of our 455 B. fragilis group species isolates were susceptible. Of note were our MIC₉₀s of metronidazole for B. fragilis, B. thetaiotaomicron, and B. caccae, 4 µg/ml, which is slightly higher than the values usually obtained. This was due to a "statistical" occurrence since the MIC₈₉ for each species was 2 µg/ml and was more usual. We retained the "accurate" but somewhat misleading value in Table 2. Overall, our data suggest that the frequency of metronidazole resistance remains low worldwide. Ceftriaxone had uniformly poor activity against B. fragilis group species. There appeared to be increasing levels of resistance of B. fragilis group species to ampicillin-sulbactam and cefoxitin compared to those in our prior survey (6).

Table 3 reports the comparative susceptibilities of the various agents studied by country when there were more than five isolates per genus or species. No differences were noted in the overall geographic susceptibilities of the anaerobes studied to MK-0826 or imipenem. B. wadsworthia isolates from the five countries from which such isolates were obtained had similar susceptibilities to all the agents studied. Appleman et al. (38th ICAAC) also noted that for 9 of the 10 strains of B. wadsworthia that they tested the MIC₉₀ of MK-0826 was 2 µg/ml and that one strain was highly resistant. Clindamycin susceptibility varied the most by geographical origin. For B. fragilis, the isolates from Canada and Mexico were relatively more susceptible than those from other locations, although the number of strains studied is relatively small. Clostridium innocuum isolates from Canada and South Africa were generally more susceptible to clindamycin than were those isolated from Brazil and Guatemala, perhaps reflecting local antibiotic usage patterns.

This study demonstrates the potent anaerobic activity of MK-0826 against a wide range of clinical anaerobic isolates from diverse locations worldwide. Clinical trials to correlate these findings are in progress.