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Journal of Hospital Infection

(2002)

51: 21±26


doi:10.1053/jhin.2002.1186, available online at http://www.idealibrary.com on


Management of an outbreak of

Enterobacter


cloacae

in a neonatal unit using simple


preventive measures


Y. v. Dijk *, E. M. Bik


y, S. Hochstenbach
-
Vernooijy, G. J. v. d. Vlistz


P. H. M. Savelkoul


x, J. A. Kaank

and R. J. A. Dieperslootk


*Department of Infection Control, Diakonessen Hospital, Utrecht,


yDepartment of Microbiology and Immunology,


St. Antonius Hospital, Nieuwege
in,


zDepartment of Paediatrics, Diakonessen Hospital, Utrecht,

xDepartment of


Microbiology and Infection Prevention, VU Medical Center, Amsterdam, and


kDepartment of Microbiology and


Immunology, Diakonessen Hospital, Utrecht, The Netherlands


Summary:

Enterobacter cloacae

is becoming an increasingly important nosocomial pathogen. Outbreaks of


E. cloacae

in intensive care units and burns units have been described frequently. In December 1999, a
neonate


with line sepsis was transferred from a university

hospital to the neonatal unit of the Diakonessen Hospital.


Blood culture yielded

E. cloacae. An outbreak of

E. cloacae

was occurring in the university hospital at that
time.


In February 2000, a second neonate in our hospital developed line sepsis caused

by

E. cloacae. Direct
measures


taken included cohorting of infected children, disinfection of incubators, thermometers and wards, and


screening patients. Of nine neonates, seven were colonized with

E. cloacae. Despite these measures, the
out
-


break con
tinued. Forty
-
one patients were screened; 15 were colonized. Environmental searches yielded


E. cloacae

in a sink and on two thermometers. Sixteen isolates were typed by arbitrarily primed PCR using
four


primers. All the patient isolates and the two isola
tes from thermometers were identical. The strain isolated


from the sink was unrelated. Amplified fragment length polymorphism typing showed that the outbreak clone


was identical to that in the university hospital. After the introduction of disposable
thermometer covers,


E. cloacae

colonization slowly decreased.


&

2002 The Hospital Infection Society


Keywords: Enterobacter cloacae; neonatal unit; thermometer; transmission; genotyping.


Introduction


Enterobacter

species, particularly

Enterobacter


clo
acae, are commensals of the gastrointestinal tract


in healthy adults, and rarely cause primary human


disease. However,

E. cloacae

is becoming an


increasingly important nosocomial pathogen.
1

It


is frequently cultured form body substances of


hospitalize
d patients, particularly those treated with


antibiotics.
2

Colonization has been associated


with blood, wound, respiratory and urinary tract


infections.
3

It has been suggested that colonization


and infection may arise endogenously and that


cross
-
infect
ion is relatively uncommon.
1

Never
-


theless, outbreaks of infections have been reported


in neonatal intensive care units (NICUs),
4±6

surgical


wards
7

and burns units,
8

mediated by factors such


as contaminated pharmaceutical products,
9,10

cross
-


contamination by healthcare workers' hands,
11

and


medical equipment.
12

Outbreaks often continue for


Received 20 June 2001; revised manuscript accepted


15 January 2002.


Author for correspondence: Dr Y. v. Dijk, Diakonessen Hospital


Utrecht, Department
of Infection Control, Bosboomstraat 1,


3582 KE Utrecht, The Netherlands. E
-
mail: yvdijk@diakhuis.nl


0195
-
6701/02/010021

1

06 $35.00/0

&

2002 The Hospital Infection Society

months

and

require

radical

measures,

such

as

ward


closure

or

changing

antibiotic

policy.
13

Here

we


report

an

outbreak

of

cross
-
colonization

and

infec
-


tion

by

E.

cloacae

with

resistance

to

third
-
generation


cephalosporins,

among

patients

in

a

neonatal

care


unit,

and

describe

the

ability

of

relatively

simple


infection

control

proced
ures

to

stop

the

outbreak.


Patients

and

methods


Characteristics

of

the

ward


The

outbreak

occurred

in

the

Diakonessen

Hospital


in

Utrecht,

The

Netherlands,

a

teaching

hospital

of


370

beds.

The

neonatal

unit

is

a

12
-
bed

unit

divided


into

a

large

room

and

one

smaller

room,

and

has


250

admissions

per

year.

Premature

neonates

<30


gestational

weeks

or

with

severe

organ

dysfunction


are

transferred

to

the

intensive

care

unit

of

a


university

hospital

for

life
-
support

treatment

and

are


returned

to

our

unit

when

stabilized.

Nurses


assigned

to

the

unit

work

in

all

the

rooms.

Nurses,


staff

and

visitors

wear

gowns

whenever

children

are


taken

out

of

the

incubator.

Nurses

and

(para
-
)


medical

personnel

are

instructed

to

disinfect


their

hands

prior

to

each

contact.

The

children

are


not

routinely

screened

for

bacterial

colonization.


First
-
line

therapy

for

suspected

sepsis

is

amoxicillin


in

combination

with

gentamicin.


Case

definition


A

case

was

defined

as

a

child

admitted

to

the

neonatal


unit,

who

had

E.

cloacae

identified

in

an

isolate

from


any

site.

Criteria

for

infection

were

as

defined

by

the


Centers

for

Disease

Control.
14

Isolates

from

speci
-


mens

not

associated

with

infection

were

classified

as


colonization.


Bacteriological

cultures


During

the

outbreak,

specimens

for

surveillance


culture

were

obtained

once

a

week

from

the

naso
-


pharynx

and

rectum

from

all

children

in

the

neonatal


unit.

Samples

were

also

obtained

from

environ
-


mental

sites,

including

sinks,

hand

basins,

bedrails,


ventilators,

thermometers,

oil

tissues,

solutions,

and


incubators.


Specimens

were

cultured

on

5%

sheep

blood


agar

and

Colombia

CNA

agar

(bioMeÂ

rieux,


Marcy
-
l'Etoile,

France)

and

incubated

at

37
?

C.

The


bacterial

isolates

were

identified

to

species

level


using

standard

methods,

and

tested

for

susceptibility


to

amoxicillin,

amoxillin/clavulanate,

gentamicin,


amikacin,

cotrimoxazole,

ceftazidime,

ceftriaxone,


ciprofloxacin

and

imipenem.

All

the

isolates

were


stored

at


?
70
?

C,

and

were

tested

for

the

presence

of


extended
-
spectrum

b
-
lactamase

using

the

E
-
test


system

(AB
-
Biodisk,

Solna,

Sweden).


Epidemiological

typing

of

bacterial

isolates


To

examine

the

relatedness

between

different


E.

cloacae

isolates,

two

molecular

typing

methods


were

used.

First,

13

patient

isolates

and

three


environmental

strains

(two

from

thermometers


and

one

from

a

hand

basin)

were

typed

using

arbi
-


trarily

primed

PCR

(AP
-
PCR).

Chromosomal

DNA


was

extracted

from

bacterial

cultures

on

sheep

agar


plates

using

the

QIAamp

DNA

mini

kit

(QIAgen,


Westburg,

The

Netherlands).

AP
-
PCR

was


performed

in

25

mL

reaction

volumes

using

primers


ERIC1,

ERIC2,

RW3A

and

AP1.
15±17

Each

reaction


mixture

contained

10

m
M

Tris
-
HCl

(pH

9.0),

50

m
M


KCl,

2.5

m
M

MgCl,

0.1%

Triton

X
-
100,

0.01%

(w/v)


gelatin,

200

m
M

of

each

of

the

four

deoxynucleotide


triphosphates,

40

pmol

primer,

0.75

U

of

SuperTaq


polymerase

(HT

Biotechnology

Ltd.,

Cambridge,


UK)

and

50

ng

of

chromosomal

DNA.

A

Perkin


Elmer

PE9600

thermocycler

was

used

for

amplifi
-


cation.

Cycling

conditions

consisted

of

the

following


steps:

5

min

at

95
?

C,

35

cycles

of

1

min

at

95
?

C,

1

min


at

25
?

C,

and

2

min

at

72
?

C,

followed

by

a

7
-
min


extension

at

72
?

C.

PCR

products

were

separated

by


electrophoresis

on

a

1%

agarose

gel,

stained

with


ethidium

bromide

and

visualized

by

UV

light.

Sec
-


ondly,

isolates

from

different

hospitals

were

com
-


pared

by

amplified

fragment

length

polymorphism


(AFLP),

using

restriction

enzymes

EcoR1

and

Mse1


as

described

previously.
18

Control

strains

from


unrelated

patients

were

included

in

both

typing


techniques.


Infection

control

measures


At

the

start

of

the

outbreak

(February

2000),

the


infection

control

measures

on

the

unit

were

reviewed


and

personnel

were

urged

to

be

careful

in

their

use,


and

incubators

and

materials

were

disinfected


(hypochlorite

300

ppm

or

70%

ethyl

alcohol).

All


infected

and

colonized

children

were

cohorted.


Cohorting

of

the

personnel

was

impossible

because


of

shortage

of

nurses.

Gloves

were

worn

to

handle


22

Y.

v.

Dijk

et

al.

colonized

children.

Contaminated

objects

and


surfaces

were

routinely

disinfected

with

hypochlo
-


rite

300

ppm

or

70%

ethyl

alcohol.

A

three
-
day


observational

study

was

conducted

to

monitor

the


infection

prevention

practice

of

healthcare

workers


and

parents.

The

results

of

the

interventions

were


discussed

during

weekly

meetings

with

representa
-


tives

of

the

neonatal

care

unit

and

the

Department


of

Infection

Prevention

to

watch

over

the

progress.


Results


Description

of

the

outbreak


In

December

1999,

a

neonate

(patient

1)

was


transferred

from

the

NICU

of

a

university

hospital

to


our

neonatal

care

unit.

The

neonate

developed

line
-


associated

sepsis

the

following

day.

Blood

culture


yielded

E.

cloacae

(Figure

1).

In

February

2000,

a


second

neonate

(patient

2)

developed

(line
-


associated)

sepsis

caused

by

E.

cloacae

(Figure

1).

At


the

time,

an

outbreak

of

E.

cloacae

involving

32


patients

was

occurring

at

the

NICU

of

the

university


hospital

from

which

the

index

chi
ld

was

transferred.


We

immediately

screened

all

neonates

by


specimen

collection

from

the

nasopharynx

and

rec
-


tum.

Of

the

nine

children

screened,

seven

were


colonized

with

E.

cloacae.

All

those

who

had

been

in


the

neonatal

unit

for

at

least

two

days

were

colonized.


Environmental

cultures

yielded

E.

cloacae

from

a


hand

basin

and

two

thermometers.

No

other


environmental

samples

were

found

to

harbour

the


organism.


Measures


In

our

weekly

meetings

with

the

neonatal

unit,

we


discussed

our

first

interventions.

All

sinks

and

hand


basins

were

disinfected

daily

with

hypochlorite


(300

ppm).

Disinfection

of

thermometers

changed


from

a

quick

rinse

with

70%

ethyl

alcohol

to

10

min


immersion

in

70%

ethyl

alcohol.

Other

measures


included

cohorting

of

the

colonized

and

infected


children,

disinfection

of

the

incubator

beds

and


wards,

reinforcement

of

handwashing

practice,

and


weekly

bacterial

screening

of

all

patients.


Observation

of

the

healthcare

workers'


handwashing

practice

indicated

good

compliance.


The

parents

turned

out

to

regularly

take

new


universal

oil

tissues

with

contaminated

hands.


Although

no

micro
-
organisms

were

isolated

from


tissues,

it

was

decided

to

give

each

child

their

own


tissues.

During

the

observation,

no

other

possible


route

of

transmission

was

noticed.

Despite

these


preventive

measures,

the

outbreak

continued.

A

total


of

41

patients

were

screened,

of

whom

15

were


colonized

with

E.

cloacae

(Figure

1).

No

other

child


developed

an

infection.


Despite

the

extended

disinfection

time,

Gram
-


negative

micro
-
organisms

were

still

isolated

from

the


thermometers.

Therefore,

all

thermometers

were


replaced

and

disposable

covers

were

introduced.


After

this

and

the

introduction

of

individual

oil


tissues,

the

number

of

new

cases

of

E.

cloacae

slowly


decreased.


One

month

and

six

months

after

the

outbreak,

all


neonates

were

screened

again.

In

July

2000,

no


E.

cloacae

was

found.

In

October

2000,

E.

cloacae


16


Jul


1


Month


P


a


t


ie


n

t



n


um

b

e

r


Dec


1999

Oct

Nov


Jan


2000

Feb

Mar

Apr

May

Jun


2


3


4


5


6


7


8


9


10


11


12


13


14


15


Figure

1

Patients

colonized

with

E.

cloacae

strains

(


bfd


)

admission

on

the

neonatal

unit;

(

l


fl


)

culture

with

E.

cloacae.


E.

cloacae

in

neonatal

unit

23

was

isolated

from

the

rectum

of

one

neonate


(patient

16).


Sensitivity

and

typing


All

the

E.

cloacae

strains

isolated

during

the

outbreak


displayed

similar

antibiograms.

All

were

resistant

to


amoxicillin,

amoxicillin/clavunalate,

ceftazidime

and


ceftriaxone.

An

extended
-
spectrum

b
-
lactamase

was


demonstrated

in

all

isolates.

Sixteen

E.

cloacae

strains


isolated

from

patients

and

environmental

sites

in

the


Diakonessen

Hospital

were

typed

genetically

using


AP
-
PCR

with

four

different

primers.

The

result


from

the

ERIC1

PCR

fingerprinting

is

shown

in


Figure

2.

Four

control

strains

(lanes

1±4)

could

all

be


distinguished

from

each

other,

confirming

the

reso
-


lution

of

this

technique.

All

E.

cloacae

strains

isolated


from

patients

2±15

from

February

to

April

2000,

and


the

two

isolates

from

thermometers

showed

identical


fingerprints

with

each

of

the

primers,

confirming

the


suspected

cluster,

and

linking

it

to

the

thermometers.


In

contrast,

the

isolate

from

the

hand

basin

and

the


strain

from

patient

16

which

was

isolated

in

October


2000,

six

months

after

the

outbreak,

both

displayed


unique

fingerprint

patterns,

indicated

that

their


genotypes

were

unrelated

(Figure

2,

lanes

19

and

20,


respectively).


AFLP

typing

showed

that

the

E.

cloacae

outbreak


strain

was

identical

to

the

outbreak

strain

at

the


university

hospital

from

which

the

index

child

was


transferred

(Figure

3).


1000 bp


Patient


16


500 bp


Thermo
-


meters

Sink


Patients


Control


strains


M

C

C C

C

2

4

5

6

7 8

9

10 11

12

13

15

T


T

S

16

N

C


Figure

2

ERIC1

PCR
-
fingerprinting

of

E.

cloacae

strains.


100


60


Outbreak strain university hospital


Outbreak strains


Diakonessenhuis


Control strain 1


Control strain 2


70

80

90


Figure

3

AFLP

typing

of

E.

cloacae

strains.


24

Y.

v.

Dijk

et

al.

Discussion


In

this

study,

we

describe

an

outbreak

of

E.

cloacae


colonization

in

a

neonatal

unit.

Newborns

are

known


to

be

at

risk

for

colonization

of

Enterobacter

spp.
19


While

neonates

initially

have

a

sterile

gastrointestinal


tract,

bacterial

colonization

occurs

rapidly

in


hospital,

the

result

of

selective

antibiotic

pressure


and

nosocomial

spread

through

inadequate


hygiene.
20

Interestingly,

the

NICU

that

transferred


the

index

case

to

our

hospital

used

a

third
-
generation


cephalosporin,

ceftriaxone,

as

routine

therapy

for


neonatal

sepsis.


In

some

hospitals,

children

become

rapidly


colonized.

In

a

multicentre

study,

Fryklund

et

al.


found

a

colonization

rate

in

various

neonatal

care


units

of

0±78%.
2

Intestinal

colonization

appears

to

be


a

precursor

of

the

widespread

colonization

and


the

invasive

infection

which

occurs

in

only

a

small


proportion

of

colonized

children.
21

A

case

record


study

of

Acoled

et

al.

showed

an

association

between


Enterobacter

septicaemia

and

short

gestation,

low


birthweight,

longer

period

of

endotracheal

intuba
-


tion,

and

length

of

hospital

stay.
22

This

might


explain

why

only

two

of

the

children

in

our

study


developed

an

E.

cloacae

infection.

Both

(patients


1

and

2;

Figure

1)

were

premature

(32

weeks

4

days


and

30

weeks

6/7

days)

and

had

low

birthweights,


1065

g

and

1140

g,

respectively.


Before

the

outbreak,

isolation

of

E.

cloacae

bac
-


teria

on

our

neonatal

ward

occurred

rarely.

The

high


increase

in

E.

cloacae

colonized

children

during

the


first

months

of

2000

raised

the

suspicion

of

an

out
-


break.

Indeed

molecular

fingerprinting

using


AP
-
PRC

and

AFLP

showed

that

all

the

E.

cloacae


strains

from

the

15

colonized

patients

were

geneti
-


cally

identical

(Figures

2

and

3).


Immediately

after

the

discovery

of

a

large

number


of

colonized

children,

the

factors

that

may

have


contributed

to

this

outbreak

were

investigated.


Previous

reports

documented

that

transmission

of


E.

cloacae

from

patient

to

patient

was

attri
buted

to


hospital

personnel,

or

use

of

contaminated

medical


devices.
5,12

In

our

hospital,

cross
-
transmission

by


hospital

personnel

was

unlikely,

since

there

was

a


strict

handwashing

routine.

Despite

an

intensive


environmental

search,

E.

cloacae

was

only

found

in

a


hand

basin

and

on

two

thermometers.

Of

these,

the


thermometers

were

colonized

with

the

outbreak


strain,

as

shown

by

DNA

fingerprinting

(Figure

2).


This

strongly

suggested

that

thermometers

were

the


routes

of

transmission.

Our

findings

correspond

to


those

from

another

Dutch

hospital

in

which

the


involvement

of

thermometers

in

an

E.

cloacae

out
-


break

in

a

NICU

is

described.
23

Outbreaks

involv
-


ing

other

micro
-
organisms

related

to

thermometers


have

also

been

published.
24


In

neonatal

units,

the

body

temperature

is

often


measured

by

rectal

thermometers.

In

contrast

with


other

units,

disposable

covers

were

not

used,

since


the

edges

of

the

standard

covers

may

harm

the

anal


sphincter

of

neonates.

Instead,

before

the

outbreak,


every

child

received

a

personal

thermometer.

After


use,

the

thermometer

was

wiped

with

gauze

soaked


in

70%

ethyl

alcohol

and

stored

in

a

personal

plastic


basket.

After

discharge

of

the

child,

the

same


disinfection

procedure

was

followed.

For

twins,


frequently

only

one

thermometer

was

used.

The

iso
-


lation

of

E.

cloacae

from

two

thermometers

shows


that

disinfection

was

inadequate.

Van

den

Berg

et

al.


also

showed

that

after

disinfection

with

80%


ethyl

alcohol,

one

out

of

10

thermometers

remained


contaminated

with

E.

cloacae.
23

Even

after

prolonged


incubation

of

thermometers

in

ethyl

alcohol,

our


outbreak

continued,

indicating

that

such

disinfection


is

inadequate.


Special

disposable

thermometer

covers

for


neonates

without

sharp

edges

are

available


(Uni
-
Instrumenten,

The

Netherlands).

When

we


introduced

these

on

the

unit,

contamination

of

the


thermometers

and

the

outbreak

stopped.


The

genetic

unrelatedness

of

the

strain

from


patient

16

(isolated

six

months

after

outbreak)

to

the


outbr
eak

clone

indicates

that

after

April

2000,

no


further

patient
-
to
-
patient

transmission

occurred,


and

that

the

use

of

disposable

thermometer

covers


and

the

other

measures

were

finally

vindicated.


Most

likely,

the

E.

cloacae

found

in

this

patient

was


coincidental.


The

E.

cloacae

outbreak

strain

was

probably


introduced

into

the

neonatal

care

unit

of

our


hospital

by

the

transfer

of

a

colonized

child.

AFLP


typing

showed

that

the

E.

cloacae

outbreak

clone

was


closely

related

to

that

of

the

NICU

of

the

university


hospital

from

which

the

index

case

was

transferred


(Figure

3).


To

our

knowledge,

this

is

the

first

report

on


hospital
-
to
-
hospital

transmission

of

an

E.

cloacae


strain.

The

fact

that

this

caused

outbreaks

in

at

least


two

neonatal

units

suggests

that

it

possesses

certain


specific

virulence

factors,

which

enables

it

to

cause


epidemic

spread.

Not

much

is

known

about

epidemic


E.

cloacae,

and

this

finding

deserves

more

investi
-


gation.

Probably

the

difference

in

their

predilection


E.

cloacae

in

neonatal

unit

25

for

survival

in

the

human

or

in

the

environment

plays


an

important

role.
20,21


In

conclusion,

an

outbreak

caused

by

E.

cloacae

in


a

neonatal

unit

was

recognized,

and

confirmed

by


molecular

fingerprinting.

Infected

thermometers


were

found

to

be

the

most

important

vector.

When

an


outbreak

of

this

kind

occurs,

stopping

further


admission

is

an

accepted

intervention.
23

Our

results


show

that

a

strict

regime

of

simple

infection

control


procedures

in

combination

with

regular

feedback

can


be

a

worthwhile

alternative.


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