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International Journal of Antimicrobial Agents 29 (2007) 89–92

Short communication

Analysis of plasmids encoding extended-spectrum �-lactamases (ESBLs)from Escherichia coli isolated from non-hospitalised patients in Seville

Carmen Velasco a,∗, Luisa Romero a, Jose Manuel Rodrıguez Martınez a,Jesus Rodrıguez-Bano b, Alvaro Pascual a

a Department of Microbiology, Universidad de Sevilla, Spainb Seccion de Enfermedades Infecciosas, Hospital Universitario Virgen Macarena, Sevilla, Spain

Received 19 June 2006; accepted 1 August 2006

bstract

Extended-spectrum �-lactamase (ESBL)-producing Escherichia coli is an emerging pathogen in the community. We investigated the specificypes of ESBLs in E. coli isolates from non-hospitalised patients and the plasmids harbouring these ESBLs. Forty-eight consecutive ESBL-roducing E. coli were studied. All the isolates were clonally unrelated. ESBLs were characterised by sequencing. Mobility of the undigestedlasmids was investigated both by conventional electrophoresis and by pulsed-field gel electrophoresis. Southern restriction fragment lengtholymorphism (RFLP) experiments were carried out by digesting plasmids with EcoRI and HindIII. CTX-M-14 was the most frequent ESBL

28 isolates), followed by SHV-12 (16 isolates). Plasmids encoding CTX-M-14 appeared homogeneous both in hybridisation profile andobility. In contrast, plasmids expressing SHV-12 were more varied in size and hybridisation patterns. Our data suggest that a single type or

ery closely related plasmids harbouring CTX-M-14 are spreading in the community among unrelated E. coli isolates in our area.2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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eywords: Escherichia coli; Plasmids; ESBL; CTX-M-14; SHV-12

. Introduction

Extended-spectrum �-lactamase (ESBL)-producingnterobacteria are resistant to penicillins, narrow- andxtended-spectrum cephalosporins and aztreonam. Theserganisms are also frequently resistant to aminoglycosides,rimethoprim/sulphamethoxazole and quinolones [1]. Untilecently, most infections caused by ESBL-producing organ-sms had been described as nosocomially acquired or nursingome related. However, in recent years, ESBL-producingscherichia coli (ESBLEC), particularly those producingSBLs from the CTX-M family, has emerged as a relevantause of infections in non-hospitalised patients worldwide

2,3]. This problem is also present throughout Spain [4].

ost of these community ESBLEC are clonally unrelated

∗ Corresponding author. Present address: Department of Microbiology,chool of Medicine, Av. Doctor Fedriani s/n, 41071 Seville, Spain.el.: +34 954 552 863; fax: +34 954 377 413.

E-mail address: cvelasco@us.es (C. Velasco).

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924-8579/$ – see front matter © 2006 Elsevier B.V. and the International Societyoi:10.1016/j.ijantimicag.2006.08.027

5–7], although clonal spread has been described in theommunity in some areas [7,8].

Our group has recently described the epidemiologynd clinical features of infections due to ESBLEC inon-hospitalised patients in our area [5–9]. These studiesescribed the emergence of ESBLEC in our area and observedhat most of the strains were CTX-M-producers. Whennvestigated by repetitive extragenic palindromic polymerasehain reaction (REP-PCR), none of the E. coli isolates werelonally related. To investigate further the molecular epidemi-logy of infections caused by ESBLEC in non-hospitalisedatients, we characterised the ESBLs and analysed the plas-ids encoding the most prevalent ESBLs in E. coli.

. Materials and methods

.1. Characterisation of ESBLs

Forty-eight ESBLEC isolated from outpatients betweenanuary 2001 and May 2002 in Seville, Spain, were studied

of Chemotherapy. All rights reserved.

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5]. Sequencing was used to characterise the ESBLs. Primerairs used both to amplify and then sequence the ESBL genesave been described previously [10,11].

.2. Plasmid analysis

Transconjugants were obtained using E. coli J53 Azr ashe recipient strain, but only those expressing one ESBL weresed for plasmid analysis.

Plasmids were extracted according to the protocolescribed by Kado and Liu [12]. Mobility of undigested plas-ids was first investigated by conventional electrophoresis on

.7% gels and by pulsed-field gel electrophoresis (PFGE) fol-owed by Southern hybridisation. Plasmids were run in PFGECHEF-DR II; Bio-Rad, Madrid, Spain): running time, 8 h;witching time, 1 s to 6 s; voltage, 6 V/cm. Supercoiled DNAEpicentre, Madison, WI) was used as a molecular weighttandard (see Fig. 2). Once separated, gels were blotted andybridised with the probes described below to facilitate loca-ion in the gels.

Southern restriction fragment length polymorphismRFLP) experiments were carried out by digesting plasmidsith EcoRI and HindIII. Digestion products were separatedy electrophoresis and then transferred to membranes fol-owing standard methodology [13]. A lambda HindIII linearNA marker was also included. PCR fragments of 856 bp

CTX-M-14) and 930 bp (SHV-12) were used as templates forabelling using DIG High Prime DNA Labelling kit (Roche,arcelona, Spain). Primer pairs used to amplify and label therobes have been described previously [10,11].

. Results and discussion

As determined by sequencing, 45 of 48 strains expressedne ESBL: CTX-M-14 (n = 26 isolates), CTX-M-9 (n = 1),

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ig. 1. Analysis of plasmids encoding CTX-M-14 extended-spectrum �-lactamasesed as the probe for hybridisation. (A1) Lanes 1–24, plasmids digested with EcoRI.3 and 2 kb). (A2) Southern hybridisation corresponding to (A1).

timicrobial Agents 29 (2007) 89–92

HV-12 (n = 15), TEM-type (n = 1) and CTX-M-type (n = 2).hree isolates expressed two ESBLs: CTX-M-9 + SHV-12

n = 1) and CTX-M-14 + TEM-type ESBL (n = 2).Transconjugants were obtained for 40 strains using E. coli

53 Azr as the recipient strain, but only those expressing oneSBL (24 encoding CTX-M-14 and 12 encoding SHV-12)ere used for plasmid analysis.All plasmids carrying blaCTX-M-14 showed very similar

obility in gel when separated by conventional electrophore-is or by PFGE, with sizes between 95 kb and 120 kb (dataot shown). In all these plasmids, Southern RFLP analy-is revealed a single 20 kb hybridisation band when usinglasmid DNA digested with EcoRI (Fig. 1). Southern RFLPigestion with HindIII was also performed, producing theame result, a single 20 kb band in all cases (data not shown).hese data indicate that plasmids carrying blaCTX-M-14re at least very closely related. In contrast, the 12 plas-ids encoding SHV-12 were much less homogeneous inobility, both by conventional electrophoresis and PFGE

Fig. 2A), with sizes ranging from 60 kb to 160 kb. More-ver, Southern RFLP analysis of these plasmids revealedeterogeneous banding patterns (Fig. 2B). These data sug-est that plasmids carrying the SHV-12 gene are structurallyiverse.

Our results agree with data from other areas of the country14] and confirm that CTX-M-14 is the most frequent ESBLroduced by E. coli in outpatients in Spain, followed by SHV-2. Clonal dissemination of ESBLEC producing ESBLs fromhe CTX-M family in the community has been describedreviously in Canada and the UK [7,8], but the explosivemergence of CTX-M-producing E. coli in our area is notxplained by horizontal transmission of strains, as all strains

n our area were clonally unrelated [5].

The high similarity of plasmids encoding CTX-M-14trongly suggests that plasmid transmission among clonallynrelated strains of CTX-M-14-producing E. coli might be

s by Southern restriction fragment length polymorphism. CTX-M-14 was; lane 25, lambda HindIII DNA molecular weight marker (23, 9.4, 6.6, 4.3,

C. Velasco et al. / International Journal of Antimicrobial Agents 29 (2007) 89–92 91

Fig. 2. Analysis of plasmids encoding SHV-12 extended-spectrum �-lactamases. Southern hybridisation of undigested plasmids encoding SHV-12 separatedby pulsed-field gel electrophoresis (PFGE) and further hybridised with the probe SHV-12 (A) and Southern restriction fragment length polymorphism (RFLP)of these plasmids digested with either EcoR1 (B1) or HindIII (B2). SHV-12 was used as the probe for all hybridisations. (A1) PFGE of undigested plasmidsand (B2) Southern hybridisation corresponding to (A1). Lanes 1–12, undigested plasmids in (A1) and (A2); lane 13, 23 kb linear molecular weight marker,i Madiso( ted withl hybridi

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ndicated by an asterisk; lane 14, supercoiled marker DNA from Epicentre (B1) Lanes 1–12, bands obtained after hybridisation of the plasmids digesambda HindIII molecular weight marker; lanes 2–13, bands obtained after

aking place in our area, whilst the diversity among plasmidsncoding SHV-12 would rule out a common source of theselasmids. Thus, our results also strongly suggest that twoifferent epidemiological situations are co-existing amongSBLEC in our area.

In summary, CTX-M-14 is the most frequent ESBL pro-uced by E. coli isolated from non-hospitalised patients inur area, followed by SHV-12. Plasmids encoding CTX-M-4 enzymes appear to be very closely related, suggesting thathey could share a common origin, whilst plasmids encodingHV-12 are more heterogeneous. These data are indicative ofubstantial epidemiological differences between both typesf ESBLs.

cknowledgments

This study was partially supported by grant 75/04 fromonsejerıa de Salud (Junta de Andalucıa, Spain) and thepanish Network for Research in Infectious Diseases (REIPI,nstituto de Salud Carlos III, Spain) CO3/14. The authorshank Dr Luis Martınez-Martınez for critical review of the

anuscript.

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