B A C T E R IO L O G Y

O R IG IN A L A R T IC LE

Faecal carriage o f extended-spectrum ^-lactamase-producing

Enterobacteriaceae is com m on 12 months after infection and is
related to strain factors
E. T ite lm a n 12, C . M . H asan2, A . Iversen21 P. N a u c le r2'2, M . Kais2, M . K alin2 and C . G . G iske2

I) D epartm ent o f Infectious Diseases, Division o f Medicine, Karolinska University Hospital, Huddinge, 2) Clinical Microbiology, Karolinska Institutet - MTC,
Karolinska University Hospital, Solna and 3) Departm ent o f Infectious Diseases, Division o f Medicine, Karolinska University Hospital, Solna,
Stockholm, Sweden

A b s tra c t

W e aimed to determine the duration o f faecal carriage o f extended-spectrum //-lactamase (ESBL) -producing Enterobacteriaceae (EPE) in
patients w ith clinical infection caused by an EPE, to study host strains during carriage, and to identify factors associated w ith prolonged
carriage. Patients (n = 61) were followed w ith faecal samples and questionnaires about antimicrobial treatm ent and risk factors fo r EPE1 1, 3,
6 and 12 months after EPE infection. The EPE isolates were subjected to ESBL genotyping, epidemiological typing w ith pulsed-field gel
electrophoresis and PCR-based replicon typing. Escherichia coli isolates were analysed w ith PCR fo r phylogrouping, detection o f pabB
(ST 131) and virulence content. Patient-related and strain-related variables were compared fo r carriers and non-carriers at 12 months.
Carriage o f EPE was observed in 51 o f 61 (84%) patients after I month, 36 o f 61 (66%) after 3 months, 31 o f 61 (55%) after 6 months and
26 o f 61 (43%) after 12 months. O f the 26 carriers at 12 months, five had previous negative samples. In 17 o f 61 patients, ESBL was found in
a new bacterial species and/or strain during carriage. Among coli, 14 o f 49 belonged to the international clone ST131. Phylogroup B2 and
CTX-M -gr.-9 were associated w ith being carriers at 12 months (OR 4.3, 95% Cl 1.1-16.3 and OR 6.4, 95% C l 1.3-30.9, respectively). In
conclusion, EPE carriage is common 12 months after infection and persisting carriage may be associated w ith coli phylogroup B2 and
CTX-M-gr.-9. The host strain frequently changes throughout carriage and negative samples do n o t imply eliminated carriage.

K e y w o rd s : C T X -M -15, extended-spectrum //-lactamase, phylogroup B2, ST131, stool colonization

O rig in a l S u b m is s io n : 30 August 2013; R evised S u b m issio n : 13 November 2013; A c c e p te d : 15 December 2013
Editor: R. Canton
A r t ic le p u b lis h e d o n lin e : 18 February 2014
Clin Microbiol Infea 2014; 20: 0 5 0 8 -0 5 1 5
I0 .IIII/I4 6 9 -0 6 9 I.I2 5 5 9

C T X -M -IS among ESBL-producing coli is largely linked to

Corresponding author: E. Titelman, Department of Infectious
Diseases, Division of Medicine, Karolinska University Hospital,

the

Huddinge, I 73, SE-1-41 -86 Stockholm, Sweden

C o m orb id itie s, use o f antim icrobial agents and hospital contact

E-mail: em ilia.titelman@ karolinska.se

are w e ll-kn o w n risk factors fo r com m unity-onset infections

internationally

spread

coli clone

S T I3 l- 0 2 5 b

[2],

w ith ESBL-producing Enterobacteriaceae (EPE) [ I ] , b u t trans

mission o f EPE w ith in

households outweighs nosocom ial

dissemination in the n o n -o u tb re a k setting [3].

In tro d u ctio n

Although the prevalence and risk factors fo r EPE carriage

have been studied, little is kn ow n about the tim e course o f

Extended-spectrum //-lactamase (ESBL) -producing Escherichia

sto o l colonization. Previous re p o rts show th a t EPE carriage

coli and Klebsiella pneumoniae have becom e im p o rta n t causes o f

often persists fo r 3 m onths and may be prolonged by a n tibiotic

nosocom ial

high

tre a tm e n t [4], In o th e r studies, 51.5% w e re EPE carriers after

num bers o f ESBL carriers among healthy individuals have been

6 m onths (22nd European Congress o f Clinical M icrobiology

re p o rte d fro m several countries [ I ] , The rapid increase o f

and Infectious Diseases, p o ste r 1673), 20% a fte r 12 m onths,

and

com m unity-acquired

infections,

and

20 1 4 T he A u th o rs
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CMI

T ite lm a n e t o/.

Duration o f faecal carriage o f ESBL

0509

b u t <5% a fte r 2 years [5 ]. Persisting carriage o f K. pneumoniae

diagnosis. T he study o utcom e was faecal carriage o f EPE.

carbapenemase-producing K. pneumoniae was recently associ

Prolonged carriage was defined as th e finding o f EPE in faeces

ated w ith ca th ete r use, lo w functional status and long-term

at 12 m onths.

care facility stay [6].

Non-ESBL coli strains differ w idely in th e ir capacity to

S tudy population

colonize the colon, and this capacity is linked to virulence

T he Karolinska U nive rsity Hospital la b o ra to ry covers 80% o f

factors associated w ith uropathogenicity [7 ]. Genes encoding P

the Stockholm C o u n ty population. In 2009 the p ro p o rtio n s o f

fim briae, type

I fim briae and o th e r virulence factors are

ESBL-producing coli and K. pneumoniae in Stockholm w e re

enriched in resident strains, and th e re seems to be an additive

3.3% and 2.8% [ 13], The incidence o f EPE infections was 40/

effect o f these factors p ro m o tin g persistence [7 -9 ]. Strains

100 000 inhabitants [ 14].

belonging to phylogroups B2 and D have an enhanced ability to

AU patients clinical isolates o f EPE detected at Karolinska

persist in th e intestinal m icro b io ta [7 ]. H o w pathogen-related

U niversity Hospital betw een February and Decem ber 2009

factors im pact th e du ra tio n o f carriage o f EPE is unknow n.

w e re identified (n = 508). Patients th a t w e re likely to be able

ESBL genes are often located on conjugative plasmids

to understand instructions in Swedish and th a t w o u ld be able

belonging to the IncF group [10,1 I], II, N and K, w ith high

to p e rfo rm self-collection and submission o f faecal samples

potential o f recom bination [12]. It is th e re fo re likely th a t ESBL

w e re eligible fo r inclusion. Since EPE was notifiable and was

enzymes are transferred fro m transient strains to resident

still rare in Sweden a t the tim e o f the study, the regional

strains in the norm al intestinal m icrobiota. H ow ever, kn o w l

research

edge about transfer o f ESBL enzymes between d iffe re n t strains

physicians w e re contacted to obtain approval to co n tact the

and species during the course o f ESBL carriage is lim ited.

patients. Reasons fo r exclusion are shown in the Supporting

ethics

co m m itte e

requested

th a t

the

patients

W e present prospective data on the duration o f faecal

info rm a tio n (Fig. S I). Seventy-one patients w e re included.

carriage o f EPE a fte r firs t-tim e EPE infection, and identify

N in e w e re lost to fo llo w -u p and one patient died. C haracter

p a tie nt and strain factors associated w ith prolonged carriage.

istics o f the patients included and n o t included in analyses are

W e also re p o rt on changes in h o st strains th ro u g h o u t the

presented in Table I.

carriage, and present data on plasmid replicon co n te n t fo r

cases fo r w hich the ESBL p ro d u ctio n was detected in a new

C ollectio n o f samples and clinical d a ta

strain o r species during the course o f carriage.

A t th e specified tim e-points, I, 3, 6 and 12 m onths a fte r the

patients diagnosis w ith EPE infection, self-collected faecal
samples w ere obtained. A t I m onth the patients filled in a

M aterials and M ethods

questionnaire about antim icrobial tre a tm e n t received and

previously described risk factors fo r EPE infection (an tim icro

S tudy design, o u tc o m e m easures and definitions

bial trea tm e nt, hospital stay, urinary catheter, travel abroad and

The

hospital admissions abroad w ith in 6 month's before the EPE

study was designed as a prospective c o h o rt study.

Sixty-one patients w e re subjected t o fo llo w up w ith faecal

infection, and abnorm alities o f th e u rina ry tra ct). A t m onths 3 ,6

samples and questionnaires about antim icrobial tre a tm e n t and

and 12 th e patients filled in a questionnaire a b o ut new acquired

risk factors fo r EPE infection at 1 , 3 , 6 and 12 m onths after

infections and recently received antim icrobial treatm ent.

T A B L E I. C h a racteristics o f 508 p atien ts w ith ex ten d ed sp ectru m /!-lactamase-producing Enterobacteriaceae detected at the clinical
microbiology laboratory at Karolinska University Hospital between February and December 2009

Female
Age (mean)
Level o f care
Hospital inpatients
Hospital outpatients
Long-term care facilities
Primary care
Culture material
Urine
Blood
O ther

A ll patients (n S08)

Patients included in
analyses (n 61)

Patients n o t included in
analyses (n = 447 )

p value

337 (66)
57.8

38 (61)
58.3

299 (67)
57.7

0.48
0.87

308 (60)
50 (10)
25(5)
125 (25)

35 (57)
14 (23)
0
12(20)

273 (61)
36(8)
25(6)
113(25)

0.002

437 (86)
24(5)
47(9)

51 (84)
5 (8 )
5 (8 )

386 (86)
19(4)
42(9)

0.39

Data presented as number and column percentage in parenthesis if not otherwise stated.

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Clinical Microbiology and Infection, Volume 20 Number 8, August 2014

Processing o f faecal samples and co n firm atio n o f ESBL

pyelonephritis (pap): fim H, fim AM T78, papAH, papC, papEF,

prod uction

papG, papG I, papG Il and papG III [21 ].

EPE in the faecal samples w e re detected w ith

ESBL agar
streaked

(bioM e'rieux,

Craponne,

tra n s p o rt

fro m patients fo r w hich w e found th e ESBL p ro d u ctio n in a

logies w e re studied w ith species identification and antim i

diffe re n t species a n d /o r additional strain w e re subjected to a

using VITEK2

Brescia,

PCR-based replicon typin g

w ith o u t previous b ro th enrichm ent. Unique co lony m o rp h o

testing,

(Copan,

Plates w ere

The infecting isolates fro m all patients, and fo llo w -u p isolates

susceptibility

swabs

France).

Italy)

crobial

w ith

C h ro m ID

(bioMe'rieux).

N o rm a lly one t o th re e colonies representing each colony

PCR-based replicon typing previously described by C a ra tto li

et al. [22],

m orp h o lo g y w e re picked. ESBL p ro d u ctio n was confirm ed

w ith

ESBL com bination disks (Becton

Dickinson, Franklin

Statistics

Lakes, NJ, USA) o r ESBL Etest (bioM erieux) according to

Clinical data w e re extracted fro m patient questionnaires and

th e m anufacturers instructions.

medical records. Patient- and strain-related factors w ere

com pared fo r carriers and non-carriers at 12 m onths using

D e te rm in a tio n o f ESBL genotype

G r a p h Pa d Software and St a t a I 1.0. Categorical variables w e re

The infecting isolates fro m all patients, and fo llo w -u p isolates

com pared using chi-squared te s t o r Fishers exact te s t fo r

fro m patients fo r which w e found th e ESBL p ro d u ctio n in a

categorical data. Students t-te s t was used fo r continuous data.

diffe re n t species a n d /o r additional strain w e re subjected to a

M ultivariate analysis was p e rfo rm e d by logistic regression.

real-tim e m in o r groove binder-probe based PCR fo r typing o f

Exposure variables w ith a p-value < 0 .1 in univariate analyses

bioc t x - m to th e phylogenetic subgroups [15]. C TX-M -negative

w e re included in the m ultivariate model.

isolates w e re subjected to C h e ck-p o in t M DR C T 102 D N A

m icro a rra y to d e tect

W ote m

and

W o Sh v

variants [16].

Escherichia c oli phylogenetic groups

Phylogrouping o f coli was based on trip le x PCR (chuA, yjaA,

The study was approved by the research ethics co m m itte e

in Stockholm , Sweden (Recordal: 2 0 0 8 /2 :7 ) .

Results

TspE4.C2) [17], and was only carried o u t w ith the infecting

isolates.

ESBL d e tec tio n and epidem io log ical typing

A fte r I m onth, 51/61 (84%) patients w e re EPE carriers, this

Id en tifica tio n o f 0 2 5 b - S T I3 l

num ber was 36/61 (66%) a fte r 3 m onths, 31/61 (55%) after

Infecting isolates o f coli w e re analysed w ith an 0 2 5 b -S T 131

6 m onths and 26/61 (43%) a fte r 12 m onths. A t 12 m onths,

allele-specific

21% o f the EPE carriers had one o r several negative faecal

PCR assay o f th e pabB gene according to

C le rm o n t et al. [ 18].

samples during fo llo w up. D u rin g fo llo w up the ESBL p ro d u c

tio n was found in a d iffe re n t bacterial species on seven

E pidem iological typin g w ith pulsed-field gel electrophoresis

occasions o r a novel strain o f coli o r K. pneumoniae on 20

Epidemiological typing o f coli and K. pneumoniae isolates

occasions in a to ta l o f 17 patients. The results fro m th e ESBL

using pulsed field gel electrophoresis (PFGE) was perform ed

detection and epidemiological typing (PFGE) are displayed in

according to the PuIseNet p ro to c o l fo r coli O I5 7 :H 7 [19],

Fig. I. Fig. 1(a) shows data fo r carriers at 12 m onths and

using Xbal fo r re strictio n o f D N A . Isolates w e re considered

Fig. I (b) shows data fo r non-carriers.

related if th e PFGE patterns had a sim ilarity index o f >0.90

(D ice co-efficient; corresponding to a difference o f at least

ESBL genotyping

th re e bands) [20], and possibly related if th e PFGE patterns

C T X -M -grouping o f all retrievable clinical isolates ( coli

had a difference o f fo u r to six bands. W ith lo w e r sim ilarity

n = 49,

indices w e classified th e strain as d istin ct fro m the original

belonged to C T X -M -g r.-l, 13/56 (23%) to C T X -M -g r.-9 . and

strain.

K. pneumoniae

n = 7)

showed

th a t

38/56

(68%)

1/56 (2%) to C T X -M -g r.-8 o r-2 5 . T w o patients o f 56 (4%)

produced sulfhydryl variable (SHV) ESBLs and tw o had an ESBL

Screening fo r virulence facto rs w ith m u ltip le x P C R

phenotype b u t no confirm ed genotype. In 10/17 patients, fo r

T he infecting coli isolates w e re screened fo r virulence

w hom the ESBL p ro d u ctio n was found in a new species o r

factors characteristic o f uropathogenic coli w ith m ultiplex

strain

PCR, using prim ers to d e tect genes encoding type I fim briae

C T X -M -g ro u p than in th e infecting strain in one o r several

(f/m) and p-fim briae, also know n as pili associated w ith

o f th e fo llo w -u p faecal samples (Fig. 2).

during the course o f carriage, w e found a n other

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CMi

T ite lm a n e t al.

(a) Carriers 12 months after EPE-infection

Infecting
isolate

H
H

H H
ns

H H

12

OO

21
23

20

24
25

26

ns

HO h o
ns

O H

H
OH H

18

HO

O
HO

Fecal iso la te (s)

O
O

27
28

29

30

31
32

34

H
H

35

33

O
O
HO

36

37

38
39
40
41

42

43

44

45

46

47

48

49

HH

H
H
O
H

ns

_
ns

16
17

Infecting

isolate(s) ! m o n t h 3 m o n th s 6 m o n th s

O
H

H H

Patient

6 m o n th s 12 m o n th s

H
HH OHO
ns
H H

051

(b) Non-carriers 12 months after EPE-infection

Fecal iso la te (s)

!m o n th 3 m o n th s

Duration o f faecal carriage o f ESBL

ns

H
H

ns

ns

H
O

50

51

52
53

54

ns

E. coli, fir s t strain

BH

H K. p n e um o n ia e , firs t strain

O CH> fo u rth strain

coli, fifth strain

N egative sam ple

58

O
Q

C- c lh non typ a b le
coli, no PFGE-result

ns

N o sa m p le

59

Possibly relate d

60

CH> second strain

coli, th ird strain

H*0 H

H
H
OH H
O
H

H
H

ns

K- pne um o n ia e , second strain

55

E nte rob a cte ria cea e , o th e r

56

K. p n e um o n ia e , no PFGE-result

57

61

H
H

O
H

H
H

F IG . I. Extended sp ectru m /i-lactam ase (ESBL) -p ro d u cin g strains d urin g fo llo w up. O v e rv ie w o f th e d u ra tio n and dynam ics o f faecal carriage o f
ESBL-producing Enterobaaeriaceae (EPE). (a) Results o f cu lture s and pulsed fie ld gel e le c tro p h o re s is (PFGE) fo r th o se w h o w e re c a rrie rs at
12 m onths, (b) results fo r n on -ca rrie rs. Each ro w shows th e results fo r o ne p atient. T h e sym bols re p re s e n t th e species and strains w h e re ESBL was
dete cte d in th e infecting isolate and in faeces a t I, 3, 6 and 12 m onths.
Escherichia c oli phylogenetic groups

and fim AM T78 as w e ll as several genes encoding P fim briae

M ost o f the clinical coli isolates belonged to phylogroup B2

(papAH, papC, papEF, papG and papGII). O f these, only tw o

(n = 19), fo llo w e d by D (n = 15), A (n = 10) and B I (n = 5).

w e re derived fro m patients w ith prolonged carriage.

Id en tifica tio n o f 0 2 5 b - S T I3 l

PCR-based replicon typin g

The pobB PCR (m a rke r o f S T 131) was positive in 14/49 cases,

The m ost freq u e n t replicon co n te n t among the clinical isolates

all B2 isolates.

o f all patients was FrepB (n = 46) fo llo w e d by FIA (n = 29) and

FIB (n = 27). Seventeen isolates contained b o th FIA and FIB
replicons. Eight isolates w e re positive fo r HI I and 11, tw o fo r

Screening fo r virulence factors

M ost coli isolates w e re positive fo r fim H (n = 48), fimAMT78

N and one was positive fo r A IC and P, respectively. W h e n

(n = 35) o r bo th (n = 33). Six isolates w e re positive fo r fim H

examining th e isolates fro m th e 17 patients in w hich ESBL

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Clinical Microbiology and lnfecdon, Volume 20 Number 8, August 2014

Patients w ith ESBL-production delected in a new species and/or an additional strain durin g follow-up

F I A . FIB, 11. FrcpB

FIA, FIB, FrepB

F I A , FIB

FIA. FIB

FIA, FIB. FrcpB -

FIA. FIB, FrepB

RA, FIB. FrepB

FrcpB

F I B . FrepB

FIA, FrepB

FIA, FrepB

F I A , FrepB O FIB, 11,

F I A , FrcpB F I A , FIB FrcpB

F I A , FrcpB FIA. FIB. FrepB

FIA, FIB, FrepB

FIA, FrcpB

FIA, FrcpB

ns

FIA, FrcpB

FrcpB

F I A , FlB

3 $ FIA, FIB

F I B . FrepB

F I A , FIB, FrepB

F I A , FIB, FrcpB

FIB, FrepB

F I A . FIB. FrepB

10

FIA, FrcpB

18

FrcpB
FIA, FrcpB

19

21

FIB. FrepB

^ F IA T lB T r e p ^ ^ ^
F I A , FIB
FIA, FIB, FrcpB

R A , FrcpB

F I A , FrepB
FIB. 11. FrepB

F I B . 11

FIB. FrcpB

FIB, FrcpB S I FlA

22

FrcpB
F I B . FrcpB

1 1 , FrepB

ns

23

FIA. FIB, FrepB

E u
F I B . FrepB

26

j FIA, FrcpB

F I A , FIB, 11. FrcpB

B FIA. Il FrcpB
F I A . FIB. FrepB

32
33

FrcpB F I A

FIB, FrepB

FrepB

S-

42
44

F I A . F lB . H I I . il

F I B , 11

F I A . FIB, FrepB

49

F I B , FrepB

FrcpB -

F I A . FIB. FrepB

FIA. FIB, FrepB

52

F I A , FIB. FrepB

FIB, FrcpB

FIA, FIB. FrcpB

FIA, FIB, FrcpB

FiA, FlB

FIA. FlB
FrepB

A FrcpB

E coli,
E. coli,
E- coli,
E. co/i,
E- coli,
E coli,
E coli,

SS- FIA, FIB. FrepB FiA, FIB. FrCpB FIA, FIB, FrepB

FIA. FlB

firs t strain, C T X -M -g r.-1

firs t strain, C T X -M -g r.-9
firs t strain, ESBL-phenotype, no confirm ed genotype
second strain, C T X - M - g r - 1

second strain, C T X -M -g r.-9

second strain, T E M

SB
EU
Q

second strain, E SBL-phenotype, no confirm ed genotype

f ib

E. coli, third strain, C T X -M -g r.E. coli, third strain, C TX -M -gr.-S

E. coli, fou rth strain, C T X -M -g r. 9
E. co/i, fifth strain, C T X -M -g r.-9
E. pneumoniae, firs t strain, C T X M -g r.-I
E. pneumoniae, firs t strain, C T X M -gr.-9
K. pneumoniae, second strain, C T X -M -g r.-l
N o PFGE- o r C T X -M -ty p in g result

F IG . I . C T X -M -g ro u p s and re plicon types o f e xte n de d sp ectru m /S-Iactamase (ESBL) -p ro d u c in g strains d urin g fo llo w up. O v e rv ie w o f th e patients
fo r w h o m w e fo u n d th e ESBL p ro d u c tio n in a n ew species o r stra in d urin g fo llo w up. Each r o w show s th e results o f th e culture s, pulsed fie ld gel
e le ctro p h o re sis (PFGE)1 C T X -M g ro u p in g and PCR-based re plicon typ ing f o r o ne patient. T h e sym bols re p re s e n t th e species and strains w h e re ESBL
p ro d u c tio n was fo u n d as w e ll as th e C T X -M -ty p e . T h e re p lico n types o f each strain can be fo u n d n e x t t o th e sym bol.

pro d u ctio n was found in a diffe re n t species (n = 7) a nd/or a

novel strain o f coli (n = 17) during fo llo w up com pared w ith

Discussion

th e beginning o f the study, w e detected an identical com bi

nation o f replicons in b o th the original and the new strains in

O u r main finding was th a t faecal carriage o f EPE often persists

only fo u r cases. The results o f th e plasmid characterization are

I year after infection, and th a t prolonged carriage is associated

shown in Fig. 2.

w ith coli phylogroup B2 and ESBL C T X -M -g r.-9 . Phylogroup

B2 is strongly associated w ith high virulence, and o u r findings

P a tie n t factors and strain facto rs o f th e in fecting isolate in

indicate th a t strain virulence could be an im p o rta n t fa c to r also

re la tio n to d u ra tio n o f carriage

fo r persistence o f ESBL-producing coli in th e intestinal

Phylogenetic group B2 o ccu rre d m ore frequently in coli

m icrobiota. H ow ever, w e did n o t find any differences in the

strains found in ca rrie rs at 12 m onths than among non-car

presence o f the uropathogenetic virulence factors pap and fim

rie rs (12/23 (52%) versus 6/26 (23%), p 0.04), w hich was also

genes between carriers and non-carriers, n e ith e r w e re there

th e case fo r C T X -M -g r.-9 (10/25 (40%) versus 3/31 (10%),

any associations between the virulence genes and coli

com m on

phylogroups. A lm o s t all infectious coli isolates w e re positive

be carriers at

fo r fim H and fim AM T78 genes, probably reflecting th a t these

12 m onths than among patients th a t w e re n o t (6/26 (23%)

isolates are viru le n t and m ostly uropathogenic by definition.

p 0.01). Initial bloodstream

infection was m ore

among patients w h o w e re la te r found to

1/35 (3%), p 0.04). The frequencies o f the various

C T X -M -g r.-1 includes th e C T X - M - 15 enzyme associated w ith

patient and strain factors in relation to the du ra tio n o f EPE

the highly viru le n t international clone S T I31 and ind irectly to

carriage are shown in Table 2. Bloodstream infection, phylo

phylogroup B2, whereas C T X -M -g r.-9 has less frequently been

group

associated

versus

B2 and C T X -M -g r.-9

w e re

included

in a logistic

w ith

such

viru le n t

clones.

Previous

studies

regression model. Phylogroup B2 and C T X -M -g r.-9 w ere

dem onstrate th a t virulence o f coli S T I31 is related w ith

associated w ith being a c a rrie r at 12 m onths (OR4.3, 95% Cl

the strain and n o t w ith the presence o f blaCTX_M. l3 [23]. O fth e

1.1-16.3 and O R 6.4, 95% Cl 1.3-30.9, respectively) whereas

I I C T X -M -g r.-9 coli isolates, nine belonged to phylogroup

bloodstream infection was no longer associated w ith p ro

B2

longed carriage in th e adjusted analysis (O R 4.7, 95% C l 0 .4 -

C T X -M -groups in this study reflects th e m olecular epidem i

50.1).

ology o f EPE in Sweden [2 4 -2 6 ].

(n = 4)

or

(n = 5)

20 1 4 The A u th o rs
C linical M icrob io lo gy and Infection 20 1 4 European Society o f Clinical M icrob io lo gy and Infectious Diseases, CM/, 20, 0 5 0 8 - 0 5 1 5

isolates.

The

d istrib u tio n

of

CMI

T ite lm a n et al.

Duration o f faecal carriage of ESBL

0513

T A B L E 2. C o m p a ris o n o f p a tie n t and strain factors o f th e infecting isolate b e tw e e n ex ten d ed sp ectru m //-lactamase carriers and
non-carriers 12 months after extended spectrum /-lactamase-producing Enterobacteriaceae infection

V a ria b le
Sex and age
Type o f Infection
Previously described risk factors fo r acquisition of
EPE

Antimicrobial treatment6

Strain factors

Women
Median age (years)
Urinary tract infection
B lo o d s tre a m in fe c tio n *
Previous antimicrobial1*
Hospital Stayb
Travel abroadb
Abnormalities In urinary tract
Urinary Catheterb
Carbapenem treatment
C IP + TMP +TS U
N IT + MEC
More than tw o antimicrobial agents
More than three antimicrobial
agents
P h y lo g ro u p B2
S T I31
C T X -M -1-group
C T X -M -9 -g ro u p
Fim
Pop

C a rrie rs a t 12 m o n th s n
()

N o n -c a rrie rs a t 12 m o n th s n
(%)

P
value

14 (54)
63
24 (92)
6 (2 3 )
IO (42)
13 (52)
I l (31)
5 (2 0 )
9(36)
5(19)
9(39)
7 (3 0 )
3(13)
2 (9 )

21 (60)
60
30 (86)

0.8
0.9
0.7
0.04
0.8
0.4
0.3
0.76

I (3)
16 (47)
14(41)
20 (60)
8 (2 4 )
12(55)
2 (3 )
11 (52)
I l (52)
5(16)
2 (6 )

1.0
0.15
0.8

1.0
1.0
1.0

6(23)
5(19)
23 (74)
3(10)
25 (96)
3(12)

12(52)
9(39)
15 (60)
10 (40)
23 (100)
14(7)

0.04
0.2
0.4

0.01
I
0.7

Bold face = p <0.05.

bSix months before ESBL Infection.
cFor the original infection with ESBL-producing bacteria (not additional treatment during follow-up).
CIP1 ciprofloxacin; EPE, extended spectrum /i-lactamase-producing Enteroboctertoceoe; ESBL, extended spectrum ^-lactamase; MEC, mecillinam; NIT, nitrofurantoin; TMP,
trimethoprim; TSU, trimethoprim-sulphamethoxazole.

In accordance w ith previous data [6 ], w e show th a t negative

in the com m unity, it is d ifficu lt to calculate th e risk o f being

faecal samples w ith in th e firs t year after ESBL infection do n o t

re-colonized. H o w e ve r, w ith <3% o f ESBL producers among

im ply elim ination o f carriage. M ost likely EPE persists in the gut

clinical isolates o f coli and K. pneumoniae [2 8 -3 0 ] w e find

b u t in lo w numbers com pared w ith susceptible Enterobacte-

this less likely, provided th a t th e re had been no new exposure

riaceae in th e intestinal m icro b io ta , fo r which reason they are

to risk factors.

n o t detected by ro u tin e m ethods. Ecological disturbances

As

previously

re p o rte d

[10]

we

com m only

detected

caused by, fo r example, antim icrobial tre a tm e n t on a later

plasmids belonging to the IncF group, especially FrepB, FIA

occasion may p ro m o te o ve rg ro w th o f the resistant bacteria

and FIB. These plasmids are freq u e n tly found in both EPE and

[2 7 ]. H ow ever, w e found no c o rre la tio n between a n tibiotic

non-EPE [12]. T h e ir presence in tw o d iffe re n t ESBL-producing

consum ption during th e fo llo w up and a shift fro m negative to

strains fro m th e same patient does n o t co n firm th a t an IncF

positive faecal samples. N e ith e r w e re specific agents n o r the

plasmid transferred the ESBL gene. F urther, isolates frequently

num ber o f received antim icrobials associated w ith prolonged

have several plasmids, w hich may have m ultiple replicons. In 4/

carriage.

17 o f th e patients w ith ESBL p ro d u ctio n in a diffe re n t strain

In 28% o f th e patients w e found the ESBL p ro d u ctio n in

and /o r species w e found th e same replicon co n te n t o f the firs t

a n o th e r o r additional species a n d /o r strain o f coli o r

and a t least one o f the new strains (Fig. 2). H o w e ve r, w e did

K. pneumoniae during the fo llo w up than at th e beginning o f

n o t establish a specific link between ESBL genes and replicons.

th e study. This finding was m ore freq u e n t among carriers at

T he main lim itation o f this study is th e fairly small num ber o f

12 m onths than non-carriers; supporting the hypothesis th a t

patients. H o w e ve r, the mean age (p 0.87), sex (p 0.48) and

persisting carriage is linked to tran sfe r o f ESBL genes by

cu ltu re m aterial (p 0.39) did n o t d iffe r fro m the to ta l group o f

plasmids fro m transient to resident strains in the norm al

patients diagnosed w ith

EPE infection during recru itm en t,

intestinal m icrobiota. H ow ever, ten o f these patients had a

indicating th a t th e results are generalizable. H o w e ve r, th e re

new C T X -M -g ro u p in one o r several o f the fo llo w -u p samples.

was a difference in the level o f care (p 0.001), w ith no patients

These patients w e re probably colonized w ith several strains

fro m long-term care facilities included. These patients m ost

producing ESBL o f d iffe re n t genotypes at the beginning o f the

likely w o u ld have had a prolonged carriage com pared w ith the

study. Either all strains w e re n o t present in all faecal samples

patients

o r w e w e re unable t o distinguish these as unique strains when

self-collected samples, tra n sp o rta tio n tim e and the detection

selecting colonies fro m th e screening plates. It cannot be

levels used by th e la b o ra to ry may also have affected the

included

in

th e

study

[6].

T he

quality

o f the

excluded th a t a new EPE was acquired during the fo llo w up.

results. C ollecting m ore than one sample at each tim e p o in t o r

Since w e have lim ited Swedish prevalence data on EPE carriage

using e nrichm ent techniques m ight have given a higher rate and

20 1 4 The A u th o rs
C linical M icrob io lo gy and Infection 20 1 4 European Society o f Clinical M icrob io lo gy and Infectious Diseases. CM I1 2 0, 0 5 0 8 - 0 5 1 5

0514

diversity o f ESBL-positive isolates. If anything, these lim itations

w o u ld co n trib u te to an underestim ation o f the carriage rates
at the various tim e-points.

persists

2. P ito u t JD. Extraintestinal pathogenic Escherichio coli: a com bination of

virulence w ith antibiotic resistance. Front Microbiol 2012; 3: 9.
3. H ilty M, Betsch BY, Bogli-Stuber K et al. Transmission dynamics o f
extended-spectrum -lactamase-producing enterobacteriaceae in the

In conclusion w e show th a t faecal carriage o f EPE strains

often

CM!

Clinical Microbiology and Infection, Volume 20 Number 8, August 2014

I year a fte r infection, and th a t prolonged

carriage m ight be associated w ith coli phylogroups B2 and

C T X -M -g r.-9 , and may be linked to the transfer o f plasmids
carrying ESBL genes fro m an infecting o r transient strain to
resident strains o f Enterobacteriaceae in the intestinal m ic ro
biota. Negative samples w ith in th e firs t year do n o t im ply
elim ination o f carriage. This knowledge may prove im p o rta n t
fo r lim iting the spread o f EPE in hospitals and long-term care

te rtia ry care hospital and the household setting. Clin Infect Dis 200S; 55:
967-975.
4. Apisarnthanarak A, Bailey T C , Fraser VJ. D uration o f stool colonization
in patients infected w ith extended-spectrum /?-lactamase-producing
Escherichia coli and Klebsiella pneumoniae. Clin Infect Dis 2008; 46: 13221323.
5. W a rre n RE, Harvey G, C a rr R, W a rd D 1 D oroshenko A. C o n tro l o f
infections due to extended-spectrum /7-lactamase-producing organisms
in hospitals and the com m unity. Clin Microbiol Infect 2008; 14 (suppl I):
124-133.
6. Feldman N, A d le r A , Molshatzki N et al. Gastrointestinal colonization
by kpc-producing Klebsiella pneumoniae follow ing hospital discharge:

facilities.

duration o f carriage and risk factors fo r persistent carriage. Clin

Microbiol Infea 2013; 19: E 190-196.
7. W o ld AE, Caugant D A , Lidin-Janson G, de Man P, Svanborg C. Resident

A ckn o w led g em en ts

colonic Escherichia coli strains frequently display uropathogenic char

acteristics. J Infea Dis 1992; 165: 46-52.
8. O stblom A , A d le rb e rth . I, W o ld AE, N ow rouzian FL. Pathogenicity

W e thank Pia Appelgren fo r help w ith planning the study and

inclusion o f patients, and N o h a Flefel fo r excellent technical

island markers, virulence determinants malx and usp, and the capacity
o f Escherichia coli to persist in infants* commensal m icrobiotas. Appl
Environ Microbiol 2011; 77: 2303-2308.

assistance. Part o f this study was presented at the 22nd

9. Melican K, Sandoval RM, Kader A et al. U ropathogenic Escherichia coli p

European Congress o f Clinical M icrobiology and Infectious

and type I fim briae act in synergy in a living host to facilitate renal
colonization leading to nephron obstruction. PLoS Pathog 2011; 7:

Diseases, P 1662, 2012.

e l 001298.
10. Coque TM , Novais A , C ara tto li A et al. Dissemination o f donally
related Escherichia coli strains expressing extended-spectrum /7-lactam

Funding

ase C T X -M -15. Emerg Infect Dis 2008; 14: 195-200.

11. Novais A , Canton R, M oreira R, Peixe L, Baquero F,

Coque TM.

Emergence and dissemination o f enterobacteriaceae isolates producing

This w o rk was supported by grants fro m the Scandinavian

Society o f A n tim icrob ia l C hem otherapy and Strama (The
Swedish Strategic Program me against A n tim icrob ia l Resistance).

C T X -M -I-Iik e enzymes in Spain are associated w ith incfii (C TX -M -15)

and broad-host-range (C T X -M -I, -3, and -32) plasmids. Antimicrob
Agents Chemother 2007; 51: 796-799.
12. C arattoli A. Plasmids and the spread o f resistance. Int J M ed Microbiol
2013; 303: 298-304.

Tran sp aren cy D eclaratio n s

_______

13. Titelm an E, Iversen A, Kais M, Kalin M, Giske CG. Efficacy

pivmecillinam

fo r

lo w e r

urinary

tra c t

infection

caused

of
by

extended-spectrum /7-lactamase-producing Escherichia coli and Klebsiella

pneumoniae. Microb Drug Resist 2012; 18: 189-192.

C hristian G. Giske has received speakers honoraria fro m

14. SWEDRES. A report on Swedish antibicrobial utilisation and resistance in

AstraZeneca and Meda. AU o th e r authors re p o rt no potential

human medicine. Swedish Institute fo r Communicable Disease C on tro l.

conflicts.

2009.

http://www.folkhalsomyndigheten.se/pagefiles/12891 /swedres-

2009.pdf.
15. B irkett C l, Ludlam H A 1W o o d fo rd N et al. Real-time taqman PCR fo r
rapid detection and typing o f genes encoding C T X -M extended-spec

S upporting In fo rm a tio n

tru m /7-lactamases. J Med Microbiol 2007; 56: 52-55.

16. Cohen Stuart J, D ie rikx C, A l Naiemi N et al. Rapid detection o f TEM,
SHV and C T X -m extended-spectrum /7-lactamases in enterobacteria

A d d itio n a l Supporting In form ation may be found in th e online

ceae using ligation-mediated amplification w ith m icroarray analysis. J

version o f this article:

Antimicrob Chemother 2010; 65: 1377-1381.

17. C le rm o nt O , Bonacorsi S, Bingen E. Rapid and simple determ ination o f

F ig u r e S I . Inclusion o f patients and reasons fo r exclusion.

th e Escherichia coli phylogenetic group. Appl Environ Microbiol 2000; 66:

4555-4558.
18. C le rm o nt O , Dhanji H, U pton M et al. Rapid detection o f the

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