Pharmacokinetics and comparative effects of telithromycin (HMR 3647) and clarithromycin on the oropharyngeal and intestinal microflora

Charlotta Edlunda,b,*, Gunnar Alvána, Lisbeth Barkholta, Françoise Vacheronc and Carl Erik Norda

a Huddinge University Hospital, Karolinska Institute, b Södertörns högskola, University College, Stockholm, Sweden; c Aventis Pharma, Hoechst Marion Roussel, Romainville, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pharmacokinetics in plasma and saliva of a new ketolide, telithromycin (HMR 3647), and the effect on the normal oropharyngeal and intestinal microflora were studied in healthy volunteers and compared with those of clarithromycin. Ten subjects received 800 mg telithromycin perorally once daily and 10 other subjects received 500 mg clarithromycin bid for 10 days. Blood, saliva and faecal specimens were collected at defined intervals before, during and after administration for pharmacokinetic and microbiological analyses. In subjects receiving telithromycin, the mean Cmax, AUC and C24 (24 h) in saliva exceeded the values obtained from plasma, while saliva and serum pharmacokinetic parameters were in the same range for the clarithromycin group. The quantitative ecological disturbances in the normal microflora during administration of telithromycin were moderate and comparable to those associated with clarithromycin administration. No overgrowth of yeasts or Clostridium difficile occurred. Emergence of resistant strains was seen in both treatment groups. Administration of both telithromycin and clarithromycin was associated with significant increases in MICs for intestinal Bacteroides isolates, which persisted 2 weeks after discontinuation of treatment. In addition, a significant emergence of highly clarithromycin-resistant {alpha}-haemolytic streptococci, intestinal enterococci and Enterobacteriaceae was detected at day 10 in the clarithromycin group. In conclusion, administration of telithromycin resulted in high drug levels in saliva, which indicates a good therapeutic profile for throat infections. Telithromycin seems to have a more favourable ecological profile compared with clarithromycin in terms of resistance development in the normal microflora.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Telithromycin (HMR 3647) belongs to the family of ketolides representing a new class of 14-membered ring macrolides.1 Ketolides are characterized by a keto function in position 3 of the erythronolide A ring, which replaces the cladinose moiety, a sugar long considered to be essential for antibacterial activity.2 Telithromycin inhibits protein synthesis acting mainly on the 50S ribosomal subunit.

Telithromycin possesses a broad antibacterial spectrum including pathogens involved in respiratory tract infections: Gram-positive cocci, including penicillin- and macrolide-resistant pneumococci, Haemophilus influenzae and Moraxella catarrhalis, as well as atypical and intracellular bacteria.3–5 The antibacterial spectrum of telithromycin also covers many anaerobic bacterial groups such as Bacteroides fragilis, clostridia and Gram-positive anaerobic cocci.3,6

Investigation of the pharmacokinetic profile of telithromycin in saliva is of interest in order to follow the adequacy of treatment and also to explore the feasibility of following kinetics in saliva rather than plasma. Therapeutic concentrations of antibiotic maintained throughout the day would be considered beneficial for the treatment of tonsillitis. Knowledge of the impact of antibiotherapy on the oropharyngeal flora is of importance since it can lead to overgrowth of yeasts, Enterobacteriaceae or streptococcal strains that may cause local or systemic infections in immunosuppressed hosts.7 Ecological disturbances in the intestinal microflora due to antimicrobial treatment can lead to adverse effects such as diarrhoea or pseudomembranous colitis caused by Clostridium difficile,8 or even to systemic infections with yeasts and aerobic Gram-negative rods in immunosuppressed patients. Finally, selection of resistant strains in the normal oropharyngeal and intestinal microflora and possible transfer of resistance genes among various bacterial strains and groups is also a serious consequence of antimicrobial therapy for the patient and for society.9

The impact of clarithromycin on the faecal flora is well documented with a decrease in enterococci and Escherichia coli, and overgrowth of resistant Enterobacteriaceae during therapy but no emergence of C. difficile or yeasts.10–12

The purpose of the present study was: (i) to investigate the pharmacokinetic profile of telithromycin and clarithromycin, respectively, in saliva and plasma for therapeutic considerations; and (ii) to assess the impact of antibiotic treatment on the oropharyngeal and intestinal microflora, including potential emergence of resistance before, during and after administration of telithromycin or clarithromycin given to healthy subjects.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects

Twenty subjects (10 women and 10 men; mean age 25.1 years, range 18.0–34.9 years) were enrolled in a randomized double-blind controlled trial. All subjects were considered healthy on the basis of their medical history, none of them had any history of significant cardiovascular, gastrointestinal, hepatic or renal diseases. None of the volunteers had taken any antibiotics during the previous 3 months. No other medication except contraceptives was allowed during the investigation period. The trial was approved by the Ethics Committee of Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden.

Drug administration

The subjects were randomized into two groups. Ten subjects received two 400 mg capsules of telithromycin (Hoechst Marion Roussel, Romainville, France) once daily (a.m.) and two placebo capsules (p.m.) once daily for 10 days. The other 10 subjects were given 500 mg (two 250 mg capsules) of clarithromycin (Sanofi-Winthrop, Paris, France) bid for 10 days.

Sampling of blood, saliva and faecal specimens

A total of 21 plasma samples and 21 saliva samples were collected from each of the 20 patients at defined intervals for pharmacokinetic analyses on days 1, 2, 5, 10, 11 and 12. Faecal samples were collected on days 2, 5, 10, 12 and 15 for assay of the antibacterial agents. Saliva and faecal samples for microbiological analyses were collected before the drug administration (day 0), during administration (days 2, 5 and 10) and after withdrawal of the agents (days 12, 15, 18 and 24). Unstimulated mixed saliva was sampled by spitting into sterile tubes. Faecal samples were collected in sterile plastic containers. All specimens were frozen within 1 h and stored at –70°C until assayed.

Assays of telithromycin and clarithromycin concentrations

The plasma and saliva concentrations of telithromycin and clarithromycin were determined microbiologically using the agar plate diffusion method. Telithromycin and clarithromycin concentrations in plasma and saliva were determined in quadruplicate in Antibiotic Medium Merck A agar and Bacillus subtilis ATCC 6633 as the test organism, based in plasma on a lower limit of quantification of 0.025 mg/L. The plates were incubated aerobically for 18 h at 32°C. Concentrations of telithromycin and clarithromycin in faeces were determined by the agar well diffusion method. The test medium was Antibiotic Medium No. 1 (Difco, Detroit, MI, USA) and the indicator strain was Micrococcus luteus ATCC 9341. Standards with known concentrations of the two drugs were prepared in a faecal suspension from a healthy volunteer and diluted 1:3 in 0.15 M phosphate buffer pH 7.2. The faecal samples were diluted in phosphate buffer (1:3) and centrifuged at 3000g for 10 min. Samples were run in duplicate and on each agar plate a standard series was inoculated. The plates were incubated for 18 h at 37°C. Plasma, saliva and faecal drug concentrations were determined in relation to the diameters of the inhibition zones caused by the known concentrations from the standard series.

Processing of saliva and faecal specimens for microbiological analyses

The microbiological analyses of the specimens were performed as described previously.12,13 The saliva and faecal specimens were suspended in pre-reduced peptone–yeast extract medium, diluted 10-fold and inoculated on non-selective and selective media. The aerobic agar plates were incubated for 24 h at 37°C and the anaerobic plates for 48 h at 37°C in anaerobic jars (GasPak; BBL, Cockeysville, MD, USA). After incubation, different colony types were counted and isolated in pure culture. All isolates were identified according to Gram's stain and biochemical tests.14 The anaerobic microorganisms were identified by gas– liquid chromatography of metabolites from glucose.14,15 The lower limit of detection was 102 microorganisms per millilitre of saliva or per gram of faeces.

Antibiotic susceptibility tests

Three representative colonies of intestinal Enterobacteriaceae, enterococci and Bacteroides, and three oral {alpha}-haemolytic streptococcal colonies were isolated from each subject on days 0, 10 and 24 in order to study the antimicrobial susceptibility during the investigation period. The MICs for telithromycin and clarithromycin were determined by the agar dilution method using PDM Antibiotics Sensitivity Medium (AB Biodisk, Solna, Sweden). E. coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Bacteroides fragilis NCTC 9343 were used as reference strains. The inoculum was 107 cfu/mL for aerobic strains, and 108 cfu/mL for Bacteroides spp. The agar plates were incubated aerobically or anaerobically at 37°C for 24 and 48 h, respectively.

Statistical analysis

Statistical analyses of the pharmacokinetic parameters were mainly descriptive. Quantitative alterations in cultivation were compared statistically within groups between day 0–10 and between day 0–24 using Wilcoxon signed rank test (P values <=0.05 were considered stastistically significant). The MICs for each species were compared within groups between day 0–10 and between day 0–24 using the Mann–Whitney U-test in order to detect significant decreases in susceptibility during and after the administration period (P values <=0.01 were considered statistically significant). P values were adjusted for multiple analyses.

Safety data

All volunteers had a physical examination before entering the study, at day 11 and after completion of the study (day 24). Vital signs (blood pressure and heart rate) and 12-lead electrocardiogram (ECG) were performed and possible adverse events were followed throughout the investigation period.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pharmacokinetics of telithromycin and clarithromycin in plasma and saliva

The plasma and saliva concentrations of telithromycin at day 1 are shown in Figure 1Go. Telithromycin was eliminated in a one-compartment model in plasma and in a twocompartment model in saliva. Table IGo shows the pharmacokinetic data for both drugs. In subjects receiving telithromycin the mean Cmax, area under the curve (AUC) and C24 in saliva exceeded the values obtained from plasma; Cmax was c. 1.2–1.5 times higher in saliva compared with plasma, AUC (0–24 h) was 1.6–1.7 times higher in saliva compared with plasma and C24 was 3–5 times higher in saliva compared with plasma. In the clarithromycin group, values of these parameters in saliva were, generally, slightly lower or equal to the corresponding plasma value. Large variations in Cmax and AUC were noted in both treatment groups.



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Figure 1. Mean concentrations (± s.e.m.) of telithromycin in plasma and saliva on day 1 ({diamondsuit}, day 1 plasma; {diamond}, day 1 saliva).

 

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Table I. Pharmacokinetic parameters of telithromycin and clarithromycin in plasma and saliva
 
Faecal concentrations of telithromycin and clarithromycin

The faecal concentrations of telithromycin and clarithromycin are shown in Table IIGo. The concentrations of telithromycin were high during the administration period, i.e. day 5–10, mean values >500 mg/kg with three subjects having levels >1000 mg/kg. The concentrations of clarithromycin in faeces were lower compared with telithromycin (Table IIGo). One subject had concentrations >500 mg/kg on days 10 and 12. Large variations in drug concentrations were noted in both treatment groups.


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Table II. Faecal concentrations of telithromycin and clarithromycin in 10 subjects receiving 800 mg telithromycin once daily for 10 days or 500 mg clarithromycin bid for 10 days, respectively
 
Effect of telithromycin and clarithromycin on the oropharyngeal microflora

There were only minor disturbances in the aerobic oropharyngeal microflora owing to the administration of telithromycin and clarithromycin, respectively. No significant alteration in the levels of {alpha}-haemolytic streptococci, micrococci, staphylococci, Haemophilus or Neisseria spp. was noticed in either of the groups during the administration period. The numbers of corynebacteria were reduced at day 10 in both treatment groups, although only significantly in the telithromycin group (P <= 0.05). Six subjects in the telithromycin group and four in the clarithromycin group were colonized by low numbers of Candida spp. A transient colonization of the oropharynx with low numbers of Enterobacteriaceae (Klebsiella spp., Enterobacter spp. or Citrobacter spp.) was recorded during or after the administration period in five subjects receiving telithromycin and in four subjects receiving clarithromycin. In the anaerobic oropharyngeal microflora, the numbers of Actinomyces and Prevotella spp. were moderately suppressed in both groups, while no other bacterial group, such as peptostreptococci, streptococci, bifidobacteria, lactobacilli and Veillonella spp., was affected to any major extent. The oropharyngeal microflora was normalized 2 weeks after discontinuation of the administration of telithromycin and clarithromycin, respectively.

Effect of telithromycin and clarithromycin on the intestinal microflora

The administration of telithromycin and clarithromycin caused similar and moderate disturbances in the aerobic intestinal microflora as shown in Figure 2Go. In both groups, the numbers of E. coli were significantly reduced at day 10 (P <= 0.05). In the telithromycin group, an overgrowth of staphylococci was recorded at day 10 (P <= 0.05), and the levels of enterococci were decreased, although not significantly, during the administration period. Overgrowth of non-E. coli Enterobacteriaceae, such as Klebsiella, Citrobacter and Enterobacter spp., occurred in five volunteers receiving telithromycin and in six subjects receiving clarithromycin during or after the administration period. No significant overgrowth of Candida spp. occurred in any of the groups.



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Figure 2. Impact of (a) telithromycin and (b) clarithromycin administration on the intestinal aerobic microflora of 10 subjects. —, median value of the logarithmic number of microorganisms/g faeces.

 
Figure 3Go shows the effect of telithromycin and clarithromycin on the anaerobic intestinal microflora. There was a marked reduction of lactobacilli and bifidobacteria on day 10 in the telithromycin group (P <= 0.05) and in the clarithromycin group (P <= 0.01), which persisted at day 24 in both groups (P <= 0.05). The numbers of peptostreptococci, streptococci, clostridia, Veillonella and Bacteroides spp., or the total number of anaerobic bacteria was not significantly affected by any of the administration regimens. None of the subjects were colonized by C. difficile during the administration period.



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Figure 3. Impact of (a) telithromycin and (b) clarithromycin administration on the intestinal anaerobic microflora of 10 subjects. —, median value of the logarithmic number of microorganisms/g faeces.

 
Antibiotic susceptibility tests

The antibiotic susceptibility of isolated oral {alpha}-haemolytic streptococci and intestinal enterococci, Enterobacteriaceae and Bacteroides spp. for telithromycin and clarithromycin, respectively, are shown in Table IIIGo. A minor increase in MICs occurred among salivary streptococci from the telithromycin group, although all isolates remained susceptible to telithromycin throughout the study period (MIC <= 1.0 mg/L). In the clarithromycin group a significant decrease in susceptibility was noted, MIC90 increased from 0.032 mg/L pretreatment to >128 mg/L at day 10 and 24 (P <= 0.001, Mann–Whitney U-test). In the telithromycin group, MICs of telithromycin against enterococci and Enterobacteriaceae at day 10 and 24 were not significantly altered compared with pretreatment values. Isolates with MIC >= 16.0 mg/L were mostly non-E. coli Enterobacteriaceae (e.g. Klebsiella and Citrobacter spp.). In the clarithromycin group, MICs against enterococci and Enterobacteriaceae (mostly E. coli, Klebsiella, Citrobacter and Enterobacter spp.) were significantly increased at day 10 (P <= 0.001) but not at day 24 compared with pre-treatment values. A selection of highly resistant Bacteroides isolates was recorded during and after treatment in both treatment groups (P <= 0.001).


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Table III. Minimum inhibitory concentrations of telithromycin and clarithromycin against oral {alpha}-streptococci, intestinal enterococci, enterobacteria and Bacteroides
 
Adverse events

Thirty-one mild to moderate adverse events considered possibly related to study medication were reported during the investigation period. With telithromycin, the most frequently reported adverse events were taste perversion and diarrhoea (three subjects each). In the clarithromycin group, the most frequently reported adverse events were taste perversion (10 subjects), abdominal pain (three subjects) and diarrhoea (two subjects). Telithromycin or clarithromycin had no clinically noteworthy effect on vital signs or ECG parameters. The duration of the QTc interval was not increased for any of the subjects.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Careful investigation of the impact of antibiotic treatment on the endogenous microflora is of importance since alteration in the balance of the flora, qualitatively and/or quantitatively, may facilitate colonization by new potentially pathogenic strains or enable overgrowth of resistant microorganisms already present in the normal flora.16,17 Treatment with clarithromycin has been associated with suppression of intestinal enterococci, Enterobacteriaceae and certain anaerobic bacteria.10–12 The effect of ketolides like telithromycin on the normal microflora has not been studied before. In the present study, the mean concentration of telithromycin in saliva was higher on average than that in plasma and was above the MIC50 (<=0.06 mg/L) for most respiratory pathogens, such as ß-haemolytic streptococci, Streptococcus pneumoniae and M. catarrhalis, up to 24 h post-dose.3,4,18 This indicates a good therapeutic profile for throat infections. The retention of {alpha}-haemolytic streptococci is favourable from an ecological point of view, since these microorganisms are known to produce bacteriocins which protect against colonization with Enterobacteriaceae and other potentially pathogenic microorganisms.19,20 The high faecal concentrations of telithromycin during the administration period, which is a direct consequence of faecal elimination of this drug, are in accordance with the alterations seen in the intestinal microflora mainly affecting enterococci and E. coli. The quantitative ecological disturbances in the normal oral and intestinal microflora during administration of telithromycin were considered moderate and comparable to those of clarithromycin administration in the present study as well as in earlier studies.10–12 However, in both treatment groups qualitative alterations in terms of emergence of resistant strains occurred, which were most pronounced in the clarithromycin group. In both treatment groups, there was an increase in MICs against oral streptococci, mostly represented by Streptococcus salivarius. In the telithromycin group, virtually all {alpha}-haemolytic streptococci were considered susceptible to telithromycin (preliminary breakpoint R >= 4.0 mg/L, according to the manufacturer) throughout the study period, while in the clarithromycin group, a shift from susceptiblity to resistance was seen during and after treatment (breakpoint R >= 1.0 mg/L).21 In the clarithromycin group, a significant selection of highly clarithromycin-resistant intestinal enterococci and Enterobacteriaceae was seen at day 10, while MICs of these bacterial groups did not increase significantly during the administration period in the telithromycin group. The results of emergence of clarithromycin resistance are in agreement with those observed in a previous study.12

In conclusion, administration of telithromycin resulted in saliva concentrations that exceeded the MICs for common respiratory pathogens, and caused moderate ecological disturbance in the normal oral and intestinal microflora comparable with that associated with clarithromycin. In terms of resistance development in the normal microflora, telithromycin appears to have a more favourable ecological profile than clarithromycin.


    Notes
 
* Correspondence address. Department of Microbiology, Pathology and Immunology, F82, Huddinge University Hospital, SE-141 86 Stockholm, Sweden. Tel: +46-8-585-878-38; Fax: +46-8-711-3918; E-mail: charlotta.edlund{at}impi.ki.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bryskier, A., Agouridas, C. & Chantot, J. F. (1997). Ketolides: semisynthetic 14-membered ring macrolides. In Expanding Indications for the New Macrolides, Azalides and Streptogramins (Zinner, S. H., Young, L. S., Acar, J. F. & Neu, H. C., Eds), pp. 39–49. Marcel Dekker, New York, NY.

2 . Douthwaite, S., Mauvais, P., Champney, S. & Bryskier, A. (2000). Structure–activity relationship of the ketolide telitromycin. Fifth international conference on the macrolides, azalides, streptogramins, ketolides and oxazolidinones, 2000, Sevilla, Spain.

3 . Boswell, F. J., Andrews, J. M., Ashby, J. P., Fogarty, C., Brenwald, N. P. & Wise, R. (1998). The in-vitro activity of HMR 3647, a new ketolide antimicrobial agent. Journal of Antimicrobial Chemotherapy 42, 703–9.[Abstract]

4 . Malathum, K., Coque, T. M., Singh, K. V. & Murray B. E. (1999). In vitro activities of two ketolides, HMR 3647 and HMR 3004, against Gram-positive bacteria. Antimicrobial Agents and Chemotherapy 43, 930–6.[Abstract/Free Full Text]

5 . Hamilton-Miller, J. M. T. & Shah, S. (1998). Comparative in-vitro activity of ketolide HMR 3647 and four macrolides against Gram-positive cocci of known erythromycin susceptibility status. Journal of Antimicrobial Chemotherapy 41, 649–53.[Abstract]

6 . Edlund, C., Sillerström, E., Wahlund, E. & Nord, C. E. (1998). In vitro activity of HMR 3647 against anaerobic bacteria. Journal of Chemotherapy 10, 280–4.[ISI][Medline]

7 . Heimdahl, A. & Nord, C. E. (1985). Colonization of the oropharynx with pathogenic microorganisms – a potential risk factor for infection in compromised patients. Chemioterapia 4, 186–91.[ISI][Medline]

8 . Fekety, R. B. & Shah, A. (1993). Diagnosis and treatment of Clostridium difficile colitis. Journal of the American Medical Association 269, 71–5.[Abstract]

9 . Davies, J. (1994). Inactivation of antibiotics and the dissemination of resistance genes. Science 264, 375–82.[ISI][Medline]

10 . Brismar, B., Edlund, C. & Nord, C. E. (1991). Comparative effects of clarithromycin and erythromycin on the normal intestinal microflora. Scandinavian Journal of Infectious Diseases 23, 635–42.[ISI][Medline]

11 . Adamsson, I., Nord, C. E., Lundquist, P., Sjöstedt, S. & Edlund, C. (1999). Comparative effects of omeprazole, amoxycillin plus metronidazole versus omeprazole, clarithromycin plus metronidazole on the oral, gastric and intestinal microflora in Helicobacter pylori infected patients. Journal of Antimicrobial Chemotherapy 44, 629–40.[Abstract/Free Full Text]

12 . Edlund, C., Beyer, G., Hiemer-Bau, M., Ziege, S., Lode, H. & Nord, C. E. (2000). Comparative effects of moxifloxacin and clarithromycin on the normal intestinal microflora. Scandinavian Journal of Infectious Diseases 32, 81–5.[ISI][Medline]

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

15 . Summanen, P., Baron, E., Citron, D., Strong, C., Wexler, H. & Finegold, S. (1993). Wadsworth Anaerobic Bacteriology Manual, 5th edn. Veterans Administration, Wadsworth Medical Center, Los Angeles, CA.

16 . Edlund, C. & Nord, C. E. (1993). Ecological impact of antimicrobial agents on human intestinal microflora. Alpe Adria Microbiology Journal 2, 137–64.

17 . Periti, P., Mazzei, T., Mini, E. & Novelli, A. (1993). Adverse effects of macrolide antibacterials. Drug Safety 9, 346–64.[ISI][Medline]

18 . Pankuch, G. A., Hoellman, D. B., Lin, G., Bajaksouzian, S., Jacobs, M. R. & Appelbaum, P. C. (1998). Activity of HMR 3647 compared to those of five agents against Haemophilus influenzae and Moraxella catarrhalis by MIC determination and time–kill assay. Antimicrobial Agents and Chemotherapy 42, 3032–4.[Abstract/Free Full Text]

19 . Sprunt, K. & Redman, W. (1968). Evidence suggesting importance of role of interbacterial inhibition in maintaining balance of normal flora. Annals of Internal Medicine 68, 579–90.[ISI][Medline]

20 . Brook, I. (1999) Bacterial interference in upper respiratory tract infections. Reviews in Medical Microbiology 10, 225–33.[ISI]

21 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 4th edn. Approved Standard M11-A4. NCCLS, Villanova, PA.

Received 21 February 2000; returned 5 June 2000; revised 26 June 2000; accepted 3 August 2000