Efficacy of a novel injectable cephalosporin, Cefclidin, on the experimental complicated urinary tract infections with urinary stones caused by Pseudomonas aeruginosa and Proteus mirabilis
Satoh, Masaru; Munakata, Kei-ichi; Takeuchi, Hideo; Yoshida, Osamu
泌尿器科紀要 (1994), 40(8): 689-694
Departmental Bulletin Paper
Acta Urol. Jpn. 40: 689-694, 1994
EFFICACY OF A NOVEL INJECTABLE CEPHALO· SPORIN, CEFCLIDIN®, ON THE EXPERIMENTAL COMPLICATED URINARY TRACT INFECTIONS WITH URINARY STONES CAUSED BY PSEUDO· MONAS AERUGINOSA AND PROTEUS MIRABILIS Masaru Satoh and Kei-ichi Munakata From the Department of Microbiology, Tokyo Research Laboratories, Eisai Co., Ltd.
Hideo Takeuchi and Osamu Yoshida From the Departmant of Urology, School of Medicine, Kyoto University
We evaluated the effects of a novel cephalosporin, cefc1idin (CFCL) and imipenen (IPM), on the eradication of bacteria from the urine, bladder stones and the kidneys, and also on the prevention of the infection stone formations, in our polymicrobial urinary tract infection model of rats associated with bladder stones using IPM-sensitive or IPM-resistant Pseudomonas aeruginosa and Proteus mirabilis as a causative pathogen. CFCL completely eradicated P. mirabilis from the urine and the stone in the short-term regimen (5 days). CFCL completely eradicated both IPM-sensitive P. aeruginosa and P. mirabilis from the urine, the stones and the kidneys as compared to IPM in the long-term regimen (II days), reflecting the superior antibacterial activity of CFCL. CFCL also significantly prevented the development of infection stones as compared to IPM in the long-term regimen. There was no significant difference in the blood urea nitrogen (BUN) values between the CFCL or IPM-treated and the non-treated groups. The cumulative recovery rate of unchanged CFCL reached 47.3% of the total dosage (20 mg/ kg) within 8 hours. (Acta Urol. Jpn. 40: 689-694, 1994) Key words: Pseudomonas aeruginosa, Urinary tract infections
INTRODUCTION Pseudomonas urinary tract infections associated with urinary stones, indwelling catheter and/or obstructive uropathy are well known refractory and resistant to various antipseudomonal chemotherapies. In terms of the bacteriological feature, most of Pseudomonas complicated urinary tract infections are noted to be polymicrobial infections. Accordingly, antipseudomonal agents with a broad spectrum are preferable in the management of Pseudomonas complicated urinary tract infections. Therefore, imipenem (IPM), which shows a very broad spectrum is one of the most useful antimicrobial agent in controlling Pseudomonas urinary tract infection 1,2). However, emergence of resistant P. aeruginosa to IMP
was recently noted on a patient with complicated urinary tract infections~). Some recent reports also indicated that resistance of P. aeruginosa to IPM was acquired in the course of the actual treatment 4 ,5), probably due to the decrease in the permeability of IPM toward the outer membrane of P.
aeruginosa 6) • CFCL, a novel injectable cephalosporin, manifests a broad antibacterial spectrum and especially exhibits prominent activity toward various clinical isolates of P. aeruginosa including IPM-resistant P. aeruginosa 7,8), Therefore, we evaluated the prospective usefulness of CFCL in the management of P. aeruginosa induced complicated urinary tract infections, using our polymicrobial urinary tract infection model assocaited with urinary stones 9).
Acta Urol. Jpn. Vol. 40, No.8, 1994
MATERIALS AND METHODS Bacteria Proteus mirabilis (E05106) , IPM-sensitive (MIC of IPM 3.13 f1g/ml) Pseudomonas aeruginosa (E030033) and IPM-resistant (MIC of IPM 6. 25 f1g/ml) Pseudomonas aeruginosa (E030400) were isolated from
patients with urinary tract infections.
Antibiotics CFCL was synthesized at Eisai Co., Ltd. and its purity was more than 98%. IPM preparation (Banyu Pham., Co., Ltd., Japan) was purchased.
Animals Female Sprague Dawley strain rats (specific pathogen free, 7 weeks old, each weighing 180 to 220 g, Charles River Japan Inc.) were used in all experiments.
The experimental procedures for the preparation of our polymicrobial infection model in rats The fundamental experimental procedures were conducted according to the method of Satoh et al. (1984). Namely, rats were intra peritoneally anesthetized with sodium pentobarbital. A sterile zinc ring (4 mm in diameter) was surgicaIly implanted into the bladder foIlowed by the administration of aminobenzyl-peniciIIin to prevent the infection associated with surgery. Seven days after surgery, the first bacterium, P. mirabilis (107 cfu/rat) was transure -theraIly inoculated through a polyethylene tube (PE-IO), and then the second test bacterium, P. aeruginosa (10 8 cfu/rat) was inoculated in the same manner 5 days after the first inoculation. Three types of experiments were performed: Experiment 1 (the short-term regimen for the IPM-sensitive P. aeruginosa (E030033) plus P. mirabilis (E05106) infection]. The minimum inhibitory concentration (MIC) of CFCL for P. mirabilis (E05106) and P. aeruginosa (E030033) was 0.4 f1g/ml and 0.8 f1g/ml, respectively, and the MIC of IPM for P. mirabilis and P. aeruginosa was 6.25 f1g/ml and 1.56 f1g/ml, respectively. CFCL was administered intramuscularly twice a day at the dosage of 20 mg/kg for
5days beginning from the 4th day after infection of P. aeruginosa. IPM was administered intramuscularly twice a day at the dosage of IOmg/kg under the same condition. Experiment 2 (the short-term regimen for the IPM resistant P. aeruginosa (E030400) plus P. mirabilis (E05106) infection]. The MIC of CFCL and IPM for P. aeruginosa (E030400) was 0.2 f1g/ml and 6.25 f1g /ml, respectively. The antibiotics were administered under the same condition as described in experiment I. Experiment 3 (the long-term regimen for the IPM-sensitive P. aeruginosa (E030 033) plus P. mirabilis (E05106) infection]. The antibiotics were administered for I I days under the same condition as described in experiment 1. AIl the tested rats were killed to make the stipulated determinations 24 hours after the final dosing.
Bacteriological determinations At the time of death, the bladder urine was aspirated with a tuberculin syringe, and then diluted with a sterile saline. Both kidneys were removed and homogenized. The bladder stones were removed from the bladder at autopsy and weighed, then immersed in a 70% alcohol solution for 10 minutes, foIlowed by twice washing with a sterile saline and cultured after grinding them. Our preliminary examinations clarified that live bacteria on the surface of the bladder stone were eradicated by the immersion in 70% alcohol for 10 minutes which had no effect on the live bacteri~ inside the bladder stone. The specimens were cultured after inoculation onto both BTB and NAC agar media (Eiken Co., Ltd., Japan).
Blood urea nitrogen (BUN) determinations At the time of death, the blood specimens from the aorta abdominals were collected to determine the blood urea nitrogen (BUN) according to the method of Seligson 11).
Determination of urinary recovery rates of antibiotics The amounts of unchanged CFCL and
Satoh, et al.: Infection, Chemotherapy
infectiom bladder stones. There was no significant difference in BUN values or the incidence of kidney abscess between the treated and non-treated groups (Table 1). In experiment 2 (the short-term regimen for the IPM- resistant P. aeruginosa plus P. mirabilis infection), CFCL more effectively eradicated IPM-resistant P. aeruginosa from the urine and the stone as compared to IPM, reffecting its superior in vitro antipseudomonal activity and CFCL also completely eradicated P. mirabilis from the urine and the stones. Furthermore, CFCL significantly prevented the development of infection bladder stones as compared to IPM. There was no significant defference in the incidence of abscess formation among the three groups and all the BUN values in the three groups were in the normal range (Table 2). In experiment 3 (the long-term regimen
IPM in the urine were determined by the thin layer disc method using Escherichia coli E01174 and Bacillus subtilis ATCC 6633 as a test bacterium.
RESULTS Effects if CFCL and IPM on the polymicrobial infection if P. aeruginosa plus P. mirabilis in rats In experiment 1 (the short-term regimen for the IPM-sensitive P. aeruginosa plus P. mirabilis infection), CFCL completely eradicated P. mirabilis from the urine and the stones, and also P. aeruginosa from the stone. IPM eradicated P. aeruginosa from the stones, but could not eradicate P. mirabilis from the urine and the stones, and P. aeruginosa from the urine. Both CFCL and IPM showed marked efficacy in preventing the development of
Table 1. Effects of the short-term regimen (5 days) with CFCL and IPM on viable cells in both the urine and the stone, and on the infection stone formation caused by IPM-sensitive P. aeruginosa + P. mirabilis Incidence of positive culture
t: 15/8 tt t[ V'~ 4/8 tt t[ 2/8
J J 11/12
a) Number of rats showing a positive culture judged by the following criteria: Urine; >2X 10' cfu/m!. Stone; >40 cfu/stone. Kidney; >40 cfu/kidneys. b) " : P
Table 2. Effects of the short-term regimen (5 days) with CFCL and IPM on viable cells in both the urine and the stone, and on the infection stone formation caused by IPM-resisitant P. aeruginosa + P. mirabilis. Incidence of positive culture a )
Group P. mirabilis
CFCL IPM Non-treated
['' J 8/9
Urine P. aeruginosa
V'~ 6/9 t
J 'I b)
0/' 1/9 6/12
.[;"'H.~ 77 .9±8.5
14.4± 1.2 16.4± 1.5
a) Number of rats showing a positive culture judged by the following criteria: Urine; >2X 10' cfu/m!. Stone; >40 cfu/stone. Kidney; >40 cfu/kidneys. b) " : P
Acta Urol. Jpn. Vol. 40, No.8, 1994 Effects of the long- term regimen (11 days) with CFCL and IPM o~ viable cells in the urine, the stone and the kidney, and on the infection stone formatIOn caused by IPMsensitive P. aeruginosa + P. mirabilis
Incidence of positive culture ll )
Urine P. mirahilir
:[ 6/II]tt tt[ 4/llltt tt[ 2/11 Jtt ttf 0/11 tt tt[ 0/11
O/IIJ" [ 24.4± 4.2]
tt ttf 2/11 tt •• - 9/10
Stone weight (mg)
47.6± 6.B •• 20.7± 0.7
a) Number of rats showing a positive culture judged by the following criteria: Urine; >2X 102 cfu/ml. Stone; >40 cfu/stone. Kidney; >40 cfu/kidneys. b) " : P
"~ " ...c:::~ >
Urinary levels of CFCL and IPM in rats with infection stones caused by P. mirabilis plus P. aeruginosa. CFCL and IPM were administered intramuscularly 13 days after infection of P. mirabilis (8 days after infection of P. aeruginosa, CFCL; 20 mg/kg, IPM; IOmg/kg).
for the IPM-sensitive P. aeruginosa plus P. mirabilis infection), CFCL completely eradicated both P. aeruginosa and P. mirabilis from the urine, the stones and the kidneys. CFCL was significantly superior to IPM in the prevention of the development of infection bladder stones (Table 3). Kidney
abscesses were observed in 6 of the 12 rats in the non-treated groups and in of 8 rats in the IPM-treated groups. Meanwhile, non of the rats in the CFCL-treated group had kidney abscesses. The cumulative recovery rate of unchanged CFCL reached 47.3% of the total dosage (20 mgj
Satoh, et al.; Infection, Chemotherapy
kg) within 8 hours, comparable to that of IPM (48.0%). Meanwhile, CFCL showed a urinary excretion pattern different from that of IPM, with a more prolonged urinary level (Fig. I). Emergence of resistant strains of P. aeruginosa toward CFCL and IPM during the present treatment was not observed.
DISCUSSIONS We evaluated the efficacy of CFCL and IPM against the polymicrobial Pseudomonas urinary tract infections, a representative refractory infection, using our polymicrobial urinary tract infection model associated with urinary stones. The short-term regimen (5 days) with CFCL was insufficient to eradicate IPM-sensitive P. aeruginosa (MIC of CFCL; 0.8 pg/ml) from the urine as compared with the corresponding long-term regimen (11 days). This suggests that the long-term regimen should be used for the satisfactory management of Pseudomonas complicated urinary tract infections, even if a potent antipseudomonal agent such as CFCL is used. Meanwhile, CFCL more effectively eradicated IPM-resistant P. aeruginosa (MIC of CFCL; 0.2 pg/ml) from the urine and stones than IPM in the short-term regimen. This may be attributable to the difference in antipseudomonal activity and the urinary excretion pattern between CFCL and IPM. Concerning the correlation between the MIC values and eradicative actions to P. aeruginosa from the urine, the kidney and the stones, an interesting finding has been reported; both the short-term and longterm regimen (11 days) with indicating MIC for the tested P. aeruginosa of 1.56 pg/ ml were insufficient to eradicate P. aeruginosa from the urine, the stone and the kidney in the same experimental conditions l2 ) Similarly, the present study showed that IPM with an MIC of 1.56pg/ml for P. aeruginosa was not sufficient for the eradication of P. aeruginosa in experimental 3 (the long-term regimen). Accordingly, the MIC values of a therapeutic agent toward P. aeruginosa must be below 0.8 pg/ml, in order to fulfill satisfactory eradication of P.
aeruginosa from the urine, the kidney and associated infection stones. In the present experiment, Enterococcus faecalis infections were observed in CFCLtreated, IPM-treated and non-treated groups, although, the rats had not been inocuated with E. faecalis. Our preliminary examinations clarified that these E. faecalis infections were inevitably induced by the transuretheral cannulation of the polyethyllene tube via the ascending route. Although IPM had potent antibacterial activity toward E. faecalis D , E. faecalis could not be eradicated from the urine, the stones or the kidneys. The insufficient eradicative effects of IPM might be attributable to its rapid excetion from the urine. Our present findings indicate that the associated P. mirabilis must primarily be eradicated for the complete eradication of P. aeruginosa because infection stones act as a sanctuary to hide bacteria from chemotherapy. Accordingly, it is basically important to remove the associated urinary stones from the urinary tract for the treatment of urinary tract infections accompanied with urinary stones. Recently, ultrasonic lithotripsy or extracorporeal shock wave lithotripsy has been widely used for the treatment of urinary stones. In lithotripsy regimen, the bacteria existing in the urinary stones are released and induce fever and bacterimia, so that the combination regimen of a powerful antibacterial agent such as CFCL and lithotripsy is strongly recommended as an intensive therapy in the management of Pseudomonas complicated urinary tract infections accompanied with urinary stones. ACKNOW LED GEMEN T We are indebted to Dr. Tamotsu Kanazawa and Mr. Masanori Kayano for their encouragement throughout this investigation.
REFERENCES I) Kropp H, Sundelof j, Kahan j, et al.; MK 0787 (N-formimidoyl thienamycin) evaluation of in vitro and in vivo activities. Antimicrob Agents Chemother 17; 993-1000, 1980 2) Clair EC and Michael LC; Safety and effi-
cacyofimipenem/cilastatinintreatmentof complicatedurinarytractinfections.AmJ Med78192-94,1985
4)NielsenDM,KatzJR,AhloyRD,cta1.: Imipenem/cilastatintherapyforseriousbact ・terialinfection
和文抄録 感 染 結 石 を 伴 うPseudomonasaeruginosaとProteUSmirabilislこ
よ る実 験 的
複 雑 性 尿 路 感 染 に 対 す る 新 規 注 射 用 セ フ ァ ロ ス ポ リ ン"セ
フ ク リ ジ ン"の
エーザイ株式会社 ・東京研究所 佐藤
京都大学医学部泌尿器科学教室(主 任:吉 田 修 教授) 竹 内 Imipenem(IPM)感
受 性 お よ び 耐 性 のPseudomo-
よる 感染 性 膀 胱
の優 れ た抗 菌 力 を反 映 して11日
間 の 長 期 投 与 でIPMと
比 較 した 時,尿,膀
結 石 を 伴 う新 規 ラ ッ ト複 数 菌 尿 路 感 染 モ デ ル を 用 い
お よび 腎 か らIPM感
aeruginosαを 完 全 に除 菌 した.ま
胱 結 石 お よび 腎 か らの 除 菌 お よ び 感 染 性 膀
胱 結 石 の 形 成 抑 制 に 対 す る 新 規 セ フ ァ ロ ス ポ リ ン ・セ フ ク ロ ジ ソ(CFCL)お
よ び チ ェ ナ ム(IPM)の
を 評 価 した. CFCLは,5日
期 投 与 でIPMと 抑 制 した.BUN値
受性 お よび耐 性 のPsetectomonas た,CFCLは,長
比 較 して,感 染 結 石 の成 長 を 有 意 に は,CFCL投
お よ び 非 投 与 群 の 間 に 有意 の 差 は な か った,ラ 間(20mg/kg,b.i.d)の
で 尿 お よ び 結 石 中 か らProteusmirabitisを
短 期投 与 完 全 に除 菌
(20mg/kg,筋 中 回収 率 は,8時
注)に おけ る未 変 化体(活 性 体)の 尿 間 で47.3%に
(泌尿 紀 要40:689-694,1994)