1 Laboratory Specialists, Inc., Cleveland, OH; 2 M. S. Hershey Medical Center, Hershey, PA; 3 R. M. Alden Research Laboratory, Santa Monica, CA, USA; 4 Institute für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig, Leipzig, Germany; 5 ADRINORD, Lille, France
Received 27 October 2003; returned 14 January 2004; revised 12 March 2004; accepted 22 March 2004
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Abstract |
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Keywords: amoxicillin/clavulanic acid, anaerobes, susceptibility data
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Introduction |
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Anaerobic bacteria are commonly found in polymicrobial infections with reported incidence ranging from 4% to 50%,1,2 however, anaerobic cultures are not routinely carried out. Therefore, it is important to include an antimicrobial agent with known efficacy against anaerobes when empirically treating an infection with a high probability of anaerobic presence.24 Like the increasing antimicrobial resistance in common aerobic bacteria, anaerobes have also showed trends towards some increasing resistance in recent years.57 Since susceptibility testing for anaerobes is often not done, except for sterile-site isolates (blood, CSF, etc.), it is especially important to know the background susceptibility patterns in the community.8 This is commonly and efficiently done through regular surveillance studies. These studies, when carried out repeatedly over time, may additionally provide researchers and clinicians with advance notice of emerging patterns of resistance.
At present, empirical treatment of an infection with a high probability of anaerobe presence involves either the use of a very broad- spectrum antimicrobial agent or combined therapy with a drug effective against the known or suspected facultative anaerobes and one known to be effective against strict anaerobes.9 Some of the drugs known to target strict anaerobes include clindamycin and metronidazole. Both drugs have good efficacy against many anaerobes but clindamycin has no effect on enteric Gram-negative facultative anaerobic bacilli and metronidazole is essentially ineffective against most other classes of bacteria. Either drug is generally given in combination with aminoglycosides or cephalosporins. Penicillin and related ß-lactam drugs are often effective against both strict anaerobes and facultative anaerobes, but are often inactivated by bacteria that produce ß-lactamases. Carbapenems, a class of ß-lactams that includes imipenem and meropenem, are also very effective against both classes of bacteria and are generally not inactivated by ß-lactamases. Since many of the strict anaerobes are ß-lactamase producers, especially Bacteroides, Prevotella and some Fusobacterium species,10 it is often preferable to use a non-ß-lactam antimicrobial agent if that genus is suspected or include a ß-lactamase inhibitor such as clavulanic acid, sulbactam, or tazobactam, in combination with a broadly effective ß-lactam antimicrobial agent.11
Co-amoxiclav (amoxicillin/clavulanic acid) was developed to enhance the activity of amoxicillin against ß-lactamase-producing bacteria by the addition of clavulanic acid, an active ß-lactamase inhibitor. In over 20 years of use as an orally administered antimicrobial agent, co-amoxiclav has maintained exceptional in vitro activity against respiratory tract pathogens. This study was conducted to provide susceptibility data for co-amoxiclav and five other antimicrobial agents against several important pathogenic anaerobic bacteria collected from 14 institutions in the USA and Europe.
The antimicrobial agents tested included amoxicillin alone, to provide background comparison for amoxicillin without the ß-lactamase inhibitor clavulanic acid. Any bacterial isolates that do not produce ß-lactamase would be expected to have similar MIC data with amoxicillin and co-amoxiclav. Both metronidazole and clindamycin were chosen as comparators because of their historical efficacy against anaerobes, but also because of reports of increasing incidence of resistance.12,13 Since fluoroquinolones and related drugs are often used in polymicrobial infections, levofloxacin was included to represent the group.14,15 Finally, imipenem, a broad-spectrum carbapenem that is often used to treat anaerobic infections, was also included. Many of the drugs used as comparators in this study have also been used in several previously published surveillance and comparison reports.1619
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Materials and methods |
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The following antimicrobial agents were tested: co-amoxiclav at a fixed clavulanic acid concentration of 2 mg/L (A/C 2), co-amoxiclav at a 2:1 amoxicillin/clavulanic acid concentration ratio (A/C 2:1), amoxicillin, clindamycin, imipenem, levofloxacin, and metronidazole. Co-amoxiclav was tested in two formulations to account for the differences in testing methods in the USA and Europe. In the USA, the concentration of amoxicillin to clavulanic acid is tested at a ratio of 2:1, whereas in parts of Europe, the drug is tested with a fixed concentration of 2 mg/L clavulanic acid. A/C 2:1 was tested by the US centres and the German centre and A/C 2 was tested by the German and French centres.
Susceptibility testing of the isolates was carried out at Université de Lille Laboratoire de Microbiologie Clinique (Lille, France), Institut für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig (Leipzig, Germany), M. S. Hershey Medical Center (Hershey, PA, USA), and R. M. Alden Research Laboratory (Santa Monica, CA, USA) by agar dilution.
Agar dilutions were carried out according to current NCCLS guidelines20 unless otherwise indicated. Each test concentration of antimicrobial agent was added to molten agar, which was mixed, poured into a Petri dish, and allowed to solidify. Standardized suspensions of isolates to be tested were inoculated onto the surface of each plate in the concentration series with the use of a replicating device. The inocula were allowed to absorb into the medium and placed in an anaerobic atmosphere within 30 min of inoculation. After plates were incubated anaerobically with an anaerobic indicator for approximately 48 h, the plates were examined visually. The lowest concentration of each antimicrobial agent that inhibited growth of an organism was reported as the MIC of the antimicrobial agent.
Quality control strains as specified by the NCCLS guidelines20 (Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741) were tested in each batch. Results for the study strains were accepted only if quality control results were within established NCCLS ranges.
MIC50s, MIC90s and the percentages of isolates susceptible, intermediate and resistant were determined. Susceptibility rates were calculated using NCCLS interpretive criteria20 and pharmacokinetic/pharmacodynamic (PK/PD) breakpoints for amoxicillin and levofloxacin.21,22 The same NCCLS breakpoints were used for interpretation of A/C 2:1 and A/C 2 based on prior studies that reported no differences in MICs.11,23,24
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Results |
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The Prevotella data were analysed to determine whether any species-specific or collecting centre-specific patterns of resistance were present that should preclude analysis of the genus as a whole. No significant pattern was discernable in the data, so the MIC50, MIC90 and susceptibility rates were not determined for each species. Of the isolates tested, there was one Prevotella oralis from France with an A/C 2 MIC of 32 mg/L and three Prevotella loeschii from France with an A/C 2 MIC of 8 mg/L. There were 20 isolates resistant to clindamycin (including six isolates from France and six from Cleveland, OH, USA). In addition, four isolates showed intermediate susceptibility to clindamycin. There were no other isolates with resistance to any of the other drugs tested. The only multidrug resistance found in Prevotella was in one isolate of P. loeschii, which had a clindamycin MIC of >256 mg/L and an A/C 2 MIC of 8 mg/L. All Prevotella isolates were susceptible to A/C 2:1, metronidazole and imipenem and 92.1% were susceptible to clindamycin. Amoxicillin activity (57.9% susceptible) was considerably less than co-amoxiclav activity, presumably due to ß-lactamase production. Levofloxacin MICs for Prevotella were higher, although 71.5% of isolates were in the susceptible range.
All agents were very active against Fusobacterium nucleatum. Amoxicillin was similar (±1 doubling dilution) in activity for most isolates to A/C 2 and A/C 2:1, but the MIC range was significantly wider (0.03>128 versus 0.062 and 0.031). Only one isolate (from Santa Monica, CA, USA) was resistant to clindamycin, six isolates were resistant to amoxicillin and four isolates were resistant to levofloxacin.
Clindamycin and metronidazole were not active against the E. corrodens isolates tested; the other agents tested showed good activity. All amoxicillin and co-amoxiclav MICs were 2 mg/L. Levofloxacin was also very active, with all isolates at
0.125 mg/L. Only one isolate from the Netherlands had an elevated imipenem MIC of 8 mg/L.
Peptostreptococci, which have previously been examined in the aggregate as Peptostreptococcus species, were identified and analysed in this study as P. anaerobius, M. micros and F. magna. P. anaerobius was significantly different in susceptibility to co-amoxiclav compared with the other two genera. Whereas M. micros and F. magna were all 100% susceptible to co-amoxiclav, 84.3% of P. anaerobius were susceptible. Amoxicillin was similar (±1 doubling dilution) in activity to co-amoxiclav for all three species. However, there were two isolates of M. micros from Leipzig with amoxicillin MICs of 128 mg/L and co-amoxiclav MICs of
0.125 mg/L. Differences in susceptibility were also present with clindamycin. Both P. anaerobius and M. micros were highly susceptible at 98.9% and 99.3%, respectively. F. magna had a relatively lower susceptibility rate of 84.7%. All peptostreptococci were susceptible to imipenem, with the exception of one P. anaerobius isolate from France (MIC of 8 mg/L). It is noteworthy, however, that the imipenem MIC90 of 1 mg/L for P. anaerobius is higher than the MIC90s for M. micros and F. magna (0.06 and 0.125 mg/L, respectively). All P. anaerobius were susceptible to metronidazole. One M. micros and three F. magna from France were resistant to metronidazole. Levofloxacin was less active against F. magna. Levofloxacin MICs for 22.4% of F. magna were >4 mg/L, compared to 3.4% and 5.4% for M. micros and P. anaerobius, respectively.
The Porphyromonas isolates included P. asaccharolytica, P. endodontalis, P. levii, P. gingivalis, and Porphyromonas spp. All isolates were susceptible to co-amoxiclav, imipenem and metronidazole. Amoxicillin was similar (±1 doubling dilution) in activity to co-amoxiclav, with the exception of five isolates that had amoxicillin MICs of 816 mg/L and co-amoxiclav MICs of 0.1250.5 mg/L. There were two P. asaccharolytica and two P. levii from Santa Monica, CA that were resistant to clindamycin (>32 mg/L). Although all isolates were susceptible to imipenem, it is noteworthy that one-third of MICs for isolates from Germany are >0.03 mg/L and all MICs for the other isolates are at or below 0.03 mg/L. Levofloxacin MICs for Porphyromonas showed moderate activity and 92.0% were within the susceptible range. There were seven isolates with levofloxacin MICs of 4 mg/L and three with MICs of 8 mg/L.
With the exception of B. fragilis and Prevotella spp., the A/C 2 MICs correlated with the A/C 2:1 MICs. A total of 77.2% of B. fragilis with A/C 2 MICs 2 mg/L had lower MICs (by 14 dilutions) with A/C 2 compared to A/C 2:1. However, 56.5% of B. fragilis with A/C 2 MICs
4 mg/L had higher MICs (by 12 dilutions) with A/C 2 compared to A/C 2:1; 64.6% of Prevotella spp. with amoxicillin MICs
0.5 mg/L had lower MICs with A/C 2 compared to A/C 2:1.
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Discussion |
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Co-amoxiclav has been a consistently active antimicrobial agent against Prevotella species, as have been metronidazole and imipenem.16,18,19,26,28 Clindamycin resistance has been variable, with one study showing resistance of greater than 10%,26 one with a low range of MIC90s (0.0150.03 mg/L) across species but with an MIC range maximum of >128 mg/L,16 and others showing full susceptibility.19,28 Prevotella have been consistently susceptible to imipenem and metronidazole in the literature and this study. The genus has showed very little or no resistance to co-amoxiclav; however, some resistance to clindamycin exists, but was not found to be over 10% in this study.
Fusobacterium are analysed and reported in the literature in aggregate as Fusobacterium species and as F. nucleatum. In a 1990 study by Appelbaum et al.,11 11.3% of ß-lactamase-positive Fusobacterium species were resistant to co-amoxiclav and none of the isolates was resistant to metronidazole. Subsequent studies16,17,29 continue to report no resistance to either metronidazole or imipenem. Studies specifically describing F. nucleatum1618,26 report high activity with co-amoxiclav, imipenem, metronidazole and clindamycin. The one study that included levofloxacin reported an MIC90 of 0.5 mg/L (range 0.061 mg/L).16 F. nucleatum susceptibility data in this study were largely consistent with that found in the literature.
Two 1998 studies combined Porphyromonas isolates with other bacteria to report susceptibilities. Ednie et al.17 examined a pool of 103 Porphyromonas and Prevotella species, finding no resistance to clindamycin and metronidazole (MIC90 = 0.03 and 4 mg/L, respectively), but reporting maximum MICs at the intermediate breakpoint for both antimicrobials (4 and 16 mg/L, respectively). Edlund et al.29 pooled Porphyromonas, Prevotella and Bacteroides species for a sample of 100 isolates. No resistance was reported to clindamycin or imipenem, and the MIC90 of metronidazole was 1.0 mg/L, well below the susceptible breakpoint of 8 mg/L. In 1999, Goldstein et al.16 reported no Porphyromonas resistant to co-amoxiclav, imipenem, or metronidazole, and high levofloxacin activity (MIC90 = 0.5 mg/L). The MIC90 reported for clindamycin was
128 mg/L, for a rate of resistance well over 10%. However, with a sample size of only 11, this could represent as few as two resistant isolates. The susceptibility data for Porphyromonas in this study were largely consistent with the 1999 Goldstein study, showing no resistance to co-amoxiclav or metronidazole and 3.2% resistance to clindamycin. There is a shift in imipenem MIC90 and MICmax from
0.015 and
0.015 mg/L in the earlier study to 0.5 and 0.5 mg/L in this study.
Peptostreptococci have often been examined in the aggregate as Peptostreptococcus species.17,18,28,29 In these prior studies, F. magna and M. micros were still considered part of the Peptostreptococcus genus. The four studies consulted that examined the genus as a whole consistently reported no resistance to metronidazole. Clindamycin was also tested and reported by Aldridge & Johnson19 to be fully active against the 17 isolates tested. In 1992, a study by Wexler et al.18 included co-amoxiclav and reported 12% resistance. That study and the 1998 study by Edlund et al.29 reported no resistance to imipenem. In 1998, Ednie et al.17 reported resistance to clindamycin. The paper reported MIC50/90 and range, with the MIC90 at the intermediate breakpoint of 4 mg/L and MICmax at >32 mg/L. When the genus is evaluated by species, the sample sizes are often small, making comparisons statistically questionable. The 1999 Goldstein et al.16 study reported on four species of Peptostreptococcus with sample sizes of 1214. All species were susceptible to imipenem; F. magna, M. micros and P. anaerobius were all susceptible to clindamycin as well; and F. magna and M. micros were also susceptible to metronidazole and co-amoxiclav. Levofloxacin had high activity against M. micros. An earlier publication by Sheikh et al.26 reported MICs for F. magna, M. micros and other Peptostreptococcus species. Imipenem was consistently active against all species tested. Metronidazole was active against F. magna, but M. micros and the other species showed some resistance. Clindamycin was the least active of the three antimicrobials tested, especially against M. micros. The major changes noted in the Peptostreptococcus/Finegoldia/Micros group in this study, compared with prior publications, are the prevalence of clindamycin resistance and the appearance of metronidazole resistance in F. magna. Also of note in this study were the significant differences seen among these three genera. In the clinical setting, it is recommended that these isolates are identified (as they are no longer grouped as peptostreptococci) in order to assure appropriate therapy for good outcomes. Clindamycin, for example, is one drug that could be used with some confidence to treat P. anaerobius or M. micros infections, but which should be used more carefully in treating F. magna. And while F. magna and M. micros were 100% susceptible to co-amoxiclav, P. anaerobius had a susceptibility rate of 84.3%.
There were some collecting centre differences in susceptibility that raise some questions about emerging resistance. Among B. fragilis, co-amoxiclav resistance was most prevalent in Cardiff, UK and the only metronidazole resistance was also observed in Cardiff. In contrast, the clindamycin resistance among B. fragilis was relatively low in Cardiff and much higher in France. The highest prevalence of elevated co-amoxiclav MICs for Prevotella was observed in France. Among Porphyromonas spp., the only clindamycin resistance was observed in Santa Monica, CA, USA and a slight shift in imipenem MICs was observed in Germany. Nine metronidazole-resistant peptostreptococci (including isolates identified as Peptostreptococcus spp., F. magna and M. micros) were also isolated from France. It would be instructive to attempt to determine whether the rates of clinical treatment failure are likewise increasing, a project perhaps to be undertaken through national registries and beyond the scope of this paper. The apparent shift in the metronidazole MIC curve against Porphyromonas also bears watching, as this, too, might represent emergence of resistance.
The comparatively large sample sizes used in this study allow some statistical confidence in the results. The lack of similarly large numbers in many of the older published studies makes longitudinal comparisons difficult, and underscores the importance of large surveillance studies. The appearance of possible shifts in MIC curves may provide advance notice of emerging resistance and should be followed closely and correlated with clinical outcomes.
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Acknowledgements |
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Footnotes |
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References |
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