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Antimicrobial Agents and Chemotherapy, November 2002, p. 3669-3675, Vol. 46, No. 11
0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.11.3669-3675.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Medicine, Immunology and Molecular Genetics, University of California Los Angeles School of Medicine, Los Angeles, California 90024,1 Research Services, Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, California 90073,2 Medical Services, Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, California 90073,3 Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles School of Medicine, Los Angeles, California 900244
Received 25 October 2001/ Returned for modification 24 January 2002/ Accepted 6 August 2002
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ABSTRACT |
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4 µg/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 µg/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were 4 µg/ml. Faropenem had MICs of 8 and 16 µg/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 µg/ml). MICs were 4 µg/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested. |
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The bacteria included in this study were recent clinical isolates from the Greater Los Angeles VA Healthcare Center. Bacteria were identified according to established procedures (5). MICs were determined by the NCCLS-approved Wadsworth agar dilution technique using 105 CFU/spot of inoculation and Brucella base-laked blood agar (6). Plates were incubated in an anaerobic chamber (Anaerobe Systems, San Jose, Calif.) for 48 h at 37°C. MICs were defined 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. The following antimicrobial agents were obtained as powders: faropenem (Bayer Corporation, West Haven, Conn.), imipenem and cefoxitin (Merck, Rahway, N.J.), amoxicillin-clavulanate (GlaxoSmithKline, West Sussex, United Kingdom), and clindamycin and metronidazole (Sigma, St. Louis, Mo.).
The presence of the metallocarbapenemase gene cfiA was detected by PCR (16). An aliquot (2.5 µl) from a boiled, centrifuged lysate of each strain to be tested was added to a 47.5-µl reaction cocktail to give a final reaction mixture containing 1x Taq buffer B, 2.5 U of Taq DNA polymerase, 3.0 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, 0.4 µM primer F677cfiA (5'-TGGGGTATGGTACCTTCCAA-3'), and 0.4 µM primer R1080cfiA (5'-AGCATACATCCGCCAAAAAG-3'). Reactions generating a band with a mobility of 403 bp indicated the presence of cfiA within the strain.
Table 1 lists the activity of faropenem compared to those of the other antimicrobial agents tested. For analytical purposes, the bacteria were grouped into species or genus groups of more than five isolates and the MIC ranges, MICs at which 50% of the isolates were inhibited (MIC50s), and MIC90s were reported (Table 1). The geometric mean was calculated when all values were within the range tested (i.e., no "greater than" values). As the breakpoint for faropenem has not yet been established, the percents susceptible, intermediate, and resistant were not reported for that agent. Overall, the MIC90 of faropenem (2 µg/ml) was twofold higher than the MIC90 of imipenem (1 µg/ml), as was the MIC50. However, in several groups of bacteria, this was not the case (see results below).
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In B. fragilis, high-level carbapenem resistance is mediated by the cfiA gene and requires an upstream insertion sequence (IS) element. Several such elements have thus far been identified: IS1186 (10), IS1187 and IS1188 (11), and IS942 (13). Other studies have also described strains that have the cfiA gene yet do not have high-level penem resistance and have shown that these strains do not have the IS element, which is associated with high-level cfiA expression (4). Additional studies to determine whether the cfiA-positive strains identified in this study have upstream ISs are under way.
Three additional strains for which faropenem had intermediate MICs did not have the cfiA gene. Faropenem had an MIC of 16 µg/ml for an additional isolate of B. fragilis. This strain was sensitive to imipenem (MIC of 1 µg/ml). Faropenem had MICs of 8 µg/ml for two additional isolates of B. fragilis (MICs of imipenem were 4 and 2 µg/ml for these isolates). The MICs of these strains were reconfirmed by repeat testing. These strains all had the cepA ß-lactamase gene, but the CepA ß-lactamase is not active against carbapenems. The mechanism of this intermediate carbapenem resistance is not yet known. In other species, synergy between efflux pumps and the permeability barrier may contribute to penem resistance (7) and may affect penem resistance selectively. For example, changes in a specific porin may affect imipenem but not other penems (8, 9; T. Kohler, C. Cherbuliez, and J. C. Pechere, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1519, p. 98, 2001). Also, efflux pumps in P. aeruginosa, for example, have preferences for certain penem substrates (J. Kohler, K. Young, R. E. Painter, J. A. Inumerable, and L. L. Silver, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1518, p. 97, 2001). Such mechanisms have not yet been described for B. fragilis.
One strain each of Porphyromonas endodontalis, Porphyromonas asaccharolytica, and Porphyromonas levii was clindamycin resistant. Four strains of Prevotella bivia and four strains of Prevotella species were clindamycin resistant. All other strains of Porphyromonas and Prevotella species were susceptible to all of the agents tested.
Bilophila wadsworthia was the third most common anaerobe isolated (after the B. fragilis group and Peptostreptococcus species) in cases of perforated or gangrenous appendicitis (2) and was found in nearly one-half of all specimens. MICs of both faropenem and imipenem were 0.5 µg/ml for all of the strains tested. All strains of Bilophila were susceptible to the other agents except for two strains that were resistant to cefoxitin. Sutterella wadsworthensis, a gram-negative anaerobic rod described in 1996 (17), was found in more than 10% of intra-abdominal specimens. The penems had MICs of 2 µg/ml for Sutterella species. Four strains of Sutterella were resistant to clindamycin, and two strains were resistant to metronidazole. All of the strains of Fusobacterium nucleatum, Fusobacterium mortiferum/varium, and other Fusobacterium species were sensitive to all agents except for seven strains of the F. mortiferum/varium group which were resistant to clindamycin. For several of the gram-negative rods tested, both the geometric mean and MIC90 of faropenem were lower than the corresponding values of imipenem (B. wadsworthia, S. wadsworthensis, and Campylobacter species). The geometric mean MIC and MIC90 for F. mortiferum/varium was threefold lower for faropenem than for imipenem (1 and 8 µg/ml, respectively); similarly, the geometric mean MIC was lower (0.4 and 1.4 µg/ml, respectively).
Faropenem had MICs of 16 and 32 µg/ml, respectively, for two strains of Clostridium difficile; these strains were also imipenem resistant. Faropenem had MICs of 8 µg/ml for two strains of Clostridium innocuum, for which imipenem MICs were 1 to 2 µg/ml. Faropenem had MICs of 4 µg/ml for all other strains of Clostridium tested.
Faropenem had an MIC of 8 µg/ml for one strain and MICs of 16 µg/ml for one strain of Peptostreptococcus anaerobius (MICs of imipenem were 1 to 2 µg/ml). These strains also had elevated MICs of amoxicillin-clavulanate (2 to 32 µg/ml versus the mean MIC of 0.5 µg/ml) and cefoxitin (8 to 16 µg/ml versus the mean MIC of 3.2 µg/ml). There are no descriptions of resistance mechanisms for Peptostreptococcus species that would account for these results. Faropenem and imipenem had MICs of <4 µg/ml for all other strains of gram-positive anaerobic cocci (55 isolates) and for all strains of non-spore-forming gram-positive rods (27 isolates).
There is limited information about the activity of faropenem against anaerobes (14). Spangler et al. studied the activity of WY-49605 (an earlier designation for faropenem) against anaerobes and found no B. fragilis group isolates with MICs of >4 µg/ml (the MIC90s for B. fragilis group isolates were 1 to 4 µg/ml, depending on the species) (15). Woodcock et al. reported an MIC90 of 4 µg/ml for 24 strains of B. fragilis; the MIC range extended to 32 µg/ml (18). No strains of resistant Peptostreptococcus (19 strains tested) were seen in that study. Strains of C. difficile with moderately high MICs have been found (MIC50 of 4 µg/ml and MIC90 of 8 µg/ml [15, 18]). MICs for Peptostreptococcus were 2 µg/ml, and the MIC90 was 1 µg/ml (15).
Faropenem is stable to many ß-lactamases, including TEM-1, SHV-1, TEM-3, and TEM-9 (18). ß-Lactamases belonging to Class-group 2f, including Imi-I from Enterobacter cloacae, hydrolyze penems and carbapenems at modest rates (12). This class of ß-lactamase has not been reported in Bacteroides. Both penems and carbapenems are also degraded by the metallo-ß-lactamases coded for by the cfiA gene (1, 19). Imipenem resistance in anaerobes is most often attributable to degradation by this enzyme, but there may be other mechanisms that are also important (3). In aerobes, both efflux pumps and permeability barriers contribute to carbapenem resistance (7). These mechanisms have not yet been described in anaerobes.
Faropenem is a promising novel oral penem agent with very good activity against a wide range of gram-negative and gram-positive anaerobic bacteria. Efficacy will need to be confirmed in clinical trials.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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