Cefclidinの 嫌 気 性 菌 に 対 す る抗 菌 力VOL.4031

S -4

ANTIBACTERIALAGAINST

ACTIVITY
OF A NEW PARENTERALCEPHEM,
CEFCLIDIN,
ANAEROBICBACTERIA
AND Gardnerellavaginalis

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 withthose 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 grouporganisms. Against recent clinical isolates, CFCL revealed moderate or weak activity
against members of the B.fragilis group as did other antimicrobials tested. CFCL showedpoor activity against Prevotella bivia and Clostridium difficile as well. Against most ofother 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 regrowth 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 48hafter starting administration of this compound. CFCL seemed to cause little overgrowthof C.difficile in a mouse model.
Key words : cefclidin, anaerobic bacteria,
Clostridium difficile
Introduction
Cefclidin (CFCL) is a novel semisynthetic parenteral cephalosporin. The molecular weight of thiscompound is 550.619.
In the present study, we extensively evaluated thein vitro and in vivo activity of CFCL in comparison
with related agents against anaerobic bacteria anda fastidious facultative anaerobe, Gardnerellavaginalis, which is suspected to be one of the causative pathogens of bacterial vaginosis. The stabilityof CFCL to hydrolysis by fl-lactamases produced byBacteroides fragilis strains and the inducibility ofovergrowth of Clostridium dzfficile in murine cecumby 7-day administration of this compound were also
evaluated.
Materials and Methods
Organisms - The 41 reference strains*(12 gen-g-lactamase,rat pouch, Bacteroides fragilis,era, 37 species) of anaerobic bacteria andfastidious
aerobic
bacterium,G. vaginalis,
obtainedtion

from the American(ATCC) , Rockville,

Anaerobic
lectionInstituteof TypeType Culture

theGifu.

(GAI) , Gifu, the National

Col-Cultures

Md.,

U.S.A.,(NCTC) , London,United Kingdom, the Virginia Polytechnic
and State University(VPI) , Blacksburg,A.,

andthewereCollec-Wadsworth

AnaerobictheInstitute
Va, U.S.

Laboratory(WAL) , Los Angeles, Calif., U.S.A. A total of 248
recent clinical isolates of anaerobic bacteria and G.
vaginalis were tested. Isolates were collected fromnumerous

clinicalsourcesin Japanat the Instituteof Anaerobic Bacteriology or GAI, Gifu. Organisms
were identified by standard methods.
Antimicrobial

agents -Laboratorystandard

CHEMOTHERAPY32SEPT.

powders for in vitro activity study were provided asfollows : CFCL from Eisai Co., Ltd., Tokyo ; ceft-fragilis -Crude ƒÀ-lactamaseazidimethree(CAZ) from Nihon Glaxo Co., LTd., Tokyo; cefotaxime
cefpiramide
cals, Tokyo
Chemical(CTX) from Hoechst Japan, Tokyo ;(CPM) from Sumitomo Pharmaceuti; and cefotiam(CTM) from Takeda

Industries,

for fl-lactamase
PharmaceuticalobtainedOsaka.

Cefazolin

assay was supplied by Fujisawa
Co., Osaka. Cefotetan (CTT) wasfrom Yamanouchi

inducibility(CEZ) usedof overgrowth

PharmaceuticalCo. forof C.difficilein murine

cecum.
Susceptibilitytesting

dilution

method.

forming

units

appliedonto

crobialwith

Sakuma

An

ence

a5%

strains

exceptfor

multipoint

Mich.,

USA)
agar

supplemented

for

Mobiluncus
inancubatedusedfor

MIC

determinations
culturesand72h.

The

visiblewereantimicrobial

GAI

GAM

broth

tions

of
under

2,4,of5562

,

1,was

2

or

anaerobic
6,and

as

4

of

eight-h

CFCL

orMIC.

Tubes

incubation.

in

old

male

Wistarrats

forhydrolysis

Pouchinolive

CO2,ingin-

the

pouch

6,9,24 ,tration.

thecolipouch.

bioassay

Four-week

oldAntimicrobials

of

tered

twicea dayof

body

20ml
oil
ofweight,ofdi.

B.fragilis

Bacterialatcountsintervalsof

antimicrobials

thefirstof

0,3,adminiswasmea-administration

methodofofusing

Escheri-

ICR

miceof

Cecum

contents

forC.difficile

countsmedium,(CCMA)

inwereadminis-

100mg

perkg

in

anofwereonbodyculturedaC.difficile

cefoxitin-cycloserine
medium,murine(ca.20g)

subcutaneously

a dosequantitatively

atC.difficile

days.

mannitolanaerobic

chamber.

Results
In

B.

vitroawhile

activity

broad
this

compoundsupplemented

againstB.fragilis

atconcentra-

varium,Eubacterium

were

subcultur-were0,In

also
general,antimicrobials

againstspectrum

into

at

by
crotonantimicrobial

werelowestculture

kg

after

by

rats
1%

strain.

used.

prevent-

countsinoculation

theovergrowth

CAZ

colony

after

after

of

were

administration

intoofselective

back

24h

9h

wascomparisonweight)

infusing

perE01174

7

thebody(200mgperformed

and

for

inof

by

72h

Inducibility

weightcompoundinfection -Four-weekonandabsorption
substrate2)

cephaloridine.

(ca.200g

Concentration
3 ,6

each

pouch

were
48

isspectro-in
eachIntraperitoneal

cultureinofratmadeinitiatedandcompounds
aobtained

followedoil3)

5562

agar

incubated

that

was

10150

I

the

changeofrate

injection

washadinoculated

condition
24h

10%Porphyromonas

agentsactivity

cecum atweredetermined

thevivoto

GAI

byofpercentage

In

airof

the

rate

astrainassayedofwithchiaTokyo)

wereaswascontrol(Hirasawa,

which

forty

containing

1/2

blood

organismshydrolysis

resistant

type

maximum

Relativeof

is highlyHydrolysis

techniqueexpressed:

ampicillin-highly-resistant.

absorptionB.
frompreviously1)

andwasphotometricantimicrobials

aN2,1, 600/ig/m1)

the7955befl-lactmases

Brucella

incubated82%

to(MIC,described1001ig/m1)

characterized

by

from
obtained

oxyiminocephalospor-

GAI(MIC,suredorganisms.

study -Afragilised

spp.

ofgrowth

Time-killas

except1%

5562

strainoxyiminocephalosporinase

Detroit,rabbitGAIofMICs

concentrations

laked

inoculated

Mobiluncus

andwerechambercomposed

48handB.fragilis

anaerobicforbloodIasproduces

produces

GAI

colum-Kingdom)

B.fragilis

0558

cefoxitind.)

Phar-spp.;

oftypeby ƒÀ-lactamases
wereantimicrobials

isolates

UnitedG.vaginalis
5%refer-(NissuiclinicalLaboratories,

withPlatesforhydrolysis

GAI

used.
agar

supplement-

agar

sheep

(Difcoanaerobicwith

givalised5%

antimi-usedfortheNo.3

atmosphere
8%H2.

for

strains

inase

was

HK

Mobiluncus

spp.

allAllinwasandwaswas

anblood

Basingstoke,

HKorganism.

Tokyo)

GAM

colonyml

Brucella

Co.,

was

agar

(Microplanter.

Tokyo)

withincluded

eachmodified

an106

containingandpeptone

ofperCo.,

supplemented
proteose

size

inoculator

sheep(Oxoid,inhibitory
byorganisms

Tokyo)

G.vaginalis

agar

andofplates

lakedmaseuticalininoculum

(CFU)
agar

minimaldetermined

Phamaceuticalwith

37•Ž

wereSeisakusho,(KyokutobiaThe(MICs)

to

Strainatconcentrations

ed

Stability

1992

highlyCFCL

reference
againstshowedgroup

weakresistanttestedororganisms,
lentum,wasstrains -CFCL
anaerobicandtoless

activity

Fusobacterium
C.difficile,

CAZ(Tables

activethanagainst

bacteria
little

these

which
1

theandotherreference2).
4

VOL.40

Cefclidinの 嫌 気 性 菌 に 対 す る抗 菌 力

S‑433

Table

1.

Antibacterial
spectrumanaerobic
bacteria

of cefclidin

againstreferencestrainsof gram-negative

Inoculum size: 106CFU/ml.

Cefclidin

Ceftazidime

Fig. 1. Killing-kinetics of cefclidin and ceftazidime against B. fmgilis
GAI 5562

CHEMOTHERAPY

34

Table

2.

Antibacterial
spectrumof cefclidin
anaerobic
bacteria
and Gardnerella

5EPT.

againstreferencevaginalisstrainsof gram-positive

3.13

and1992

inoculum size: 106cfu/ml.

strains.
In vitro activity against clinical isolates-The
ranges of MICs and MICs for 50 and 80% of theclinical isolates tested (MIC50 and MIC80, respectively) are given in Tables 3 and 4. CFCL showed
activity similar to those of CTM against B.fragilis,
with MIC50 and MIC80 of 50 and>
200pg/ml,respectively. CFCL was also comparable in activity
to CTM against Bacteroides unzformis, Porphyromonas spp. and Prevotella bivia. Although CFCL
revealed moderate activity against 3 species of thegenus Peptostreptococcus, its strong activity wasobserved against Mobiluncus spp. and G.vaginalis

withMIC80activityof
whichAgainstshowed

CFCL
Fig.1.

CAZ

CFCL

noparable
Stability
fragilis -CFCL

1/2

regrowthof

thekinetics

weretohydrolysis
wasdidB.fragilis

and

more

wasofillustrated

inactivity

of

the

seen

24h

after

or

more

of

MIC
obtainedfor

MIC

stabletoat(501./g/exposure
CFCL.

to
Com-CAZ.

by ƒÀ-lactamases
more

CTM.

kinetics

bactericidal
orCAZ.

Cdifficile,

CAZ

areanofbactericidal

againstofconcentrations

thoseandasdemonstrated

concentrations
andactivity

study-Theandrespectively,
thanthetaiotaomicron
littleTime-kill1.56pg/ml,
higherBacteroides

CFCLml)wasfromhydrolysis

B.
byallCefclidinの 嫌 気 性 菌 に 対 す る 抗 菌 力VOL.40S‑435Table3.

a) Pigmented
P. corporis

Comparative
activities
isolatesof gram-negativegram-negativeand 2 isolates

Bacteria

Fig.

2.

used:

MIC:

Cefclidin,

Effectofcefclidinof cefclidin
anaerobicrods spp. consistedof P. asaccharolytica.

B.

fragilis

50ug/ml;
andGAIand otherbacteriaof 12 isolates

agentsagainstof P. intermedia,7clinicalisolatesof5562.

Ceftazidime,ceftazidime

related12.5ƒÊg/ml.
ontheB.

fragilis

rat

pouch

infection

CHEMOTHERAPY

36

Comparative
activities
isolatesof gram-positiveof cefclidin
anaerobic

SEPT.

Table

4.

and other related agentsbacteria
and Gardnerella

againstclinicalvaginaris

Table

5. Stability of cefclidin, ceftazidime, cefpiramide, cefotaxime and cefotiam to thehydrolysis by g-lactamase
dervied from B. fragilis as compared with that ofcefazolina)cefazolin=100.

Table

6.

Appearanceof C.difficile in murine
cefclidin,cefotaxime
and cefotetana)Number (CFU/g)b)Not tested.

of C. difficile detected.

cecum

after

the 7 days

dosingof1992

VOL.40

ƒÀ-lactamases
less37testedthanstable

than

CAZ

vivo

activityinIn
CFCL

had

owth

was;incontrast,

seen

rat

CAZ

and

9h

after

persistedincentration

theofofCFCL

ism

by

6h

first

pouch

by

9h

after

administration,
theofCdifficile,ofofC.difficile

5days,

respectively,

CFCL,

Although

alday

after

in

for

producing

and

and

CTT

wasdetectedceasing

ent

organ-pouchofinawasof10

is expectedtoGram-negative
againstof

inthisstudyof

dosageof

CFCL

eradication

miceItcefpirome,oped;

properties
in

ity.

In

contrast,

ateor

weakactivity

group4-6).

revealed

weak

organisms

asshowedsuch

didrecentyears,

just

CPM,

CAZ,

results(CFPM),

have

been

devel-fromof

have

which

oftenagainst

members

theotheranti-pseudomonal

anactivity
activity

Againstmostofobic

bacteria,

CFCL

ciallyagainst

Mobiluncus

show

was
theothershowedthestudy,

proved

K.,

Ueno,

K.

: Biochemicalof

2.

Lorian,
ed.,

C.

otic

inactivating

as(NEU,andCFPM
ofvaginalis.

anaerespe-3.
Ueno,
tal

Muto,andV.:K.
animalmodel

We
CFCL

didlongintheexaminedafter.

Theberelatively

freecolitis

anddiarrhea.

Watanabe,K.,
andKatoh,

35:
in

andN.,

purification

Bacteroides

Wilkins,fragilis.
371-372

laboratory, 1991
medicine,

Baltimore,

20

bacterial

resistance

antibi-1986

Ohashi,

Usefulness

cause

C.difficile
wasproperties

pp.757-789,
N.,

cancitedY.,

enzymes

H.C.)
Kato,

andwouldChemother.

anddemonincreasedoforAntibiotics

Williams

vitro

diarrhea.

offromAgentsinpouch.

C.difficile

metallo-fl-lactamase

Antimicrob.

CFCL

activity,G.

B.

Bandoh,organismadministration

CFCL

1.

the

good

fl-lactams

medication
that

in

depression
the

many

C.diffici/e-associated

2nd

in

inof

adminis-

the

that

constant

whenceasingfor

rat

regrowth

B.fragilis

appearanceevensuggestft-lactams.

members

andmodel,anaerobic

P.bivia

good
spp.

moderofthese

againstobviousoflevelsuggestcolitisgenerate

after

againstthediffer-

the

CFCL

may

that

that

in

However,organism

known

some

antibiotic

Literature

activ-

present
against

well.

well

polymi-Treatment

startingofshows
theespeciallyanti-P.aeruginosa

activity

poor

is

mouse

asfi-lactams

InnotIn

strongthese

fragilisdifficile;vitro

againstparticularly

suchcefepimetheiremphasized

CFCL

bacteria,

antibiotics,andactivity

aeruginosa.

13-lactam

of

in
among

expectThe

CFCL

contrastofresultedadministration.

kinetics

a/3-lactamase-highly

after

MIC

in

diseasestreatment

CFCL

theisB.

difference

littlemaycompound.

below

this

agents.

48hI
byconsequences

we

antibiotic-associatedstrongaerobic
Pseudomonas

somethisdemonstrated

have

orCTX,

infectious

too

the

thesestrated

wasbeforkilling

6).

may

with

theI

CPM,

ofofincludingandDiscussion
CFCL

after

to

type

produceddissimilar

over-1-day

mouse(TableIt

organism

pouch

C.difficile

bring

infection

ofstable

type

meaningamongtested6h

comparingtreatment

B.fragilis

trationcausedtooutcome

cecum
was

the

infections

murine

CTT

The
for

antimicrobials

whileadministration

numberCFCL

inmurineCTX,

ceasinggiven

con-MIC

theC.difficile

CTX

low

the

crobialincommon ƒÀ-lactamase

explained.

stabilitythewhichafter

antimicrobials,

for

not

the
these(Fig.3).

overgrowthoftheMIC

administration

growthwereCFCL;

used

dosingdetected

than

astrains.

CAZ

that

B.fragilis

stability

isof

tendency

covered

7days

only

relatively

Oxyiminocephalosporinaseasfragilisthe(Fig.2).

measured

pouch

of

thewasoxyiminocephalosporinase

CTM.

known

administration
abelow

CFCL
byfromandcompound

pouch

waslongerlowered

regr-keeping

theafterexamined.

thein

Inducibility

which

the

also,

at
the

pouch

wascecum-Appearance
after

toin

the

Enzymatically,

butderivedvivo

CAZ

organismthatineffective

There

CEZ

infection-Althoughnumberinandhydrolysis

exposure

waslow

antimicrobials.

thepouch

after

concentration
6,, CTX5).

potency

48hin

Drug

CPM

(Tablebactericidalorganism

3,

Cefclidinの 嫌 気 性 菌 に 対 す る抗 菌 力

S‑4ofH.,

Muto,Y.,

Watanabe,ratpouchforanofanaerobicexperimeninfections.

K.

CHEMOTHERAPY

38

Kenkiseikin-kansenshou-kenkyu

17 : 94-98,1987

4. Isono, M., Yamada, H., Aoki, M., Sawa, K.,
Watanabe, K., Ueno, K.: In vitro and in vivo activity of cefpiramide
bacteria.

(SM-1642)

Chemotherapy

againstanaerobic

31 (S-1) : 48-56,1983

5. Watanabe, K., Katoh, N., Muto, Y., Bandoh, K.,
Ueno, K. : In vitro antibacterial
activity of cefepime

SEPT.

against anaerobic: 40-45, 1991
6.

Kato,bacteria.

N., Bando,

Chemotherapy

1992

39 (S-1)

K., Muto, Y., Watanabe,

Ueno, K. : In vitro activityof cefepime,

an new

parenteralcephalosporin,
against anaerobic
ria. Chemotherapy
39 (S-2) : 43-51,1991

新 注 射 用 セ フ ァ ロ ス ポ リ ンcefclidinの 嫌 気 性 菌 お よ びGardnerellavaginalisに

嫌 気 性 菌 お よ びGardnerellavaginalis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,PmoteJlabivia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Mobiluncus

spp.に は 強 い 抗 菌 力 が み ら れ た。CFCLはB.fragilisのphalosporinaseI型に 対 してCTX,CPM,CTMよ

あ っ た 。増 殖 曲 線 に 対 す るCFCLのりは 安 定 で,CAZよ

効 果 の検 討 で は,2MICも殖 の み られ な い 殺 菌 的 効 果 が 認 め られ た 。CFCLはと にGvaginaJisと産 生 す るoxyiminoce‑
りは わ ず か に 不 安 定 でし くは そ れ 以 上 の 薬 剤 濃 度 で 再 増

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

bacte-

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K.,