Doi:10.1016/j.jhin.2004.01.007

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: mbenbachir@wanadoo.net.ma 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.
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