Journal of Hospital Infection (2004) 57, 202–208
Molecular biology of extended-spectrumb-lactamase-producing Enterobacteriaceaeresponsible for digestive tract colonization
N. Moustaouia, A. Soukrib, N. Elmdaghria,c, M. Boudoumad,M. Benbachira,c,*
aMicrobiology laboratory, IbnRochd University Hospital, Casablanca, MoroccobFaculte´ des Sciences AinChock, Casablanca, MoroccocFaculte´ de Me´decine, Laboratoire de Microbiologie, Casablanca, MoroccodFaculte´ des Sciences BenMsick, Casablanca, Morocco
Received 29 October 2003; accepted 8 January 2004
Summary Twenty-nine extended-spectrum b-lactamase (ESBL)-producing
Enterobacteriaceae strains (14 Klebsiella pneumoniae, 10 Escherichia coli
and five Citrobacter diversus) isolated from April to July 1996 from faecal
carriers in a surgical intensive care unit at the university hospital ofCasablanca (Morocco) were studied. Plasmid content and DNA macrorestric-tion polymorphism determined by pulsed-field gel electrophoresis (PFGE)were used to compare the strains. Restriction profiles of total genomic DNAscleaved by XbaI and compared by PFGE revealed nine, four and two clones inK. pneumoniae, E. coli and C. diversus, respectively. Plasmid profile analysisof ESBL-producing strains of K. pneumoniae showed that only seven of 14isolates had a plasmid; four different plasmid profiles were observed. Threedifferent plasmid profiles were observed in E. coli and two in C. diversus. Plasmids responsible for ESBL production could be transferred by conjugationto E. coli K12 J53-2 from all E. coli isolates and from four of sevenK. pneumoniae. No plasmid transfer could be obtained from C. diversusstrains. Restriction enzyme digests of plasmids from transconjugants (fourtransconjugants of K. pneumoniae and five transconjugants of E. coli) showeddifferent patterns. In the surgical intensive care unit where the survey wasconducted, the dissemination of ESBLs was due to a mix of strain spread andstrain diversity rather than to plasmid dissemination.
Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rightsreserved.
*Corresponding author. Address: Faculte´ de Me´decine,
Laboratoire de Microbiologie, 19 rue Tarik Bnou Zyad, BP 9154
Extended-spectrum b-lactamase-producing Entero-
Casablanca, Morocco. Tel.: þ212-22-26-90-57.
E-mail address: firstname.lastname@example.org
bacteriaceae (ESBLPE) are important agents of
0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.01.007
Molecular biology of ESBL producing Enterobacteriaceae
nosocomial infections and are responsible for out-
the authors (NM) at the microbiology laboratory
breaks, which occur mainly in intensive care
of the Ramon y Cajal Hospital of Madrid, Spain
units.The dissemination of ESBL is due to clonal
spread,to plasmid dissemination orbetween species,or to both mechanisms.
Plasmid isolation and resistance transfer
Genes encoding for ESBL may also be found ontransposons.
Plasmids were extracted by the method of Kado and
Digestive tract colonization is a prerequisite for
Liu,the alkaline lysis method as described by
infection by ESBLPIn Casablanca a prospective
Sambrook et aland by the High Pure Plasmid
study conducted in 1996 in an intensive care unit of
Isolation Kit (Boehringer Mannheim, Germany).
the IbnRochd University Hospitalrevealed that
Transfer of plasmids by conjugation was performed
digestive tract colonization with ESBLPE was fre-
as described previously.A rifampicin-resistant
quent, occurred shortly after admission to the unit,
strain of E. coli K12 J53-2, was used as recipient.
and was transient. Bacteria responsible for this
Transconjugants were selected on MacConkey agar
colonization belonged to five species; Klebsiella
containing rifampicin (250 mg/L) and amoxicillin
pneumoniae was the most frequent (42.4%) fol-
lowed by Escherichia coli (30.3%) and Citrobacterdiversus (15.2%).
To analyse the mode of spread of ESBLs (plasmid
and/or clone spread) in the intensive care unit, we
Plasmid DNA from the transconjugants was digested
studied the ESBPLE isolates by macrorestriction
with EcoRI (Sigma Bio-Sciences, St Louis, MO, USA)
polymorphism of DNA determined by pulsed-field
and Hind III (Roche Molecular Biochemicals, Mann-
gel electrophoresis (PFGE) and plasmid DNA
heim, Germany) according to the manufacturer’s
The antibiotic susceptibilities were determined by
disk diffusion on Mueller-Hinton agar. The followingagents were tested: gentamicin, tobramycin, netil-
The most prevalent species of the 33 ESBLPE
micin, amikacin, trimethoprim-sulfamethoxazole,
isolated in the colonization survey were studied:
tetracycline, chloramphenicol and imipenem. The
K. pneumoniae ðN ¼ 14Þ; E. coli ðN ¼ 10Þ and
minimal inhibitory concentrations (MICs) of cefo-
C. diversus ðN ¼ 5Þ: Three of the 10 E. coli isolates
taxime and ceftazidime alone and combined with
were isolated sequentially from the same patient.
clavulanic acid (4 mg/L) were determined by agar
Four patients were simultaneously colonized by two
dilution with an inoculum of 104 cfu/spot.E. coli
species: K. pneumoniae þ E. coli (two patients),
ATCC 25922 and E. coli ATCC 35218 were used as
K. pneumoniae þ Klebsiella oxytoca (one patient),
K. pneumoniae þ C. diversus (one patient).
Agarose plugs containing chromosomal DNA were
Macrorestriction polymorphism of DNA determined
prepared by a method similar to that described by
by PFGE distinguished nine clones among the 14
Yuan et al.The chromosomal DNA was digested
K. pneumoniae strains (The 10 E. coli and
overnight with 30 U of XbaI (Boehringer, Mannheim,
five C. diversus isolates divided into four and two
Germany). The PFGE was run in a CHEF-DRIII
clones, respectively (In each of the three
apparatus (Bio-Rad, Richmond, CA, USA) at
species, some pulsotypes were detected only once
6 V/cm, 13.5 8C during 26 h for E. coli and 28 h for
whereas others were shared by two or three
K. pneumoniae and C. diversus. The pulse times
were 5 – 40 s. The banding patterns were analysed
For each of the three species, patients carrying
with the Biogene software (Vilbert-Lourmat,
the same clone were analysed in an attempt to
determine the links between them. Cases carrying a
according to the criteria of Tenover et al.
given clone were either imported from another
The PFGE experiments were performed by one of
ward or due to the presence in the ICU of a patient
PFGE of Xba1-digested total DNA from ESBL-
producing K. pneumoniae responsible for digestive tractcolonization in 14 patients.
EcoRI restriction patterns of plasmids from
already colonized. In some instances no link could
transconjugants from ESBL-producing K. pneumoniae.
be established between cases with the same clone.
Lane 1, lamda DNA/EcoRI þ Hind III. Lanes 2 and 3
During the study five new clones were introduced in
transconjugants from clone C (isolates 9 and 11). Lane 4
the ICU by patients transferred from three different
transconjugant from clone F (isolate 6). Lane 5 transcon-jugant from clone G (isolate 10).
hospital wards. Three clones detected in patientson admission to the unit were already present in it.
Plasmid DNA analysis of ESBL producing isolates
of K. pneumoniae showed that only seven of 14
The antibiotics most frequently involved in resist-
isolates had a plasmid and four plasmid profiles
ance were gentamicin, tobramycin, cotrimoxazole,
were found. Three clones (C, F, I) had the same
tetracycline and chloramphenicol. The MICs of
plasmid profile, but the restriction enzyme studies
ceftazidime were frequently higher than those of
performed in clones C and F revealed different
cefotaxime suggesting a ceftazidimase activity
patterns. Three different plasmid profiles were
of the ESBLs.Clavulanic acid at a concentration
observed in E. coli and two in C. diversus. Three
of 4 mg/L substantially reduced the MICs of both
clones of E. coli (J, K, and M) shared a common
plasmid pattern, but the enzyme restriction profiles
Plasmid transfer by conjugation to E. coli K12
J53-2 occurred with all the E. coli isolates. This
transfer was observed for only four of sevenK. pneumoniae isolates. Plasmid restriction analysis
Molecular biology methods have improved the
from the transconjugants obtained with four
understanding of ESBL epidemiology.Specific
K. pneumoniae and five E. coli strains showed
identification of the enzyme subtype has revealed
three and five patterns, respectively (). No
the considerable diversity of ESBLs,their spread
plasmids could be transferred from the C. diversus
between bacterial species,the coexistence and
Different antibiotic resistance profiles were
variable distribution according to geographic
noted in isolates belonging to the same pulsotype
area.Precise identification of ESBL variant is
(pulsotype L of E. coli and pulsotype A of
very challengingas the reference method is
K. pneumoniae). The two isolates of E. coli
nucleotide sequencingFor the investigation of
pulsotype L had a different plasmid restriction
strain relatedness, PFGE is usually used.
The analysis of ESBLPE responsible for digestive
Among the 29 ESBLPE isolates, 10 phenotypes
tract colonization by DNA macrorestriction poly-
of resistance were detected. Most isolates, except
morphism determined by PFGE and plasmid analysis
Characteristics of extended-spectrum b-lactamase-producing Klebsiella pneumoniae, Escherichia coli and Citrobacter diversus responsible for digestive tract colonization
CTX,cefotaxime; CTX-CLA, cefotaxime þ clavulanic acid; CAZ, ceftazidime; CAZ-CLA, ceftazidime þ clavulanic acid; Ge, gentamicin; To, tobramycin; Ne, netilmicin; Ak, amikacin; Ts,trimethoprim-sulfamethoxazole; Ch, chloramphenicol; Te, tetracycline; NPD, no plasmid detected; ND, not determined.
allowed the study of the mode of spread of the
This circulation of multiple clones at one time was
ESBLs in an intensive care unit during the four-
month study, in which seven of 29 isolates had no
Essack et alwho reported on multiple strains
detectable plasmid and eight isolates failed to
prevalent simultaneously in single ICUs.
transfer their plasmids by conjugation. Although
Colonization by ESBLPE could be due to clones
only a limited number of plasmids could be
already present in the unit, or to imported clones
analyzed, the prevalent mechanism of ESBL dis-
from other hospital wards. ESBL production may
semination appeared to be the existence of
have appeared de novo in the unit, although their
multiple clones and the limited spread of a few
pre-existence cannot be ruled out as screening for
clones rather than a plasmid spread as shown by the
ESBLPE was not performed in the patients who were
different plasmid digestion profiles observed for
present at the beginning of the survey. Five new
each species. Outbreaks caused by ESBLPE have
clones were introduced in the ICU by patients
been reported as due to the dissemination of a
transferred from three separate wards, demon-
single strain,to horizontal transfer of plasmidor
strating the spread of ESBLPE in our hospital. This
to concurrent dissemination of plasmids and
finding confirms the observations that the problem
of ESBLPE is usually of hospital-wide importance
The frequencies, origin and the temporal distri-
and that spread occurs through transfer of patients
bution of the observed clones were studied. The
between wards.These results support the recom-
high number of clones suggests that ESBL pro-
mendations for screening of patients from at risk
duction has become endemic rather than epidemic.
areas on admissionand documenting the carriage
For each species studied some clones were
of multi-resistant bacteria in case of transfer of
observed only once whereas a few were isolated
from two or three patients. This finding may be
Temporal analysis of cases due to the same clone
explained by differences in isolation procedures
was used to identify the link between patients. In
among colonized patients and by the fact that the
some cases no link could be found between
colonization was transient.Similar studies of
the cases raising the question of persistence of
ESBPLE responsible for digestive tract colonization
the agents within the unit. The sensitivity of the
revealed different results depending on the epide-
method used for the detection of the ESBPLE may
miology in the unit. During an outbreak the same
have been insufficient and may explain the failure
strain was responsible for most infections and for
to detect a colonized patient who could have served
digestive tract colonization.After an outbreak of
as a reservoir. Although the environment is not
K. oxytoca infections in an intensive care unit,a
considered a major reservoir for ESBLPE, one
screening programme of gastrointestinal coloniz-
outbreak strain was isolated from equipment.
ation identified by PFGE a predominant clone and15 unrelated isolates. Cukier et alreported that
Transmission from asymptomatic colonized staff is
the ESBL-producing E. coli strain responsible for an
outbreak of urinary tract infections in a geriatric
Isolates belonging to the same clone may have a
ward had a unique ribotype, whereas after the
different plasmid digestion profile (e.g. clone L of
outbreak ESBL-producing E. coli strains responsible
E. coli), which can be explained by the possible
for colonization belonged to four different
existence of transposons or integrons or an instable
plasmid that may change easily.Conversely,
The number of clones detected in this study was
isolates of the same clone may have different
higher in K. pneumoniae (nine clones in 14 isolates)
antibiotic resistance phenotypewhich may be
than in E. coli (four clones in 10 isolates) and in
due to gain, or loss of plasmids or fragments of
C. diversus (two clones in five isolates). This finding
plasmids.This finding confirms that the antibiotic
confirms that the spread of ESBL production in
resistance profile is not a valuable marker.
K. pneumoniae is due to multiple clones rather than
In this study, despite using three methods of
to the spread of a single epidemic clone. A multi-
extraction, plasmids could be detected in only
centre study of K. pneumoniae with ESBLs con-
seven of 14 K. pneumoniae strains, and of these,
ducted in European intensive care unitsreported
only four transferred their plasmids by conjugation.
85 distinct strains in 220 isolates. Major variations
In these isolates, the conjugative plasmid was
in antibiotic resistance and plasmid patterns were
responsible for ESBL production. The absence of
noticed within strains, with some intra-strain vari-
ations in b-lactamase This result is also
reported,although in other studies all isolates
supported by the important variety of plasmids and
tested had detectable plasmidsor could transfer
types of ESBL reported in K. pneumoniae.
their plasmids.The absence of plasmids in
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