ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2000, p. 2222–2224 0066-4804/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 8
In Vitro Activities of MK-826 (L-749,345) against 363 Strains of Anaerobic Bacteria HANNAH M. WEXLER,1,2* DENISE MOLITORIS,1 3
AND
SYDNEY M. FINEGOLD1,2,3,4
1
Medical and Research Services, Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, California 90073, and Departments of Medicine2 and Microbiology and Immunology,4 UCLA School of Medicine, Los Angeles, California 90024 Received 31 January 2000/Returned for modification 22 February 2000/Accepted 2 May 2000
The activity of MK-826 was compared to the activities of cefoxitin, ceftriaxone, imipenem, and meropenem against 363 gram-negative and gram-positive anaerobes by using NCCLS procedures. At least 98% of the strains were susceptible to the carbapenems. All strains of Clostridium perfringens, Fusobacterium nucleatum, Peptostreptococcus, and Sutterella wadsworthensis were susceptible to all agents tested. B. fragilis strains were inhibited by all the carbapenems at ⱕ4 g/ml. Cefoxitin inhibited 96% of strains at 16 g/ml and 98% of strains at 32 g/ml. Ceftriaxone was less active against these strains, inhibiting 42% at 16 g/ml and 70% at 32 g/ml. A similar pattern was seen with other B. fragilis group species: all strains were inhibited by the carbapenems at ⱕ8 g/ml, and cefoxitin and ceftriaxone inhibited 97 and 44%, respectively, at 32 g/ml. For the B. fragilis group species other than B. fragilis, MK-826 tended to have higher MICs for B. thetaiotaomicron (60% of MICs were ⱖ1 g/ml) than for the other species (16% of MICs were ⱖ1 g/ml). Bilophila wadsworthia, a recently described gram-negative anaerobe, was the third most common anaerobe isolated from cases of perforated or gangrenous appendicitis (2). All the agents tested inhibited all strains of Bilophila wadsworthia; the carbapenems had MIC90s of 0.12 g/ml or less. Prevotella and Porphyromonas species were also very susceptible to the carbapenem agents, with MIC90s ranging from 0.062 to 0.25 g/ ml. There was very little difference in carbapenem MICs between different species of Porphyromonas and Prevotella. Prevotella bivia and Prevotella buccae were more resistant to ceftriaxone than the other Prevotella species tested. Ceftriaxone had MICs of 64 g/ml for one strain each of Prevotella buccae and Prevotella bivia; the MICs of cefoxitin for these strains were 2 and 1 g/ml, respectively. Ceftriaxone had MICs of 32 g/ml for an additional three strains of these two species and an MIC of 256 g/ml for one strain of Porphyromonas gingivalis (the cefoxitin MIC was 4 g/ml). Campylobacter gracilis was inhibited by the carbapenems at ⱕ0.5 g/ml and by cefoxitin at ⱕ16 g/ml. One strain of Campylobacter gracilis was resistant to ceftriaxone. Sutterella wadsworthensis, a gramnegative anaerobic rod described in 1996 (11), is found in more than 10% of intraabdominal specimens. S. wadsworthensis, Fusobacterium nucleatum, and other Fusobacterium species were all susceptible to all of the agents tested (other than Fusobacterium mortiferum and ceftriaxone). MICs of the carbapenems did not vary significantly among the different species of Fusobacterium tested. Five strains of F. mortiferum were resistant to ⱖ128 g of ceftriaxone/ml; additionally, cefoxitin MICs tended to be two twofold dilutions lower for F. necrophorum than for the F. mortiferum/varium group. Clostridium difficile was inhibited by all of the carbapenems at ⱕ8 g/ml. At 4 g/ml, 100, 94, and 53% of strains, respectively, were susceptible to meropenem, imipenem, and MK826. The variation among the agents may be significant, or it may be a function of the MICs clustering at breakpoint con-
Antimicrobial resistance in anaerobes has been found for virtually all classes of antimicrobial agents. Resistance to betalactams is generally due to the production of beta-lactamases; resistance to beta-lactam–beta-lactamase combinations, clindamycin, macrolides, tetracyclines, and 5-nitroimidazoles has also been seen (9). Imipenem resistance is generally due to metallo-beta-lactamase enzymes, which hydrolyze the antimicrobial agent (1, 3). MK-826 (ertapenem; formerly L-749,345) is a potent 1-beta-methyl carbapenem with a long half-life and a broad spectrum of activity (5). MK-826, like other carbapenems, exerts its activity by binding to penicillin-binding proteins and inhibiting cell wall synthesis; MK-826 showed highaffinity binding to the essential penicillin-binding proteins of Escherichia coli (7). MK-826 was shown to be very potent against extended-spectrum and broad-spectrum beta-lactamase-producing gram-negative pathogens (7). MK-826 has been evaluated against many aerobes (4, 5, 6), but its antianaerobic spectrum has not been evaluated. This study was designed to evaluate the efficacy of MK-826 and four comparative agents against anaerobic bacteria. The bacteria included in this study were recent clinical isolates from the Greater Los Angeles Veterans Administration Healthcare Center. Bacteria were identified according to established procedures (10). MICs were determined by the NCCLSapproved Wadsworth agar dilution technique, using 105 CFU of inoculum per spot and brucella base–laked-blood agar (8). Plates were incubated in an anaerobic chamber (Anaerobe Systems, San Jose, Calif.) for 48 h at 37°C. MICs were defined as the lowest concentration of antimicrobial resulting in a marked change in the appearance of growth as compared to the control plate, as described in the NCCLS protocol. Reference strains of Bacteroides fragilis (ATCC 25285) and Bacteroides thetaiotaomicron (ATCC 29741) were used as controls in each test. Antimicrobial agents were obtained as powders from the following companies: cefoxitin, imipenem, and MK-826 (Merck, Rahway, N.J.), ceftriaxone (Sigma, St. Louis, Mo.), and meropenem (AstraZeneca, Wilmington, Del.). For analysis, the bacteria tested were placed into species or genus groups with more than five isolates, and MIC ranges and the MICs at which 50 or 90% of the isolates were inhibited (MIC50 and MIC90) were determined (Table 1). * Corresponding author. Mailing address: Microbial Diseases Research Laboratory, Bldg. 304, Room E3-224, VAGLAHS 691/151J, Los Angeles, CA 90073. Phone: (310) 268-3404. Fax: (310) 268-4458. E-mail:
[email protected]. 2222
TABLE 1. Activity of MK-826 against anaerobic bacteria Organism (no. of isolates) and drug
MIC (g/ml)
Organism (no. of isolates) and drug
MIC (g/ml)
Geometric mean
50%
90%
Range
Bacteroides fragilis (50) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
5.6 20.6 0.1 0.3 0.2
4 32 0.062 0.25 0.12
8 ⬎256 0.50 2 2
1–128 2–⬎256 0.06–1 0.12–4 0.06–4
Other B. fragilis group species (70)a Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
Fusobacterium nucleatum (14) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1 1.5 0.1 0.06 0.06
1 2 0.062 0.062 0.062
8.9 37.6 0.4 0.5 0.2
16 64 0.50 0.50 0.25
32 256 0.50 2 0.50
1–128 1–⬎256 0.06–8 0.06–4 0.06–4
Other Fusobacterium species (15)e Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
2.3 8.7 0.33 0.08 0.11
2 4 0.5 0.06 0.06
8 128 0.25 0.12 0.50
1–8 1–128 0.06–0.25 0.06–0.12 0.06–0.50
Clostridium difficile (17) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1–32 1–2 0.06–0.25 0.06–0.06 0.06–0.06
Clostridium perfringens (15) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1 1.3 0.1 0.1 0.06
1 1 0.062 0.062 0.062
Clostridium ramosum (10) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
5.3 2.3 0.3 0.8 0.6
2 1 0.25 0.50 0.50
1–8 1–64 0.06–0.25 0.06–0.25 0.06–0.12
Peptostreptococcus species (29)f Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1 1.5 0.06 0.1 0.06
1 1 0.062 0.062 0.062
1 4 0.062 0.12 0.062
1–2 1–4 0.06–0.06 0.06–0.25 0.06–0.06
1–2 1–1 0.06–1 0.06–0.06 0.06–0.06
Non-spore-forming grampositive rods (31)g Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1.7 1.6 0.1 0.2 0.1
1 1 0.062 0.12 0.062
8 8 0.25 1 0.25
1–16 1–64 0.06–0.50 0.06–2 0.06–0.50
1–16 1–⬎256 0.06–0.50 0.06–0.25 0.06–0.25
Total (363) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
3.2 5.3 0.2 0.2 0.1
2 4 0.12 0.12 0.062
16 128 0.50 1 0.50
Other Bacteroides species (9)b Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1.4 2.3 0.1 0.1 0.1
1 1 0.062 0.062 0.062
Bilophila wadsworthia (14) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
4 1.2 0.1 0.06 0.06
4 1 0.12 0.062 0.062
Porphyromonas species (10)c Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem Prevotella species (50)d Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1.1 1.7 0.1 0.1 0.1 1.5 3.2 0.1 0.1 0.1
1 1 0.062 0.062 0.062 1 1 0.062 0.12 0.062
Sutterella wadsworthensis (15) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
1 1 0.4 0.06 0.06
1 1 0.50 0.062 0.062
Campylobacter gracilis (14) Cefoxitin Ceftriaxone Imipenem MK-826 Meropenem
0.4 1.9 0.3 0.1 0.1
2 1 0.25 0.062 0.062
8 2 0.12 0.062 0.062
1 1 0.062 0.062 0.062 4 16 0.062 0.25 0.12
1 1 1 0.062 0.062 16 ⬎256 0.50 0.12 0.25
1–4 1–256 0.06–0.25 0.06–0.25 0.06–0.12
Geometric mean
66.7 37.7 3.5 5.1 1.7
50%
64 32 4 4 2
90%
1 2 0.12 0.062 0.062
Range
1–1 1–2 0.06–0.12 0.06–0.06 0.06–0.06
4 256 1 0.12 0.25
1–8 1–256 0.06–1 0.06–0.25 0.06–0.50
128 64 4 8 2
4–128 16–128 0.50–8 1–8 1–4
1 4 0.12 0.062 0.062 32 8 0.50 1 1
1–1 1–8 0.06–0.25 0.06–0.12 0.06–0.06 2–32 1–128 0.12–1 0.50–2 0.50–1
1–128 1–⬎256 0.06–8 0.06–8 0.06–4
a Bacteroides caccae (5 isolates); Bacteroides distasonis (10); Bacteroides ovatus (10); Bacteroides stercoris (2); Bacteroides thetaiotaomicron (22); Bacteroides uniformis (10); Bacteroides vulgatus (11). b Bacteroides capillosus (2 isolates); Bacteroides ureolyticus (3); Bacteroides splanchnicus (1); Bacteroides levii (2); Bacteroides species (1). c Porphyromonas asaccharolytica (4 isolates); Porphyromonas endodontalis (2); Porphyromonas gingivalis (4). d Prevotella bivia (9 isolates); Prevotella buccae (8); Prevotella disiens (4); Prevotella intermedia (10); Prevotella loescheii (2); Prevotella melaninogenica (9); Prevotella oralis (2); Prevotella oris (6). e Fusobacterium mortiferum (6 isolates); Fusobacterium varium (5); Fusobacterium necrophorum (4). f Peptostreptococcus anaerobius (9 isolates); Peptostreptococcus magnus (5); Peptostreptococcus micros (15). g Actinomyces israelii (2 isolates); Actinomyces naeslundii (1); Actinomyces odontolyticus (3); Actinomyces viscosus (1); Eubacterium lentum (7); Eubacterium limosum (2); Lactobacillus catenaforme (2); Lactobacillus jensenii (1); Lactobacillus species (1); Propionibacterium acnes (11).
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NOTES
ANTIMICROB. AGENTS CHEMOTHER.
centrations (since all strains were susceptible at 8 g/ml). Neither cefoxitin nor ceftriaxone was very active against C. difficile. All strains of Clostridium perfringens were inhibited by all agents at ⱕ1 g/ml, except for three strains of C. perfringens for which ceftriaxone had MICs of 2, 4, and 8 g/ml. One strain of Clostridium ramosum was resistant to ceftriaxone; all strains were susceptible to the carbapenems at ⱕ2 g/ml. All of the gram-positive cocci tested were susceptible to all of the agents at ⱕ4 g/ml. Among the gram-positive nonspore-forming rods, two strains were resistant to ceftriaxone; all other strains were susceptible to the agents tested (to the carbapenems at ⱕ2 g/ml and to cefoxitin at ⱕ16 g/ml). Carbapenems are generally stable to most beta-lactamases, although imipenem resistance has been observed in a number of B. fragilis isolates in Japan (up to 6%) and is usually attributed to imipenem-hydrolyzing metallo-beta-lactamases (1) (coded for by the cfiA gene [12]). The resistance is most often attributed to degradation by this enzyme, but there may be other mechanisms which are also important (3). The carbapenem MK-826 appears to have excellent activity against anaerobic bacteria (with the possible exception of C. difficile), as do the other carbapenem agents. Clinical studies are needed to assess the clinical utility of this agent in infections involving anaerobes. This study was funded in part by Greater Los Angeles Veterans Administration Medical Center research funds and in part by Merck Sharp and Dohme (Rahway, N.J.). REFERENCES 1. Bandoh, K., K. Ueno, K. Watanabe, and N. Kato. 1993. Susceptibility patterns and resistance to imipenem in the Bacteroides fragilis group species in Japan: a 4-year study. Clin. Infect. Dis. 16(Suppl. 4):S382–S386.
2. Bennion, R. S., E. J. Baron, J. E. Thompson, Jr., J. Downes, P. Summanen, D. A. Talan, and S. M. Finegold. 1990. The bacteriology of gangrenous and perforated appendicitis—revisited. Ann. Surg. 211:165–171. 3. Edwards, R., and D. Greenwood. 1996. Mechanisms responsible for reduced susceptibility to imipenem in Bacteroides fragilis. J. Antimicrob. Chemother. 38:941–951. 4. Fuchs, P. C., A. L. Barry, and S. D. Brown. 1999. In-vitro antimicrobial activity of a carbapenem, MK-0826 (L-749,345), and provisional interpretive criteria for disc tests. J. Antimicrob. Chemother. 43:703–706. 5. Gill, C. J., J. J. Jackson, L. S. Gerckens, B. A. Pelak, R. K. Thompson, J. G. Sundelof, H. Kropp, and H. Rosen. 1998. In vivo activity and pharmacokinetic evaluation of a novel long-acting carbapenem antibiotic, MK-826 (L749,345). Antimicrob. Agents Chemother. 42:1996–2001. 6. Jacoby, G., P. Han, and J. Tran. 1997. Comparative in vitro activities of carbapenem L-749,345 and other antimicrobials against multiresistant gramnegative clinical pathogens. Antimicrob. Agents Chemother. 41:1830–1831. 7. Kohler, J., K. L. Dorso, K. Young, G. G. Hammond, H. Rosen, H. Kropp, and L. L. Silver. 1999. In vitro activities of the potent, broad-spectrum carbapenem MK-0826 (L-749,345) against broad-spectrum beta-lactamase- and extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli clinical isolates. Antimicrob. Agents Chemother. 43:1170– 1176. 8. National Committee for Clinical Laboratory Standards. 1997. Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard, fourth ed. NCCLS document M11-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. 9. Rasmussen, B. A., K. Bush, and F. P. Tally. 1997. Antimicrobial resistance in anaerobes. Clin. Infect. Dis. 24(Suppl. 1):S110–20. 10. Summanen, P., E. J. Baron, D. Citron, C. Strong, H. M. Wexler, and S. M. Finegold. 1993. Wadsworth anaerobic bacteriology manual. Star Publishing Co., Belmont, Calif. 11. Wexler, H. M., D. Reeves, P. H. Summanen, E. Molitoris, M. McTeague, J. Duncan, K. H. Wilson, and S. M. Finegold. 1996. Sutterella wadsworthensis gen. nov., sp. nov., bile-resistant microaerophilic Campylobacter gracilis-like clinical isolates. Int. J. Syst. Bacteriol. 46:252–258. 12. Yamazoe, K., N. Kato, H. Kato, K. Tanaka, Y. Katagiri, and K. Watanabe. 1999. Distribution of the cfiA gene among Bacteroides fragilis strains in Japan and relatedness of cfiA to imipenem resistance. Antimicrob. Agents Chemother. 43:2808–2810.
