Comparative in vitro activity of ertapenem and 11 other antimicrobial agents against aerobic and anaerobic pathogens isolated from skin and soft tissue animal and human bite wound infections

Ellie J. C. Goldsteina,b,*, Diane M. Citrona, C. Vreni Merriama, Yumi A. Warrena, Kerin Tyrrella and Helen Fernandeza

a R. M. Alden Research Laboratory, Santa Monica-UCLA Medical Center, 2021 Santa Monica Boulevard, Suite 740, East Santa Monica, CA 90404; b UCLA School of Medicine, Los Angeles, CA 90073, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We studied the comparative in vitro activity of ertapenem, a new carbapenem, against 240 aerobic and 180 anaerobic recent clinical bite isolates using an agar dilution method and an inoculum of 104 cfu/spot for aerobes and 105 cfu/spot for anaerobes. Ertapenem inhibited 410/420 (98%) of the isolates tested at 4 mg/L with only 4/5 Campylobacter gracilis and 1/3 Campylobacter rectus strains requiring 16 mg/L for inhibition. Ertapenem was only moderately active (MIC 8 mg/L) against 4/6 Enterococcus faecalis and 1/11 Staphylococcus epidermidis strains. All Pasteurella multocida, Pasteurella septica, Pasteurella canis, Pasteurella dagmatis, Moraxella spp. and EF-4 isolates were inhibited at 0.015 mg/L. MIC90s for other aerobic genera and species were as follows: Corynebacterium spp., 4 mg/L; Staphylococcus aureus, 0.25 mg/L; Staphylococcus epidermidis, 4 mg/L; other coagulasenegative staphylococci, 0.25 mg/L; Streptococcus milleri group, 0.5 mg/L; Eikenella corrodens, 0.03 mg/L; and Bergeyella zoohelcum, 0.5 mg/L. For anaerobes the range of MICs and MIC90s were: Prevotella ssp., 0.015–0.5, 0.125 mg/L; Porphyromonas spp., 0.015–0.03, 0.015 mg/L; Fusobacterium spp., 0.015–0.125, 0.03 mg/L; Bacteroides tectum, 0.03–0.125, 0.125 mg/L; and Peptostreptococcus spp., 0.01–2, 1 mg/L. Ertapenem showed excellent potency against the full range of animal and human bite wound pathogens.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Many of the 4.5 million Americans bitten by animals or humans each year require either therapeutic or prophylactic antimicrobial therapy.1,2 Approximately 30 000 patients will visit an emergency department for care and 10 000 will be hospitalized2 and require intravenous antimicrobial therapy. Recent studies have shown the importance of both the aerobic and anaerobic flora of the biting animal or human in the pathogenesis of more serious wound infections and their complications.2,3

Ertapenem (MK-0826) is a new parenteral carbapenem that is highly resistant to inactivation by a wide variety of ß-lactamases and has been shown to have a broad spectrum of antimicrobial activity against both aerobes and anaerobes.4–10 These studies have focused on more typical isolates, especially those associated with nosocomial infection and intra-abdominal infections,4,7,8,11 and do not evaluate the in vitro activity against the specific range of bacteria typically found in human and animal bite wound infections. To evaluate the expanded activity of ertapenem, we determined its comparative activity along with 11 other antimicrobial agents against 420 aerobic and anaerobic strains recently isolated from infected skin and soft tissue bite wounds in humans.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All isolates were identified by standard criteria.12–17 The specific sources were: dog bites, 117; cat bites, 156; human bites, 132; and other animal bites, 15. The numbers and species of isolates tested are given in the Table. Standard laboratory powders were supplied as follows: ertapenem, Merck & Co, West Point, PA, USA; co-amoxiclav and ticarcillin/clavulanate, SmithKline Beecham Pharmaceuticals, Philadelphia, PA, USA; ampicillin/sulbactam, Pfizer Inc., New York, NY, USA; piperacillin/tazobactam, Wyeth-Ayerst Pharmaceuticals, Philadelphia, PA, USA; ceftriaxone, Roche Pharmaceuticals, Nutley, NJ, USA; ciprofloxacin, Bayer Inc., West Haven, CT, USA; cefepime, Dura Pharmaceuticals, San Diego, CA, USA; cefotetan, Astra Zeneca Pharmaceuticals, Wilmington, DE, USA; levofloxacin, Ortho-McNeil Pharmaceuticals, Raritan, NJ, USA; and doxycycline and oxacillin, Sigma Chemical Co., St Louis, MO, USA. Antimicrobial agents were reconstituted according to the manufacturers' instructions. Serial two-fold dilutions of antimicrobial agents were prepared on the day of the test and added to the media.

To ensure purity and good growth, frozen cultures of aerobic bacteria were transferred twice on trypticase soy agar (TSA) supplemented with 5% sheep blood, or chocolate agar (Hardy Diagnostics, Santa Maria, CA, USA) and anaerobic bacteria were cultured on Brucella agar supplemented with haemin, vitamin K1 and 5% sheep blood (Anaerobe Systems, Morgan Hill, CA, USA). Susceptibility testing was performed according to NCCLS standards.18,19 Brucella agar supplemented with haemin, vitamin K1 and 5% laked sheep blood was the basal medium used for anaerobic species, and for Eikenella corrodens and Bergeyella zoohelcum. 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, USA). The inoculum used for aerobic bacteria was 104 cfu/spot, and the inoculum used for E. corrodens and anaerobic bacteria was 105 cfu/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 18–20 h and then examined. E. corrodens and streptococci were incubated in 5% CO2 for 42–44 h, and plates with anaerobes were incubated in an anaerobic chamber (Anaerobe Systems) at 35°C for 48 h.

The control strains 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 as compared with the growth control plate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The full results of the study are noted in the Table. Ertapenem inhibited 98% (410/420) of the isolates studied at <=4 mg/L. Pasteurella multocida, Pasteurella septica and other Pasteurella species were inhibited by <=0.015 mg/L. Ertapenem was also potent against unusual isolates such as E. corrodens (MIC100 <= 0.03 mg/L) and B. zoohelcum (MIC 100 <= 0.5 mg/L). In accordance with Fuchs et al.,5 who noted that ertapenem had an MIC range of 8–>16 mg/L against 30 E. faecalis isolates, all six of our strains of E. faecalis required 4–8 mg/L of ertapenem for inhibition. One of 11 strains of Staphylococcus epidermidis was inhibited only at 8 mg/L.

Against anaerobes, ertapenem was active against 170/180 isolates at <=0.05 mg/L (overall MIC 90 <= 0.5 mg/L), including strains of Prevotella heparinolytica, other Prevotella spp., Porphyromonas spp., Bacteroides tectum and Peptostreptococcus spp.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Previous studies10,11 have reported that Fusobacterium nucleatum is susceptible to <=0.06 mg/L of ertapenem, as it was in our study. Of particular interest is that F. nucleatum species (MIC50/90 1/8 mg/L) and Fusobacterium russii (MIC > 8 mg/L) of animal origin were more resistant to levofloxacin than those isolated from human bites (MIC50/90 0.25/1 mg/L). This disparity was also seen with ciprofloxacin, but to a more modest extent of one doubling dilution, since the fusobacteria are generally more resistant to ciprofloxacin. One could speculate that this might be associated with the use of fluoroquinolones for veterinary infections as well as growth promotion. In addition, 3/12 of the F. nucleatum animal isolates were ß-lactamase producers, while none of the 14 human isolates produced ß-lactamase.

Wexler et al.10 reported that ertapenem (MK-0826) inhibited 14 strains of Campylobacter gracilis at <=0.25 mg/L. However, in our study, the only isolates tested that required >=16 mg/L of ertapenem for inhibition were four of five C. gracilis and one of three Campylobacter rectus strains. The reason for this disparity is unclear, as we used the same methods, agar medium and inoculum as Wexler et al.10 Our Campylobacter strains were also resistant to all other ß-lactam agents tested, but susceptible to the fluoroquinolones (levofloxacin, ciprofloxacin) and doxycycline.

As expected, oxacillin was relatively inactive against E. corrodens, EF-4a and EF-4b isolates, Neisseria spp., Moraxella spp. and Veillonella spp., and often 2–4 mg/L was required to inhibit B. zoohelcum; it also required 1–4 mg/L of oxacillin to inhibit the various Pasteurella species, making it of limited clinical value in the therapy of bite wound infections. Cefotetan had limited activity against B. zoohelcum and streptococci.

Ertapenem has an excellent broad spectrum of activity and consequently merits further evaluation as a therapeutic alternative in serious animal and human bite wound infections.


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Table. Comparative and in vitro activity of ertapenem against 240 aerobic and 180 anaerobic bite wound isolates
 

    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We wish to thank Judee H. Knight and Alice E. Goldstein for assistance. This study was partially supported by a grant from Merck & Co.


    Notes
 
* Corresponding author. Tel: +1-310-315-1511; Fax: +1-310-315-3662; E-mail: EJCGMD{at}aol.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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5 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (1999). In-vitro antimicrobial activity of a carbapenem, MK-0826 (L-749,345) and provisional interpretive criteria for disc tests. Journal of Antimicrobial Chemotherapy 43, 703–6.[Abstract/Free Full Text]

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8 . Koehler, J., Dorso, K. L., Young, K., Hammond, G. G., Rosen, H., Kropp, H. et al. (1999). In vitro activities of the potent, broadspectrum carbapenem MK-0826 (L-749,345) against broad-spectrum ß-lactamase- and extended-spectrum ß-lactamase-producing Klebsiella pneumoniae and Escherichia coli clinical isolates. Antimicrobial Agents and Chemotherapy 43, 1170–6.[Abstract/Free Full Text]

9 . Sundelof, J. G., Hajdu, R., Gill, C. J., Thompson, R., Rosen, H. & Kropp, H. (1997). Pharmacokinetics of L-749,345, a long-acting carbapenem antibiotic, in primates. Antimicrobial Agents and Chemotherapy 41, 1743–8.[Abstract]

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11 . Goldstein, E. J. C., Citron, D. M., Merriam, C. V., Warren, Y. & Tyrrell, K. (2000). Activity of ertapenem (MK-0826) against 1,001 anaerobes isolated from human intra-abdominal infections. Antimicrobial Agents and Chemotherapy 44, 2389–94.[Abstract/Free Full Text]

12 . Alexander, C. J., Citron, D. M., Gerardo, S. H., Claros, M. C., Talan, D. & Goldstein, E. J. C. (1997). Characterization of saccharolytic Bacteroides and Prevotella isolates from infected dog and cat bite wounds in humans. Journal of Clinical Microbiology 35, 406–11.[Abstract]

13 . Hudspeth, M. K., Gerardo, S. H., Citron, D. M. & Goldstein, E. J. C. (1997). Growth characteristics and a novel method for identification (the Wee Tab system) of Porphyromonas species isolated from infected dog and cat bite wounds in humans. Journal of Clinical Microbiology 35, 2450–3.[Abstract]

14 . Hudspeth, M. K., Gerardo, S. H., Citron, D. M. & Goldstein, E. J. C. (1998). Evaluation of RapID CB Plus System for identification of Corynebacterium species and other gram-positive rods. Journal of Clinical Microbiology 36, 543–7.[Abstract/Free Full Text]

15 . Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H. (1999). Manual of Clinical Microbiology, 7th edn. American Society for Microbiology, Washington, DC.

16 . Mutters, R., Ihm, P., Pohl, S., Frederiksen, W. & Mannheim, W. (1985). Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa. International Journal of Systematic Bacteriology 35, 309–22.

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18 . National Committee for Clinical Laboratory Standards. (1998). Method for Dilution Antimicrobial Susceptibility Testing for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

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Received 9 May 2001; returned 12 July 2001; revised 27 July 2001; accepted 3 August 2001