Impact of a new quinolone, DU-6859a, and two oral carbapenems, CS-834 and L-084, on the rat and mouse caecal microflora

Cheng-Xu Liua, Naoki Katoa,*, Kunitomo Watanabea, Tadashi Sakatab and Tsutomu Kanekob

a Institute of Anaerobic Bacteriology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705 and b Central Research Institute, Meiji Milk Product Co., Tokyo 189-8530, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We determined the influence of a new quinolone, DU-6859a, and two oral carbapenems, CS-834 and L-084, on the rat and mouse caecal microflora. The caecum–skin fistula-implanted rats and conventional mice were given oral antimicrobials at doses of 30 mg/kg bid for 5 days. DU-6859a generated a marked decrease in the numbers of caecal flora except for enterococci. CS-834 and L-084 had little impact on the rat caecal flora. CS-834 caused a great decrease in the numbers of mouse caecal flora but L-084 did not. In vitro studies suggest that the difference is due to the extension of inactivation of antimicrobials by caecum contents.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
New quinolones such as DU-6859a1,2 and oral carbapenems such as CS-8343,4 and L-084,5,6 which are prodrugs of the active metabolites, R-95867 and L-036, respectively, are oral agents which have good in vitro activity against both anaerobic and aerobic bacteria. Because of their activity against anaerobic bacteria (MIC90s <= 2 mg/L),2,4,6 the predominant species in the intestinal flora, severe or frequent gastrointestinal effects such as diarrhoea are a major concern.

We evaluated the impact of DU-6859a, CS-834 and L-084 on caecal microflora in two animal models; caecum–skin fistula-implanted rats,7 from which caecal samples can be taken repeatedly without killing, and conventional mice.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
DU-6859a (Daiich Pharmaceutical, Tokyo, Japan), CS-834 and its active metabolite, R-95867 (Sankyo, Tokyo, Japan), and L-084 and its active metabolite, L-036 (Wyeth Lederle Japan, Tokyo, Japan), were supplied as standard powders of known potency by the manufacturers.

All animal experiments were conducted under the guidelines for animal experiments of Gifu University School of Medicine. Three male Wistar rats weighing c. 350 g were used for each antimicrobial treatment. After being fed a standard diet (Japan Clea, Tokyo, Japan) for 1 month, rats were implanted with a plastic fistula device in the caecum.7 After the operation, the diet was switched to one containing 40% protein (Japan Clea). Male ddY mice weighing c.25 g were fed 40% protein throughout the experiment.

Antimicrobials were suspended homogeneously in 5% carboxymethyl cellulose solution using a glass blender and were given orally to experimental animals at a dose of 30 mg/kg bid for 5 days.

Quantitative culture of caecal contents was carried out by serial 10-fold dilution with pre-reduced anaerobe broth MIC medium (Becton Dickinson). The media used for the isolation of anaerobes and aerobes and culture conditions are described elsewhere.8,9 Anaerobes and aerobes were identified to the genus or species level for Staphylococcus spp., Enterococcus spp., Enterobacteriaceae and the Bacteroides fragilis group by standard methods8,9 and with API identification kits (bioMérieux Viteck, Marcy-l’Etoile, France).

To assess the inactivation of antimicrobials, 25 µL of antibiotic solution (20 mg/mL) was incubated anaerobically for up to 48 h at 37°C with 500 mg of rat caecal contents, and 25 µL of antibiotic solution (100 mg/mL) was added to 2.5 mL of a bacterial suspension (108 cfu/mL) in brain–heart infusion broth of the major anaerobic and aerobic isolates from rat caecum before and after treatment. Antimicrobial activity of caecal contents or broth was measured by bioassay. All 28 caecal isolates used were assayed for ß-lactamase production by the cefinase test (Becton Dickinson).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The mean log10 counts of the caecal flora in the rats given DU-6859a, CS-834, L-084 and 5% carboxymethyl cellulose alone (controls) are summarized in Table IGo. The caecal flora was markedly affected by oral DU-6859a but returned to pretreatment levels 15 days after administration. CS-834 and L-084 had little impact on the caecal flora whereas the numbers of some organisms such as Enterobacteriaceae were reduced by over 2 log10 1 day after the end of treatment with CS-834 and L-084. The microflora returned to pretreatment levels 15 days after treatment.


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Table I. Caecal flora of three rats given oral DU-6859a, CS-834 or L-084 at a dose of 30 mg/kg bid for 5 days
 
Among Staphylococcus spp. and Enterococcus spp., Staphylococcus aureus and Enterococcus faecalis were the most predominant species, respectively, in the rat caecum. Escherichia coli, always found in the caecal flora, was markedly affected by the DU-6859a treatment but not by CS-834 or L-084. Bacteroides distasonis and Bacteroides uniformis were almost always found in the rat caecal flora and were eradicated tentatively by treatment with DU-6859a but not by CS-834 or L-084 treatment.

The numbers of anaerobes detected from the mouse flora were much lower than those in rats (Table IIGo). A significant impact on the aerobic caecal flora was seen in mice treated with DU-6859a. A marked effect on the anaerobic caecal flora was seen in mice given DU-6859a and CS-834. In contrast, there was little impact in mice given L-084.


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Table II. Caecal flora of the control mice and the mice given oral DU-6859a, CS-834 and L-084 at a dose of 30 mg/kg bid for 5 days
 
Unidentified Staphylococcus spp., E. faecalis, E. coli, Bacteroides vulgatus, Bacteroides ovatus and Bacteroides thetaiotaomicron were the dominant species in the mouse caecal flora. It was interesting that Enterococcus gallinarum appeared 1 day after the CS-834 treatment and E. gallinarum and Proteus mirabilis 1 day after the L-084 treatment.

Mice given CS-834 or L-084 had extremely low levels of antimicrobial activity (0.69 ± 0.01 and 0.16 ± 0.24 mg/kg of the mouse caecum contents, respectively) in the caecum 12 h after the 5 day treatment, while DU-6859a was present at concentrations of 173 ± 46.2 mg/kg of mouse caecal content 12 h after the treatment. R-95867 and L-036 were apparently inactivated by the 24 h incubation with the rat caecal contents but not DU-6859a, CS-834 or L-084 when compared with antimicrobials in saline as controls. In contrast, only L-084 and L-036 were inactivated after 24 h incubation with the mouse caecal contents.

None of the strains tested reduced the antimicrobial activity of CS-834, R-95867, L-084 or L-036, compared with the drugs in saline, during 24 h incubation (data not shown). All anaerobic and aerobic strains used were positive for ß-lactamase by the cefinase test.

The three oral antimicrobials, DU-6859a, CS-834 and L-084, used in this study have good in vitro activity against most anaerobes and aerobes. Thus, we presumed that oral administration of these agents would have a significant impact on the caecal flora of rats and mice. In fact, DU-6859a generated a marked decrease in the number (>3-log10) of both anaerobes and aerobes in the rat and mouse models. These results are consistent with a previous study on the influence of oral DU-6859a on faecal flora in human volunteers.10 CS-834 and L-084, however, had only a slight effect on the caecal flora of rats and L-084 had only a slight influence on the caecal flora of mice.

In in vitro inactivation studies, CS-834 and L-084 were found to be stable in the caecal contents of rats and mice but their active metabolites were unstable in the caecal contents of rats. The mechanism(s) by which the antibacterial activity of CS-834, L-084 and their metabolites is reduced in the caecum remains unknown. One possibility is that ß-lactamases produced by the caecal flora inactivate these carbapenems retained in the gut lumen. However, we did not find any caecal isolates that might hydrolyse these agents.


    Notes
 
* Corresponding author. Fax: +81-58-265-9001; E-mail: nk19{at}cc.gifu-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Sato, K., Hoshino, K., Tanaka, M., Hayakawa, I. & Osada, Y. (1992). Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates. Antimicrobial Agents and Chemotherapy 36, 1491–8.[Abstract]

2 . Kato, N., Kato, H., Tanaka-Bando, K., Watanabe, K. & Ueno, K. (1996). Comparison of in vitro activities of DU-6859a and other fluoroquinolones against Japanese isolates of anaerobic bacteria. Clinical Infectious Diseases 23, Suppl. 1, S31–5.[ISI][Medline]

3 . Fukuoka, T., Ohya, S., Utsui, Y., Domon, H., Takenouchi, T., Koga, T. et al. (1997). In vitro and in vivo antibacterial activities of CS-834, a novel oral carbapenem. Antimicrobial Agents and Chemotherapy 41, 2652–63.[Abstract]

4 . Kato, N., Tanaka, K., Kato, H. & Watanabe, K. (2000). In vitro activity of R-95867, the active metabolite of a new oral carbapenem, CS-834, against anaerobic bacteria. Journal of Antimicrobial Chemotherapy 45, 357–61.[Abstract/Free Full Text]

5 . Hikida, M., Itahashi, K., Igarashi, A., Shiba, T. & Kitamura, M. (1999). In vitro antibacterial activity of LJC 11,036, an active metabolite of L-084, a new oral carbapenem antibiotic with potent antipneumococcal activity. Antimicrobial Agents and Chemotherapy 43, 2010–6.

6 . Kato, N., Kato, H., Tanaka, K. & Watanabe, K. (1998). L-084, a new oral carbapenem: in vitro activity against anaerobic bacteria. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract F-70, p. 258. American Society for Microbiology, Washington, DC.

7 . Sakata, T., Tsuchita, H., Ishii, K., Hojo, K., Isawa, K., Satomi, K. et al. (1999). Blood biochemical characteristics, cecal microbiota and short-chain fatty acid composition in fistula implanted rats. Experimental Animals 48, 95–100.[ISI][Medline]

8 . Summanen, P., Baron, E. J., Citron, D., Strong, C., Wexler, H. M. & Finegold, S. M. (1993). Wadsworth Anaerobic Bacteriology Manual, 5th edn. Star Publishing, Belmont, CA.

9 . Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H. (1995). Manual of Clinical Microbiology, 6th edn. American Society for Microbiology Press, Washington, DC.

10 . Inagaki, Y., Yamamoto, N., Chida, T., Okamura, N. & Tanaka, M. (1995). The effect of DU-6859a, a new potent fluoroquinolone, on fecal microflora in human volunteers. Japanese Journal of Antibiotics 48, 368–79.[Medline]

Received 14 March 2000; returned 24 May 2000; revised 12 June 2000; accepted 10 July 2000





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