CHEMOTHERAPY

190

The

morphologicalresponse

HirotoDapartment

Washida,wasHideki
Yamadag/ml),

twostrains
theofurineofinjection,

offourandtheTheurinaryinwallcontourfrom

cellWeespecially

KeyPBPs

words:

lighturinary

ceftazidime,

were

GNB

(CAZ)

lactamases,

hasandshows

onPseudomonas

apotent

ofstrongly

negativedoubleblind

newantibiotics

safety1).

shown
ied

good

CAZ
as

used

CAZ

for

ourclinicalmorphologicalbacterialmechanism

In

theof

not

especiallyaafterCAZ

in

MATERIALS

AND

Five

between

April,
thewithweresubject

1989andof

thisMarch,study;

inthe first

4thinjection.

MIC,

fromcells

weremarkedly

dissociation

wereofobserved

thetheundercellelectronproteins(PBPs),changes

twostrainsofforboth)wereisolated

from

sents

of

were

and

was

the

treatment
andit(MIC:andofpractice

cepaciaboth),

thedevelopmentthepatients.

explained

towerethepatients

checked,anda negative

confirmed

prior

2.
A

catheterUrinary

to

we

stud-

nectedtoanti-

beforethe first

drugsampleobtained

ataclinical

a4

after
hours

at

complicatedadministration.

ourdepartment

onward,1991

werepled

immediately

samplewasonestrainchosenofclosed

theirƒÊ

wasinformed

con-allergy

histories

reactiontoCAZ

andcon-administration.

systemimmediatelyadministration.
this

was

time

The
was

for

the first

drugadministration,

2-hourintervals
thenumberedobtained

8

hours

after
third

12andhours

2afterasat

2-hourthesecondthenadministration

administrations

beforeurine

regarded

done

From
odd

transurethrally

drainage

Sampling

intervals

samples

procedure

skinplaced

theon

urine

50

sampling
wasstudy,urine

(MIC:

from

drug

CAZ

for

marcescens

andTheirofS.

two

1.56ƒÊg/ml

examination

obtained.

has0.39ƒÊg/ml),(MIC:Theclarify

monomicrobial
toto

aeruginosainMETHODS

admittedƒÊ
two

theP.

forpatients
who

2ndofoftype

Subjects

UTI,

andexcreted

both)strainsdrug

utility

to

0.39

after

penicillin-binding

control.

1.

theregardlessMorphologicalnewsetting.

as

tog/mlandhighpresent

responsesof ƒÀ-marcescens

theitsresults.

the

forof

inprogressive

bacteriolysis

bacteria,

spectrum,reference

test

because
have

is

a

comparison

UTI

variousSerratia

servedWecomplicatedtoeffects,

aeruginosa,

hasweretract

(MIC:

GNB.

anti-bacterialProteus.

butforefficiently

structures,

binds

urinary

wasPseudomonas

resistant

wide

CFU

ceftazidime

cepaciagg/m1

Vacuole-likedebristo1.56

The filamentous

and

Gram

anti-bacterial

indole-positivedrug,is(GNB)

CAZ

CAZ.

CAZ

INTRODUCTION
Ceftazidimeboth).

eliminatedby1992)

Pseudomonas

observed,

microscopy.

thatofurinaryurinary

membrane,

3,ofto ceftazidime

complicatedof(MIC:

forbacteria

thespeculatestrain50 ƒÊg/ml

decrease10,

monomicrobialonecharacteristicallyofunderthemicroscopy.

withwere

1993

Anjo, Aichi 446, Japan

bacteria

aeruginosa

viableresponses

Novembergram-negativepatients(MIC:wasmorphologicalurinary

Pseudomonas

patients.

12-38 Miyukihonmachi,

1992•EAcceptedbacteria

marcescens

Prominent filamentation

unevenoftarget

strains

Serratia

19,

in five

The

Honma,Tozawa,response(UTI).

bacteria

YasuyukiJune

investigatedinfections

negative

Keiichiof Urology, Anjo Kosei Hospital,morphological(CAZ)

gram

Kiho Kan and(ReceivedTheof urinary

FEB

werehourstheafter.
last

samFinal

adminis.

aVOL.41

NO.2

Morphologicalchangesof urinary

tration. Morphological changes of urinary bacteria
were observed up until 2 hours after the third administration. Part of each sample was frozen immediately after collection and stored for determinationof drug concentration.
3. Measurement of MIC and urinary drug concentration
The MICs of the GNB were determined according
to the standard method proposed by the Japan
Society of Chemotherapy2), and the urinary CAZ
concentrations were measured using a bioassay
method with Proteus mirabilis ATCC 21100 as thetest organism.
4. Measurement of urinary CFU
The CFU were quantitatively measured by theagar plate dilution method.
5. Light microscopy
The urine samples were centrifuged at 1,000 rpm/
minute for 10 minutes. A drop of urinary sediment
was spread over a thin film of Tryptocase soy agar
medium (BBN) on a slide glass and covered with acover glass which was then fixed with liquidparaffin . The morphological changes were observed
and recorded photographically by means of adifferential interference
contrast
microscope(Olympus).

Fig. 1. Bacteriologicalazidime (arrows).

bacteria
6.

ElectronThesediments

aldehyde

and

Epon

microscopy

812,

stainedwere
1%further fixed

osmiumcutwithinto

a3%

glutar-embedded

insections,

acetateunder(Hitachiwithtetroxide,ultra-thinuranylphotographed
scope

191

to ceftazidime

and

lead

transmission

doublecitrate,andelectron

micro-H-300).
RESULTS

1.

Changesof

CFU

andurinary

excretionof

CAZ
Changesofnary

excretion

Table

1,

tion,

one

strainare.

of

Afterandfour.

Urinary

could(a68-year-old,

anceof76.5

fromthekidney.

inand
in

Fig.

the first

strains

onedecreasedwas. The

showedacreatinine

ml/minute:

CAZ

is

mainlywere

becauseof thepatients,

toeliminate

neededinreasonexplained

administrations

instepexcellentbeall fiveuri-

1.56 ƒÊg/ml)

man)

In

the
1 andadministra-(MIC:excretion

poor
not

patients

shownlinearly

andexcretion

theaeruginosapatients

naryinCAZ

P.

fashionfour

CFU

ofrespectively

decreased-wise

theinforthepoorpatient
clearexcreted
twodrug

theuri-

GNB.
2.

Light

microscopy

Prominent filamentation
werecharacteristicallyeffects in urine after intravenous

withnoticed

administration

irregularintheof ceft-contours
majorityofTableFig.

FEB.1993

CHEMOTHERAPY

192

2.

Light

microscopic

phological
in

view

responseurine

beforeceftazidime

and
(bars

A:

Pseudomonas

ofceftazidime.

of

1. Urinaryof

gramnegativeafter

administration

represent
cepaciathelevels of ceftazidime

Fig.

mor-

3.
of

bacteria

Transmission
theelectronmicroscopic

morphologicalaeruginosaofinceftazidime

10 ƒÊm).
before(mg/ml)of

after

administration

urine(barrepresents

Spratt's-lactam

report",antibiotics

andtheinteraction

PBPs

has

the strains
filamentous(Fig. 2-B) . Spheroplasts
and shortcells were observed in one strain of P.

tion

theantibiotics'

As

isgenerally

known,

PBPs

aeruginosa,

which

to

cell

lengthextension,

administration
3. Electron

after

CAZ

(Fig. 2-C) .
microscopy

instudies

septumgicalcytoplasmic contents flowed out (Fig. 3) . Vacuole-like structures emerged in the cytoplasma as welledcells

aretoPBPs

3.

from the surface struc-The

shapeofthemechanism.

2 and3 are

cells
be

their

affmities

tomainlyobserved

thebest ƒÀ-lactam

a potential

actionThemayinaffinity

PBPs

treatedattributed
eachantibiotics
toatten-related

determination,

bacterial

antibiotics

differences1,

between ƒÀ
attracted

respectively.

changeslactamdetachingofformation,

The following process of bacteriolysis
was observed. The cell surface structure
was ruptured andas the cytoplasma
ture (Fig. 4) .

of1ƒÊm).

DISCUSSION
Since

linearly

Pseudomonas

A gomandized
filamentous
cell showing
bacteriolysis: rupture of cell wall and leakage
of cytoplasm(arrow); peusopodiumof aphagocyte(arrowhead)
6 hours after.

administration

B: Prominent
filamentous
response,withPseudomonas cepacia showing irregular forms
2 hours after.
C: "Rabbit-ear"
response (arrow) , spheroplast of Pseudomonas aeruginosa 2 hours after.

decreasedview

response14)

PBPs;

andmorpholowith ƒÀtothe

filament-antibiotics

bindingarerequired

ideallyRe-growthoftheVOL.41

NO.2

Morphologicalchangesof urinary

bacteria

withato ceftazidime

step-wisebacteriolysis
plastformed

thesedecrease,

wasmembranemorethanfilamentsof the morphologicalsible

thistoouter

betweenmakeclearchangewastheimpos-study.

In one patient
CAZ,

The

be difference

However,difference
fromThesphelo-formed.

would

strains.

respectively.
in case whenintensepenetration

two193decreasewith poor

urinary

excretionof CFU and filamentous

ofcells wereunexpectedly observed at a concentration
below theMIC, in this study. Antibacterial
agents do notFig.

4.

Transmission

oftheelectronmicroscopic

morphologicalmarcescens

inceftazidime(barsview

response

urine

afterof

Serratia

administration

representof

begin to act only when their concentration

exceeds

theatMIC; theirconcentrations.
bacteria

1 ƒÊm).

A: A spherical pole form on one side of a rod-shaped cell with vacuole-likestructure
2
hours after.
B: Filamentous
cells with vacuole-likestructure (arrow): detachment of the bacterial cellwall and cytoplasmic
membrane(arrowhead) 6 hours.

actions

mayThis

to

cause

on

bacteria

is considered

subinhibitory

in bacterial

by Fujii et al5) We havecarried out investigations

similar to this study using

other antibiotics6-9)
Thefilamentous cells have taken

antibiotics
formingpoor clinical effects:

The authors

thisstudy,

theviable

eliminatedfromtheurine

bacterial
through

cells

were

ticallyof urinary

GNB

excellent1)

technical

filamentous

cells

with

help; Mr.

Kumazawa

J,

studyofCefepime

ment

ofcomplicatedBacteriolysisof the filamentous

bymicroscopic

electronNakao

et al reported

cells

JellyJ

observation.

minimumG:
incefmenoxime

in theurinary

Similarly,

Philosophica

5)examined.
theKonno

shapeofEscherichia

cephalosporinof viableP.

Thisof the patientin thecould

be explained

difference

morphologicalspheroplasts

differentchangesand filaments
witha linearinwereurinary
formeddecreaseNat.

shapeAcad.

ofSci.

of ƒÀ-lactam

issueofresistanceM,

Ubrkataonthemechanism

toantibiotics)

5: 669•`675,

coli

1986

K:

The

filamentous

withpenicillin

anditstreatedanalogues
I.

com-J. J. A.

MedicinaInf.

orclinicalD.

44:62•`71Hirao

N,HachisukaOnclinical,1970

aer-2 patients

WashidaY,from

H,E.

Tsugaya

Takeuchicefotiambacteria:

coli

onin

M,

K,

InuzukaK:

acutecystitisandUrine.

Acta

Urol.

changes

ineffects

offormofJpn.

29:1373•`1380,

Hirao

N,

Hachisuka

1983

in the urine

and the patient(SpecialC
partproteinsandproteins.

Philosophica
R,Proc.

mechanisms

acquisition

Fujii

binding

1975

penicillin-binding

The

assay

concentration.

1981

12.

on

(CMX)10)countK

pounds6)

markedlycoli

treat-1992

elongation,

2999•`3003,

significance

weredivision,confirmed

vitro11)
Changesthetheinfections.

Rerevisedpenicillin

Actionaffinity of CAZ to PBPs was, similarly to CMX ,high in the descending order of PBPs 3, 1 and 2, inuginosa

Distinct5

tract

inhibitoryM:the death of the filamentous

withforComparativefor

Chemotherapy:

29: 76•`79,

B

72:

institues):

54: 970-985,ofMedicina

cells treatedUrol.

methodof

Shiosaka

Ceftazidime
urinaryofUSA4)

vsSocietySpratt

(54

Konno

was

Kiha-CITED

al.

Japan

involved

shape.

et.

Chemotherapy
3)characteris-irregularmorphology')

preparation.

Nishinihon2)

morphologicalby CAZ wereofdrugs

would like to thank Professor

Escherichia

responses

ofchiro Shimizu, Tokyo Women's Medical College,
for valuable advice; Mr. Kazuhisa
Inuzukaforthey merely elongated bacterial cells and could not
eliminated the bacteria from the urine.
In

lower

exposure

ACKNOWLEDGEMENTS

LITERATURE

cells reported

that

concentration

alterations

manuscript
filamentous

begin

7)

WashidaH,

TsugayaM,

CHEMOTHERAPY

194

8)

Y,

Takeuchicaltrials

K,

Washida

H,

9)

K,

StudiesM,
Inuzukaof AC-1370.

H,

Sakagami

TakeuchiK,

Inuzuka

Escherichia

coli

acutesimple

cystitis

Acta

Urol.

Nakao

andclini-electron

32(S-2):

response

Hirao

N,

M,

mictoscopy

K:

FundamentalandChemotherapy

Jpn.
Kondo

in

H,

Iwase

K:

Morphological

theY,

urine

3211)

(S

treatedwithS,

1987

Tsuchiya

K:

Light

CeftazidimeにEscherichia

Cefmenoxime

1993

of

the

morphologicalcoli

andSerratia(SCE-1365)

cefalosporin.

J.

Marces.

, a new

Antibiotics

Okumura

K,

Kato

H,

YokotaBasicstudiesonthevivo

efficacy

ofceftazidime

31

LorianV,(S-3):

22•`29,AtkinsonT,

antibacterial

producedby64:

678•`688.

1975

和 志 田裕 人 ・戸 澤

啓 一 ・本 間姜泰之401).

Chemo-

1983

antibiotics,

forms

Am.

J,ofClin,

秀樹

安城更生病院泌尿器科。に つ い てceftazidime(CAZ)投の変 化 と尿 中細 菌 の形 態 変化 を検討 した。 Pseudomonas
Pseudomonas(MICは2株(MICはaeruginosa

い ず れ も50μg/ml)で菌 数 は第1回

い ず れ も1.56μg/m1),

あ っ た。CAZの尿 中 排 泄 は,4例の 投 与 に よ り経 時 的 に 減 少 し た が,消の 形 態 は細 菌 のMICに

失 に は2〜4回1株(MIC;3.13μg/ml)

Serratia

marcescens

1株に は良好 で あ った。尿 中生の投 与 を必 要 とした。細 菌か か わ らず フ ィ ラ メ ン トを形 成 す る傾 向 が 強 か っ た 。 多 くの フ ィラ メン トは光 顕 で は 凹 凸 が 著 明 で あ り,電 顕 で は空 胞 形 成,細胞 壁 と細 胞 質 膜 の 解 離,細胞 質 膜 の残 骸 お よ び それ ら の 崩 壊 像 が 観 察 さ れ た。CAZは,尿く結 合 す る こ とが 示 唆 され た 。
*安 城 市 御 幸 本 町12‑38

与後 の経時 的尿 中生菌 数

cepacia

中 で もPBP特R:and(SNB: Abnormalria

Sekiguchi

よ る尿 中 グ ラ ム 陰 性 桿 菌 の 形 態 変 化

複 雑 性 尿 路 感 染 症 単 独 感 染 例5例

1046•`

activityand瑛 鏑 ・山田

broad-34:

1981

therapy12)

with

cefbuperazone.

33: 1712•`1717,
M,

Sasakipatients

to1054,studiesofofspectrum

Hachisuka, 1984

Washidaof10)

Tsugaya

Takeuchi-9):654•`662

Laboratory

Chemotherapy

cens

1984

ClinicalK:

TA-058.

58•`597,

Y,

Inuzuka

with

FEB,

胞 壁 と細にPBP3に

強bactePatho,in