In vitro and in vivo antibacterial activities of a new injectable carbapenem, S-4661, against gynaecological pathogens

Hiroshige Mikamo*, Koji Izumi, Yin Xiang Hua, Yoh Hayasaki, Yasumasa Sato and Teruhiko Tamaya

Department of Obstetrics and Gynaecology, School of Medicine, Gifu University, Tsukasa-machi, Gifu City, Gifu 500-8705, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Carbapenems are often used to treat infections in obstetrics and gynaecology because of their activity against anaerobes. S-4661, a new injectable carbapenem antibiotic, has favourable pharmacokinetic properties and is not hydrolysed by dehydropeptidase I. We investigated the in vitro and in vivo antibacterial activities of S-4661 against strains of Streptococcus agalactiae, Escherichia coli, Peptostreptococcus magnus, Bacteroides fragilis and Prevotella bivia, which are major pathogens in the fields of obstetrics and gynaecology. The MIC50 and MIC90 of S-4661 for these strains were 0.25 and 1 mg/L, respectively. The in vivo efficacy of S-4661 was evaluated in a rat model of intrauterine infection, namely pyometra caused by E. coli and B. fragilis. The accumulation of neutrophils in the uterus in the S-4661-treated group was less marked and the number of bacteria significantly lower than those in the untreated group. These results suggest that S-4661 may be useful for treating polymicrobial infections in the fields of obstetrics and gynaecology.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Carbapenems are broad-spectrum antimicrobial agents with strong bactericidal activity; many have been developed and are widely used for clinical treatment.1 S-4661 is a new carbapenem developed by Shionogi Co. Ltd, Osaka, Japan.2,3 Preliminary reports indicate that S-4661 has activity against a broad bacterial spectrum, has favourable pharmacokinetic properties and is not hydrolysed by dehydropeptidase I (DHP-I).4 Since S-4661 is not degraded by DHP-I, the clinical use of this drug does not require the addition of a DHP-I inhibitor such as cilastatin.4 The objective of this study was to examine the in vitro and in vivo activities of S-4661 against pathogens responsible for many infections in obstetrics and gynaecology.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In vitro study

Antimicrobial agents.
S-4661 was provided by Shionogi Co. Ltd, Osaka, Japan; imipenem by Banyu Pharmaceutical Co. Ltd, Tokyo, Japan; panipenem by Sankyo Co. Ltd, Tokyo, Japan; meropenem by Sumitomo Pharmaceutical Co. Ltd, Tokyo, Japan; and ceftazidime by Nippon Glaxo Co. Ltd, Tokyo, Japan.

Strains tested.
A total of 135 recent clinical isolates (64 strains of aerobes and 71 of anaerobes) was collected from patients in the Department of Obstetrics and Gynaecology, School of Medicine, Gifu University between January 1997 and December 1998. The organisms were identified with the API STREP identification system (bioMérieux SA, Marçy l'Etoile, France), the Enterotube II identification system (Becton Dickinson, Cockeysville, MD, USA) and the Oxi/Ferm Tube II system (Becton Dickinson) for aerobic bacteria, and with the RAP ID ANA system II (Innovative Diagnosis System, Norcross, GA, USA) for anaerobic bacteria. Organisms tested were as follows: 33 isolates of Streptococcus agalactiae, 31 of Escherichia coli, 21 of Peptostreptococcus magnus, 22 of Bacteroides fragilis and 28 of Prevotella bivia.

Susceptibility tests.
MICs were determined by an agar dilution method.5,6 E. coli were grown on Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA); S. agalactiae on Mueller–Hinton agar (Difco) supplemented with 5% sheep serum (Irvine Scientific, Santa Ana, CA, USA); P. magnus and B. fragilis on modified Gifu anaerobic medium (GAM) agar (Nissui Pharmaceutical Co., Tokyo, Japan); and P. bivia on Brucella HK agar (Kyokuto Pharmaceutical Co., Tokyo, Japan) supplemented with 5% laked sheep blood. They were then suspended in Mueller– Hinton broth (Difco) or GAM broth at c. 5 x 108 cfu/mL. After 200-fold dilution of the suspension, the bacteria were inoculated on to appropriate agar plates containing each antimicrobial agent (at concentrations ranging from 0.0020 to 256 mg/L) with a multipoint inoculator (Microplanter, Sakuma Seisakusho, Tokyo, Japan) providing an inoculum of c. 2.5 x 106 cfu/spot. All aerobic cultures were incubated at 37°C for 24 h and all anaerobic cultures at 37°C for 48 h in an Anaero Pack (Mitsubishi Gas Chemical Co., Tokyo, Japan). MICs were defined as the lowest concentrations of antimicrobial agents that prevented visible growth of organisms.

In vivo study

Animals.
Female Sprague–Dawley rats (specific pathogen free, 6 weeks old, weighing 155–170 g) (Nippon Bio-Supply Centre, Tokyo, Japan) were used.

Organisms.
Organisms were clinical isolates from patients with uterine endometritis. The MICs of S-4661 and imipenem for E. coli GOG 0020 were both 0.0125 mg/L, while those for B. fragilis GOG 3102 were both 0.10 mg/L.

Experimental animal model.7,8
Rats were anaesthetized with sodium pentobarbital 25 mg/kg ip. Abdominal and flank hair was shaved, then the abdominal wall was thoroughly swabbed with povidone–iodine. A small vertical incision was made in the prepared abdominal wall and the uterus and adnexa were exposed. The uterine cervix was ligated with 1-0 silk. A 24 h culture of E. coli and 48 h culture of B. fragilis on Mueller–Hinton agar and modified GAM agar, respectively, and GAM broth as an inoculating medium, were used for inoculation. With a disposable sterile tuberculin syringe and a 27-gauge needle, 0.05 mL of bacterial suspension (4.3 x 105 cfu/rat of E. coli and 4.1 x 105 cfu/rat of B. fragilis) was injected into the right side of the uterine cavity. The abdominal wall was closed with 1-0 silk suture. After the operation, the abdominal wound was disinfected daily with povidone–iodine.

Therapeutic study.
Sixteen hours after bacterial inoculation, the rats were treated with S-4661 10 mg/kg iv tds for 5 days or with imipenem 10 mg/kg supplemented with cilastatin 10 mg/kg iv tds for 5 days; they were then compared with an untreated control group (six rats in each group). At 136 h after bacterial inoculation, the rats were anaesthetized with sodium pentobarbital, the peritoneal cavity was opened aseptically and bacterial counts of the uterine contents were performed using BTB agar (Eiken Chemical Co. Ltd,Tokyo, Japan) for E. coli and modified GAM agar for B. fragilis.

Statistical analysis.
Values are reported as mean ± s.d. All results were analysed using Fisher's protected least significance difference test and P values of <0.05 were considered significant.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Table IGo shows MIC ranges, MIC50s and MIC90s of S-4661, imipenem, meropenem, panipenem and ceftazidime. S-4661 inhibited >90% of clinical isolates of S. agalactiae, E. coli, P. magnus, B. fragilis and P. bivia at a concentration of 1 mg/L.


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Table I. In vitro antimicrobial activity of S-4661, imipenem, meropenem, panipenem and ceftazidime against clinical isolates studied
 
Table IIGo shows the bacteriological effects of S-4661 and imipenem/cilastatin against polymicrobial infections caused by E. coli and B. fragilis in the rat pyometra model. The bacteriological response was significantly greater for the S-4661 group than for the imipenem/cilastatin and untreated groups. The effects of S-466110 mg/kg iv tds were equivalent to those of imipenem/cilastatin 10 mg/kg iv tds. S-4661 has shown similar efficacy in animal models of localized infections.9,10 The accumulation of neutrophils in the uterus in the S-4661 (1596.6 ± 368.4 cells/mm2) and imipenem/cilastatin (1625.6 ± 409.0 cells/mm3) groups was less marked than that in the untreated group (3508.2 ± 390.0 cells/mm3).


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Table II. Therapeutic effects of S-4661 and imipenem/cilastatin against polymicrobial infections (Escherichia coli and Bacteroides fragilis) in the rat pyometra model. Viable counts in the uterine contents are expressed as log10 cfu/mL
 
These results suggest that S-4661 may be useful for treating polymicrobial infections in the fields of obstetrics and gynaecology.


    Notes
 
* Corresponding author. Tel: +81-58-267-2631; Fax: +81-58-265-9006; E-mail: mikamo{at}cc.gifu-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Malanoski, G. J., Collins, L., Wennersten, C., Moellering, R. C. & Eliopoulos, G. M. (1993). In vitro activity of biapenem against clinical isolates of gram-positive and gram-negative bacteria. Antimicrobial Agents and Chemotherapy 37, 2009–16.[Abstract]

2 . Iso, Y., Irie, T., Nishino, Y., Motokawa, K. & Nishitani, Y. (1996). A novel 1ß-methylcarbapenem antibiotic, S-4661. Synthesis and structure–activity relationships of 2-(5-substituted pyrrolidin-3-ylthio)-1ß-methylcarbapenems. Journal of Antibiotics 49, 199–209.[ISI][Medline]

3 . Iso, Y., Irie, T., Iwaki, T., Kii, M., Sendo, Y., Motokawa, K. et al. (1996). Synthesis and modification of a novel 1ß-methyl carbapenem antibiotic, S-4661. Journal of Antibiotics 49, 478–84.[ISI][Medline]

4 . Mori, M., Hikida, M., Nishihara, T., Nasu, T. & Mitsuhashi, S. (1996). Comparative stability of carbapenem and penem antibiotics to human recombinant dehydropeptidase-I. Journal of Antimicrobial Chemotherapy 37, 1034–6.[ISI][Medline]

5 . National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria that Grow Aerobically—Second Edition: Approved Standard M7-A2. NCCLS, Villanova, PA.

6 . National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Second Edition: Approved Standard M11-A2. NCCLS, Villanova, PA.

7 . Mikamo, H., Kawazoe, K., Sato, Y., Izumi, K. & Tamaya, T. (1998). Studies on the clinical implications of anaerobes, especially Prevotella bivia, in obstetrics and gynecology. Journal of Infection and Chemotherapy 4, 177–87.

8 . Mikamo, H., Kawazoe, K. & Izumi, K. (1997). Therapeutic effects of cefluprenam (CFLP) on polymicrobial infections associated with Enterococcus faecalis in rat pyometra model. International Journal of Antimicrobial Agents 8, 7–11.[ISI]

9 . Tsuji, M., Ishii, Y., Ohno, A., Miyazaki, S. & Yamaguchi, K. (1998). In vitro and in vivo antibacterial activities of S-4661, a new carbapenem. Antimicrobial Agents and Chemotherapy 42, 94–9.[Abstract/Free Full Text]

10 . Yano, Y., Oguma, T., Nagata, H. & Sasaki, S. (1998). Application of logistic growth model to pharmacodynamic analysis of in vitro bactericidal kinetics. Journal of Pharmaceutical Science 87, 1177–83.[ISI]

Received 1 December 1999; returned 3 March 2000; revised 20 April 2000; accepted 19 May 2000