Cefclidinの 嫌 気 性 菌 に 対 す る抗 菌 力 VOL.40 S -4 31

ANTIBACTERIAL ACTIVITY OF A NEW PARENTERAL CEPHEM, CEFCLIDIN, AGAINST ANAEROBIC BACTERIA AND Gardnerella vaginalis

Naoki Kato, Kakuyo Sawa, Yoshinori Muto, Kunitomo Watanabe and Kazue Ueno Institute of Anaerobic Bacteriology, Gifu University School of Medicine.

Abstract The activity of a new parenteral cephalosporin, cefclidin (CFCL) , was compared with those of ceftazidime (CAZ) , cefotaxime (CTX) , cefpiramide (CPM) , and cefotiam (CTM) against anaerobic bacteria and Gardnerella vaginalis. In general, CFCL showed a broad spectrum against Gram-positive and -negative reference strains of anaerobic bacteria although weak activity of this compound was seen against Bacteroides fragilis group organisms. Against recent clinical isolates, CFCL revealed moderate or weak activity against members of the B.fragilis group as did other antimicrobials tested. CFCL showed poor activity against Prevotella bivia and Clostridium difficile as well. Against most of other members of anaerobic bacteria, CFCL showed good activity, especially against Mobiluncus spp. and G. vaginalis. Enzymatically, CFCL was more stable than CPM, CTX, and CTM, but less stable than CAZ to hydrolysis by oxyiminocephalosporinases type I derived from B.fragilis. Time-kill study demonstrated bactericidal activity without regr- owth at concentrations of two times or more the MIC of CFCL for B.fragilis. Although CFCL had bactericidal potency in rat pouch infection with B.fragilis, regrowth was seen 48h after starting administration of this compound. CFCL seemed to cause little overgrowth of C.difficile in a mouse model.

Key words : cefclidin, anaerobic bacteria, g-lactamase, rat pouch, Bacteroides fragilis, Clostridium difficile

Introduction Cefclidin (CFCL) is a novel semisynthetic par- era, 37 species) of anaerobic bacteria and enteral cephalosporin. The molecular weight of this fastidious aerobic bacterium, G. vaginalis, were compound is 550.619. obtained from the American Type Culture Collec- In the present study, we extensively evaluated the tion (ATCC) , Rockville, Md., U.S.A., the Gifu. in vitro and in vivo activity of CFCL in comparison Anaerobic Institute (GAI) , Gifu, the National Col- with related agents against anaerobic bacteria and lection of Type Cultures (NCTC) , London, the a fastidious facultative anaerobe, Gardnerella United Kingdom, the Virginia Polytechnic Institute vaginalis, which is suspected to be one of the causa- and State University (VPI) , Blacksburg, Va, U.S. tive pathogens of bacterial vaginosis. The stability A., and the Wadsworth Anaerobic Laboratory of CFCL to hydrolysis by fl-lactamases produced by (WAL) , Los Angeles, Calif., U.S.A. A total of 248 Bacteroides fragilis strains and the inducibility of recent clinical isolates of anaerobic bacteria and G. overgrowth of Clostridium dzfficile in murine cecum vaginalis were tested. Isolates were collected from by 7-day administration of this compound were also numerous clinical sources in Japan at the Institute evaluated. of Anaerobic Bacteriology or GAI, Gifu. Organisms Materials and Methods were identified by standard methods. Organisms - The 41 reference strains (12 gen- Antimicrobial agents - Laboratory standard

* 32 CHEMOTHERAPY SEPT. 1992 powders for in vitro activity study were provided as Stability to hydrolysis by ƒÀ-lactamases from B. follows : CFCL from Eisai Co., Ltd., Tokyo ; ceft- fragilis -Crude ƒÀ-lactamase were obtained from azidime (CAZ) from Nihon Glaxo Co., LTd., Tokyo three strains of B.fragilis as described previously1) :

; cefotaxime (CTX) from Hoechst Japan, Tokyo ; Strain GAI 0558 produces oxyiminocephalospor- cefpiramide (CPM) from Sumitomo Pharmaceuti- inase type I strain GAI 7955 is highly resistant to cals, Tokyo ; and cefotiam (CTM) from Takeda cefoxitin (MIC, 1001ig/m1) and strain GAI 10150

Chemical Industries, Osaka. Cefazolin (CEZ) used produces oxyiminocephalosporinase type I and is for fl-lactamase assay was supplied by Fujisawa characterized to be ampicillin-highly-resistant

Pharmaceutical Co., Osaka. Cefotetan (CTT) was (MIC, 1, 600/ig/m1) . Hydrolysis of the compounds obtained from Yamanouchi Pharmaceutical Co. for by fl-lactmases was assayed by a spectro- inducibility of overgrowth of C.difficile in murine photometric technique of the change in absorption cecum. at the absorption maximum of each substrate2)

Susceptibility testing The minimal inhibitory Relative hydrolysis rate of each compound was concentrations (MICs) were determined by an agar expressed as a percentage obtained in comparison dilution method. An inoculum size of 106 colony with the hydrolysis rate of cephaloridine. forming units (CFU) of organisms per ml was In vivo activity in rat pouch infection -Four-week applied onto agar plates containing each antimi- old male Wistar rats (ca.200g of body weight) were crobial with a multipoint inoculator (Microplanter used. Pouch was made on the back of rats by 20 ml

Sakuma Seisakusho, Tokyo) . Brucella HK agar of air injection followed by infusing 1% croton oil

(Kyokuto Phamaceutical Co., Tokyo) supplement- in olive oil3) Intraperitoneal administration of ed with 5% laked sheep blood was used for refer- antimicrobials (200mg per kg of body weight, di. ence strains and modified GAM agar (Nissui Phar- d.) was initiated 24h after inoculation of B.fragilis maseutical Co., Tokyo) was for clinical isolates GAI 5562 culture into the pouch. Bacterial counts except for G.vaginalis and Mobiluncus spp.; colum- in the pouch were performed at intervals of 0, 3, bia agar (Oxoid, Basingstoke, the United Kingdom) 6, 9, 24 , 48 and 72h after antimicrobial adminis- supplemented with 5% sheep blood and 1% tration. Concentration of antimicrobials was mea- proteose peptone No.3 (Difco Laboratories, Detroit, sured 3 , 6 and 9h after the first administration of

Mich., USA) was used for G.vaginalis and Brucella antimicrobials by bioassay method using Escheri-

HK agar supplemented with 5% laked rabbit blood chia coli E01174 strain. was for Mobiluncus spp. B.fragilis GAI 5562 was Inducibility of overgrowth of C.difficile in murine included in all MIC determinations as a control cecum - Four-week old ICR mice (ca.20g) were organism. All anaerobic cultures were incubated at used. Antimicrobials were subcutaneously adminis-

37•Ž in an anaerobic chamber (Hirasawa, Tokyo) tered twice a day at a dose of 100mg per kg of body in an atmosphere composed of 82% N2, 10% CO2, weight for 7 days. Cecum contents were cultured and 8%H2. Plates inoculated organisms were in- quantitatively for C.difficile counts on a C.difficile cubated with for 48h except Porphyromonas gin- selective medium, cefoxitin-cycloserine mannitol givalis and Mobiluncus spp. which were incubated agar (CCMA) medium, in an anaerobic chamber. for 72h. The MICs were determined as the lowest Results concentrations of antimicrobial agents that prevent- In vitro activity against reference strains -CFCL ed visible growth of organisms. had a broad spectrum against anaerobic bacteria

Time-kill study -A forty eight-h culture of B. while this compound showed weak or little activity fragilis GAI 5562 was inoculated into supplemented against B.fragilis group organisms, Fusobacterium

GAM broth containing CFCL or CAZ at concentra- varium, Eubacterium lentum, and C.difficile, which tions of 1/2 , 1, 2 or 4 MIC. Tubes were subcultur- were also highly resistant to CAZ (Tables 1 and 2). ed under anaerobic condition for colony counts at 0, In general, CFCL was less active than the other 4

2, 4, 6, and 24h of incubation. antimicrobials tested against these reference Cefclidinの 嫌 気 性 菌 に 対 す る抗 菌 力 VOL.40 S-4 33

Table 1. Antibacterial spectrum of cefclidin against reference strains of gram-negative anaerobic bacteria

Inoculum size: 106CFU/ml.

Cefclidin Ceftazidime

Fig. 1. Killing-kinetics of cefclidin and ceftazidime against B. fmgilis GAI 5562 CHEMOTHERAPY 34 5EPT. 1992

Table 2. Antibacterial spectrum of cefclidin against reference strains of gram-positive anaerobic bacteria and Gardnerella vaginalis

inoculum size: 106cfu/ml.

strains. with MIC80 of 3.13 and 1.56pg/ml, respectively, an In vitro activity against clinical isolates-The activity which was higher than those of CAZ. ranges of MICs and MICs for 50 and 80% of the Against Bacteroides thetaiotaomicron and Cdifficile, clinical isolates tested (MIC50 and MIC80, respec- CFCL showed little activity as did CAZ and CTM. tively) are given in Tables 3 and 4. CFCL showed Time-kill study-The bactericidal kinetics of activity similar to those of CTM against B.fragilis, CFCL and CAZ against B.fragilis are illustrated in with MIC50 and MIC80 of 50 and> 200pg/ml, Fig.1. CFCL demonstrated bactericidal activity at respectively. CFCL was also comparable in activity concentrations of 1/2 or more of the MIC (501./g/ to CTM against Bacteroides unzformis, Porphyr- ml) and no regrowth was seen 24h after exposure to omonas spp. and Prevotella bivia. Although CFCL concentrations of the MIC or more of CFCL. Com- revealed moderate activity against 3 species of the parable kinetics were obtained for CAZ. genus Peptostreptococcus, its strong activity was Stability to hydrolysis by ƒÀ-lactamases from B. observed against Mobiluncus spp. and G. vaginalis fragilis -CFCL was more stable to hydrolysis by all Cefclidinの 嫌 気 性 菌 に 対 す る 抗 菌 力 VOL.40 S-4 35

Table 3. Comparative activities of cefclidin and other related agents against clinical isolates of gram-negative anaerobic bacteria

a) Pigmented gram-negative rods spp. consisted of 12 isolates of P. intermedia,7 isolates of P. corporis and 2 isolates of P. asaccharolytica.

Bacteria used: B. fragilis GAI 5562. MIC: Cefclidin, 50ug/ml; Ceftazidime, 12.5ƒÊg/ml.

Fig. 2. Effect of cefclidin and ceftazidime on the B. fragilis rat pouch infection CHEMOTHERAPY 36 SEPT. 1992

Table 4. Comparative activities of cefclidin and other related agents against clinical isolates of gram-positive anaerobic bacteria and Gardnerella vaginaris

Table 5. Stability of cefclidin, ceftazidime, cefpiramide, cefotaxime and cefotiam to the hydrolysis by g-lactamase dervied from B. fragilis as compared with that of cefazolin

a)cefazolin=100.

Table 6. Appearance of C.difficile in murine cecum after the 7 days dosing of cefclidin, cefotaxime and cefotetan

a)Number (CFU/g) of C. difficile detected. b)Not tested. Cefclidinの 嫌 気 性 菌 に 対 す る抗 菌 力 VOL.40 S-4 37

ƒÀ-lactamases tested than CPM , CTX and CEZ but Enzymatically, CFCL was relatively stable to

less stable than CAZ (Table 5). hydrolysis by oxyiminocephalosporinase type I

In vivo activity in rat pouch infection-Although derived from B.fragilis in comparing CPM, CTX,

CFCL had bactericidal potency in vivo also, regr- and CTM. Oxyiminocephalosporinase type I is

owth was seen 48h after exposure to the compound known as a common ƒÀ-lactamase produced by B.

;in contrast, CAZ was effective at keeping the fragilis strains. The meaning of this difference in

organism in low number in the pouch (Fig.2). the stability for the treatment of infectious diseases

Drug concentration in the pouch was measured only is not explained. It may be too little a contrast in

3, 6, and 9h after the first administration of the stability to bring dissimilar consequences among

antimicrobials. There was a tendency that CAZ these antimicrobials for the treatment of polymi-

persisted in the pouch longer than CFCL; the con- crobial infections including /3-lactamase-highly

centration of CAZ in the pouch covered the MIC for producing B.fragilis or we may expect some differ-

the organism used by 9h after administration, while ent outcome among these agents. Treatment of rat

that of CFCL lowered below the MIC for the organ- pouch infection with CFCL resulted in regrowth of

ism by 6h after administration (Fig.3). the tested organism 48h after starting the adminis-

Inducibility of overgrowth of C.difficile in murine tration of this compound. The antibiotic level in the

cecum-Appearance of C.difficile in murine cecum pouch was below the MIC of CFCL for the organism

after 7days dosing of CFCL, CTX, and CTT was 6h after the administration. However, good in vitro

examined. Although CTX and CTT caused over- killing kinetics of CFCL against B.fragilis demon-

growth of Cdifficile, which was detected 1-day and strated in this study may suggest that increased

5days, respectively, after ceasing administration of dosage of CFCL shows constant depression and

antimicrobials, a low number of C.difficile was eradication of the organism in the pouch.

detected lday after ceasing in a mouse of 10 mice It is well known that many fl-lactams can cause

which were given CFCL (Table 6). antibiotic-associated colitis and diarrhea. We

Discussion demonstrated that the administration of CFCL did

CFCL is expected to have strong activity against not generate obvious appearance of C.difficile in the

Gram-negative aerobic bacteria, particularly mouse model, even when C.difficile was examined

against Pseudomonas aeruginosa. In recent years, just after ceasing medication or long after. The

some 13-lactam antibiotics, such as CPM, CAZ, results suggest that CFCL would be relatively free

cefpirome, and cefepime (CFPM), have been devel- from C.diffici/e-associated colitis and diarrhea.

oped; their properties of which have especially Literature cited

emphasized in vitro strong anti-P.aeruginosa activ- 1. Bandoh, K., Muto, Y., Watanabe, K., Katoh, N.,

ity. In contrast, these fi-lactams often show moder- Ueno, K. : Biochemical properties and purification

ate or weak activity against members of the B. of metallo-fl-lactamase from Bacteroides fragilis. fragilis group4-6). In the present study, CFCL Antimicrob. Agents Chemother. 35: 371-372 , 1991

revealed weak activity against these anaerobic 2. Lorian, V.: Antibiotics in laboratory medicine,

organisms as did other anti-pseudomonal ft-lactams. 2nd ed., Williams and Wilkins, Baltimore, 20 antibi-

CFCL showed poor activity against P.bivia and C. otic inactivating enzymes and bacterial resistance

difficile; such an activity was proved in CFPM as (NEU, H.C.) pp.757-789, 1986

well. Against most of the other members of anaer- 3. Kato, N., Ohashi, H., Muto, Y., Watanabe, K.

obic bacteria, CFCL showed good activity, espe- Ueno, K. Usefulness of rat pouch for an experimen-

cially against Mobiluncus spp. and G. vaginalis. tal animal model of anaerobic infections. CHEMOTHERAPY 38 SEPT. 1992

Kenkiseikin-kansenshou-kenkyu 17 : 94-98,1987 against anaerobic bacteria. Chemotherapy 39 (S-1) 4. Isono, M., Yamada, H., Aoki, M., Sawa, K., : 40-45, 1991 Watanabe, K., Ueno, K.: In vitro and in vivo activ- 6. Kato, N., Bando, K., Muto, Y., Watanabe, K., ity of cefpiramide (SM-1642) against anaerobic Ueno, K. : In vitro activity of cefepime, an new bacteria. Chemotherapy 31 (S-1) : 48-56,1983 parenteral cephalosporin, against anaerobic bacte- 5. Watanabe, K., Katoh, N., Muto, Y., Bandoh, K., ria. Chemotherapy 39 (S-2) : 43-51,1991 Ueno, K. : In vitro antibacterial activity of cefepime

新 注射 用 セ フ ァロ ス ポ リンcefclidinの 嫌 気性 菌 お よびGardnerella vaginalisに 対す る抗 菌 力

加 藤直樹,沢 赫代,武 藤吉徳 渡辺邦 友,上 野一 恵 岐阜大学医学部附属嫌気性菌実験施設

新 注 射 用 セ フ ァ ロ ス ポ リンcefclidin(CFCL)の 嫌 気 性 菌 お よ びGardnerella vaginalisに 対 す る抗 菌 力 をceftazidime(CAZ),cefotaxime(CTX),cefpiramide(CPM),ceftotiam(CTM)と 比 較 し て検 討 した 。CFCLはBactenides fragilis groupの 嫌 気 性 菌 に 対 して 弱 い 抗 菌 力 しか 示 さ なか っ た が,一 般 的 に は グ ラ ム 陽 性 お よ び 陰 性 の 嫌 気 性 菌 に 対 して 幅 広 い 抗 菌 力 を有 し て い た。 新 鮮 臨 床 分 離 株 に 対 して は,他 の 比 較 薬 剤 と同 様 にB.fragilis groupに 対 し て は 中程 度 か 弱 い 抗 菌 力 し か 示 さ な か っ た 。 また,PmoteJla biviaとClostndium difficileに対 して は 抗 菌 力 は あ ま りみ ら れ なか っ た。 そ の 他 の 嫌 気 性 菌 に 対 して は よ い抗 菌 力 が 認 め られ,こ と にGvaginaJisと Mobiluncus spp.に は 強 い 抗 菌 力 が み ら れ た。CFCLはB.fragilisの 産 生 す るoxyiminoce- phalosporinaseI型 に 対 してCTX,CPM,CTMよ りは 安 定 で,CAZよ りは わ ず か に 不 安 定 で あ っ た 。増 殖 曲 線 に 対 す るCFCLの 効 果 の検 討 で は,2MICも し くは そ れ 以 上 の 薬 剤 濃 度 で 再 増 殖 の み られ な い 殺 菌 的 効 果 が 認 め られ た 。CFCLは ラ ッ ト・パ ウ チ を 用 い た 感 染 実 験 に お い て も B.fragilisに 対 して 殺 菌 的 効 果 が 認 め られ た が,48時 間 後 に は 菌 の 再 増 殖 が み ら れ た。CFCLは マ ウ ス実 験 モ デ ル を 用 い た 検 討 に お い て,Cdifficileの 腸 管 内 異 常 増 殖 は 引 き起 こ し に くい 薬 剤 で あ る と思 わ れ た。

*〒500岐 阜 市 司 町40