Characterization of the highly variable region surrounding the blaCTX-M-9 gene in non-related Escherichia coli from Barcelona

Aurora García1,2, Ferran Navarro1,2,*, Elisenda Miró1, Beatriz Mirelis1,2, Susana Campoy3 and Pere Coll1,2

1 Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; 2 Unitat de Microbiologia, Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; 3 Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona-Institut de Recerca i Tecnologia Agroalimentària, 08193 Bellaterra, Spain


* Correspondence address. Departament de Microbiologia, Hospital de la Santa Creu i Sant Pau, Avda Sant Antoni Ma Claret, 167, 08025 Barcelona, Spain. Tel: +34-93-291-9071; Fax: +34-93-291-9070; E-mail: fnavarror{at}santpau.es

Received 24 March 2005; returned 30 June 2005; revised 18 July 2005; accepted 30 August 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The dispersion of a clone, a plasmid or a mobile element carrying the blaCTX-M-9 gene was evaluated in 30 Escherichia coli strains isolated in Barcelona between 1996 and 1999. The presence of the previously described orf513-bearing class 1 integron, In60, carrying the blaCTX-M-9 gene, was also studied.

Methods: The clonality was analysed by pulsed-field gel electrophoresis. Plasmid analysis was performed by S1 digestion and hybridization with the CTX-M-9 probe. PCR mapping using specific designed primers was used to study the presence of In60 and In60-like structures.

Results: The clonality between the 30 strains was minor. The size of blaCTX-M-9 carrying plasmids ranged between ~80 and 430 kb. One strain produced only a chromosome-encoded CTX-M-9 ß-lactamase. Thirty-six per cent of the strains showed differences with respect to the In60 structure due to an insertion or deletion events.

Conclusions: These findings suggest that the bla CTX-M-9 gene may be carried by a mobile element that disperses it between plasmids. The fast dispersion of the CTX-M-9 enzyme could therefore be due to both diffusion of plasmids and mobile elements.

Keywords: ß-lactamases , plasmids , integrons


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The rapid dissemination of antibiotic-resistance genes and the evidence of plasmid transfer from normal gut flora to pathogenic strains1 gives the impression that the bacterial community has access to a large pool of genetically diverse plasmids. Various studies have inferred the role of integron cassette movements,2,3 transposition,4,5 homologous recombination6 and co-integration7 in plasmid evolution.

Integrons are genetic units containing elements for site-specific recombination, capture and mobilization of gene cassettes. The integrons that are most frequently found in antibiotic-resistant bacteria belong to class 1. Conserved segments flank the cassette region usually encoding resistance to various antimicrobials. The 5'-conserved region contains essential elements for insertion and mobilization of gene cassettes: the intI1 gene encoding the integrase which catalyses site-specific recombination of adjacent gene cassettes; attI, the specific gene cassette insertion site; and Pc, a promoter common for transcription of cassette resistance genes.2 The 3'-conserved segment usually contains the truncated qacE (qacE{Delta}1) gene encoding low-level resistance to disinfectants based on quaternary ammonium compounds, the sul1 gene encoding sulfonamide resistance and sometimes an open reading frame (ORF5) of unknown function.8

A novel group of orf513-bearing class 1 integrons, also called complex or unusual class 1 integrons, have been identified. These integrons contain the classic integron structure and an additional second copy of the 3'-CS. Between the two copies of the 3'-CS lies a region of 2.1 kb, called the common region,6 which is nearly identical in In6, In7 and the integron from pSAL-1, and is followed by a variable region that contains resistance genes.8,9 The frequency of integrons among clinically significant Gram-negative isolates demonstrates that these genetic structures are widespread among isolates from independent sources around the world, in diverse species, and also within the hospital environment.10 These genetic elements are generally found as part of a transposon.11,12 Most class 1 integrons are defective transposon derivatives. They present the inverted repeats but have lost part of the tni module required for transposition.1315

In 1996, an Escherichia coli strain (785-D) containing the extended-spectrum ß-lactamase (ESBL) CTX-M-9 (CTX-M-9 group) was isolated in our laboratory.16 The region surrounding the blaCTX-M-9 gene in the plasmid pMSP071 present in the 785-D transconjugant strain (MSP492) was analysed. The blaCTX-M-9 gene was located in an orf513-bearing class 1 integron, named In60 (accession number AF174129).17 Between the 5'-CS and the first 3'-CS region, the In60 presents two gene cassettes, the drfA16 and the aadA2, and between the 3'-CS regions there is a 6.4 kb DNA fragment that includes the blaCTX-M-9 gene, the orf3-like region, and the orf1005 gene (transposase-like region) within IS3000.

The aim of the present work was to determine whether the In60 was a variable or a stable structure and whether the blaCTX-M-9 gene was always carried in the same plasmid. This study was performed with thirty CTX-M-9 ß-lactamase-carrying strains collected over a 4 year period (1996–99) in our laboratory.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The present study included 30 E. coli strains carrying the CTX-M-9 ß-lactamase. They were obtained between 1996 and 1999 from a study performed in our hospital based on the detection and characterization of ESBLs. Over this period, the ß-lactamase CTX-M-9 represented 41% of the total ESBLs and these 30 E. coli made up 0.29% of total E. coli isolated. The strains were isolated from 23 urine samples, two blood samples, one catheter, three wound exudates and one abdominal drainage exudate, from 30 patients (19 inpatients and 11 outpatients) admitted to Hospital de la Santa Creu i Sant Pau, Barcelona.

Conjugation

Filter-mating experiments were performed with all 30 strains and E. coli HB10118,19 (kanamycin-resistant due to the insertion of a Tn5-derived transposon, lactose-negative and plasmid-free) was the recipient. Donor and recipient strains at the logarithmic phase were grown in TSB (trypticase soy broth) and were mixed and incubated on solid medium at 37°C for 20 h. Transconjugants were selected on the Mueller–Hinton medium supplemented with 100 mg/L ampicillin (Sigma, Madrid, Spain) and 50 mg/L kanamycin (Sigma, Madrid, Spain).

Strain typing

Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with XbaI using orthogonal field alternating gel electrophoresis (Amersham Biosciences, Little Chalfont, England) was carried out as previously described.20 The running conditions were as follows: 24 h at constant voltage of 250 V and temperature of 12°C, with a pulse time of 20, 10 and 2 s for 8, 10 and 6 h, respectively. Lambda ladder PFGE marker (New England Biolabs, Beverley, MA, USA) was used as a standard marker.

Plasmid DNA analysis

PFGE with S1 nuclease (Roche Diagnostics GmbH, Mannheim, Germany) digestion of whole genomic DNA was used to detect large plasmids in E. coli strains as described by Barton et al.21 S1-digested slices were applied into wells in agarose gel and run in a CHEF-DRIII device (Bio-Rad, Hercules, USA) with a pulse time of 5–25 s for 6 h, and 30–45 s for 18 h at 6 V/cm, and temperature of 14°C. Lambda ladder PFGE marker (New England Biolabs) was used as a standard marker. The approximate plasmid size was determined by comparison with linear DNA markers and analysed by Whole Band Analyzer System (Bio Image Applications, Millipore, MI, USA); each band was considered a unit length linear plasmid.

Restriction and hybridization patterns

The PFGE gels with XbaI-digested DNA were blotted onto nylon membranes (Hybond N+; Amersham Biosciences) and hybridized with a PCR-generated 855 bp probe for the blaCTX-M-9 gene (CTX-M-9 probe) (using primers IATG and ISTOP, Table 1). The PFGE gels with S1-digested DNA were transferred and hybridized with two different probes, the CTX-M-9 probe and a PCR-generated 315 bp probe for the blaTEM gene (TEM probe) (primers P5 and P6, Table 1). The ECL kit (Amersham Biosciences) was used for probe labelling, hybridization and detection of results following the manufacturer's protocol.


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Table 1. Primer sequences used for PCR analysis of In60

 
Chromosomal location of the blaCTX-M-9 gene

To identify the possible chromosomal location of the blaCTX-M-9 gene, endonuclease I-CeuI (New England Biolabs) was used to cut a 26 bp site in the rrn genes, coding for the 23S large subunit rRNA. Digested DNA plugs were analysed on a CHEF-DRIII device, as described previously.22 The fragments generated with I-CeuI were determined by comparison with those of a bacteriophage lambda DNA molecular weight marker (New England Biolabs). The restricted fragments were transferred onto a nylon membrane that was hybridized with internal fragments obtained by PCRs specific for the 16S rRNA gene (with primers 16S-27f and 16S-907f) and with the CTX-M-9 probe, mentioned above (Table 1).

In60 PCR amplification

PCR mapping of the integron was carried out on total DNA in an iCycler (Bio-Rad) thermal cycler. Oligonucleotides used for PCR mapping and their annealing temperatures are listed in Table 1. Overlapping amplification products were analysed on a 0.8% agarose gel and subsequently exposed to UV light in the presence of 0.5% ethidium bromide.

DNA sequencing and sequence analysis

Sequencing was performed by Macrogen (Macrogen, Inc., Seoul, Korea). The nucleotide and the deduced protein sequences were analysed using the Lasergene DNASTAR software package (GATC Biotech, Cambridge, UK) and the software available over the Internet at the National Centre of Biotechnology Information web site http://www.ncbi.nlm.nih.gov.

Nucleotide sequence accession numbers

The sequences of partial aadA2, ISEc8 and IS26 insertions were deposited in the GenBank nucleotide sequence database under accession numbers AY512972, AY557606 and AY557605, respectively.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Conjugation

Transconjugants were obtained for 13 of the 30 evaluated strains. The range of conjugation frequencies was of 1.4 x 10–3 and 2.7 x 10–9 transconjugants per donor cell. All 13 transconjugants produced the cefotaxime-hydrolysing ß-lactamase CTX-M-9.

Strain typing

Isolates with more than six differences in the restriction pattern were assigned to a specific XbaI PFGE type. A total of 27 distinct DNA types were observed among the 30 E. coli strains studied (Table 2). Two couples of strains differed in six bands, and were considered as probably related (named as 2a, 2b and 13a, 13b) according to Tenover et al.23 (Table 2). One E. coli isolate could not be typed by PFGE due to degradation of the genomic DNA during preparation of the agarose plugs, as described by other authors.2426


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Table 2. PFGE and hybridization results

 
Restriction and hybridization patterns

When the PFGE gels, with the XbaI-digested genomic DNA, were blotted and hybridized with the CTX-M-9 probe, a high variability of band size was observed. The fragment size with a positive signal ranged from ~60 to 310 kb. One sample (836-D) presented two hybridization bands of ~80 and 180 kb.

Analysis of plasmid and chromosomal blaCTX-M-9 gene location

For size determinations, the DNA plugs for the PFGE analysis were treated with S1 nuclease to linearize circular plasmids. Almost all tested E. coli strains harboured 1–4 plasmids with sizes of ~50–430 kb. In order to localize the gene encoding the CTX-M-9 ß-lactamase the S1-digested DNAs run using PFGE were hybridized with the CTX-M-9 probe (Figure 1 and Table 2). A high variability was observed. Plasmids, considered as blaCTX-M-9 hybridization bands, ranged between ~80 and 430 kb. Two strains, 858-D and 1213-D, presented two hybridizing bands, and the DNA of the 1277-D strain did not hybridize (Table 2). This strain contained the blaCTX-M-9 gene according to the susceptibility pattern, isoelectric focusing (data not shown), PCR and sequencing study. The same approximation was performed with three transconjugant strains (MSP492, MSP508 and MSP509). All these showed the same donor hybridizing band with the exception of transconjugant MSP492, which presented a higher band than its donor.



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Figure 1. (a) Mobility of plasmids from several strains carrying the blaCTX-M-9 gene after S1-digestion and PFGE. (b) Blotting and hybridization with the CTX-M-9 probe. (c) Blotting and hybridization with the TEM probe. Arrows indicate a condensed band of total DNA.

 
These S1-digested DNAs were also hybridized with the TEM probe (Figure 1 and Table 2). Eight strains (836-D, 858-D, 909-D, 1104-D, 1129-D, 1130-D, 1213-D and 1334-D) presented two hybridization bands, three strains (814-D, 1277-D and 1406-D) presented three bands and DNA of three strains (785-D, 1068-D and 1292-D) did not hybridize with this probe. As expected according to their donor strains, the transconjugants MSP508 and MSP509 hybridized with the TEM probe but MSP492 did not. DNA restriction with I-CeuI enzyme was used to search for a chromosomal location of the blaCTX-M-9 gene (data not shown). A PFGE with the I-CeuI-digested DNA was performed and transferred onto a nylon membrane. All restriction fragments of all strains hybridized with the 16S rRNA-specific probe, whereas only one of the observed fragments, that corresponding to chromosomal DNA, also hybridized with the CTX-M-9 probe in the case of the 1277-D strain.

PCR amplification and sequencing analysis of the In60

Using primers 5–7 (Table 1) the PCR for blaCTX-M-9 gene was positive in all samples and 100% identical to blaCTX-M-9 gene (accession number AF174129) after sequencing. Analysis of the In60 structure (Figure 2a) by PCR mapping using the primers 1 and 2, 3–7, 8 and 9, 10 and 11, 19 and 20 (Table 1) determined that 19 of 30 strains had the expected regions. The remaining 11 isolates (1104-D, 1334-D, 1226-D, 1383-D, 1185-D, 1249-D, 1252-D, 1266-D, 1290-D, 1292-D and 1327-D) were PCR-negative or had an unexpected amplicon for some of these overlapping fragments (Figure 2b). The 1104-D strain had an insertion of 2442 bp which was amplified and sequenced, and was 99% identical to ISEc8.27 The 1334-D strain had an insertion of 820 bp, situated within IS3000, whose nucleotide sequence was 100% identical to IS26.28 The 1226-D strain had an additional partial aadA2 gene cassette before the second copy of 3'-CS. This insertion was amplified and sequenced. The fragment obtained remained 100% homologous with the aadA2 gene stored under accession number AY259085.29 In the case of the 1383-D strain, the region downstream of the orf513 gene was conserved, including the recognition site for the putative transposase Orf513, whereas the upstream region of this hot spot was missing. The seven remaining strains (1185-D, 1249-D, 1252-D, 1266-D, 1290-D, 1292-D and 1327-D) (Figure 2b) had the In60 structure downstream of the blaCTX-M-9 gene. However, upstream of this bla gene, the In60 was truncated somewhere inside the orf513 gene. The only exception was the 1185-D strain in which the orf513 gene was complete (PCR 3–7 positive, Table 1). In these strains the blaCTX-M-9 gene is probably not included in an orf513-bearing class 1 integron.



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Figure 2. (a) Schematic presentation of integron In60 carrying the blaCTX-M-9 gene in MSP492 strain (accession number AF174129). (b) Schematic representation of strains differing in their structure (numbers indicate the strain reference). Open arrows, ORFs; open box, the region showing high similarity to orf3 of K. ascorbata; filled black circles, 59-be; vertical black box, the recombinant site attI1; grey arrows, insertions; filled black diamonds, hypothetical recombination region. Superscript ‘a’ indicates presence of orf5 and orf6 genes downstream of the sul1 gene. Superscript ‘b’ indicates this strain presented the complete orf513 gene.

 
The second copy of 3'-CS was analysed by PCR by using primers 12–21 (Table 1). Only one strain, MSP492 (785-D transconjugant), obtained the expected 1269 bp amplicon. Another strain (1226-D) presented an unexpectedly larger 1664 bp PCR fragment. In the other 28 strains, the amplicon size was smaller than expected (900 versus 1269 bp). A preliminary study of the region surrounding In60 was performed. Overlapping PCRs were tested between IS3000 and orf5, orf6 and IS6100 (orf513-bearing class 1 integron-related genes) with primers 12–22, 12–23 and 12–24, respectively (Table 1). Only the 1226-D strain was positive for 12–22 and 12–23 PCRs, suggesting the presence of orf5 and orf6. The PCR between IS3000 and IS6100 was negative in all strains in the study.


    Discussion
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 Materials and methods
 Results
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 References
 
Over a 4 year period, a total of 30 E. coli CTX-M-9 ß-lactamase-producing strains were collected in our hospital. With the exception of two Salmonella enterica strains30 (not included in this study), no CTX-M-9 ß-lactamase-producing species other than E. coli were detected. Sixty-three per cent of these isolates were obtained from hospitalized patients in 13 different departments and the rest were from outpatients. There were no epidemiological relationships between these isolates and this was confirmed by the lack of similarity in their PFGE patterns. This suggests that a plasmid and perhaps a mobile element (transposon) are involved in the fast spread of the blaCTX-M-9 gene. The present study showed that blaCTX-M-9 carrying plasmids could be conjugatively transferred. The blaCTX-M-9 gene has been isolated from E. coli in France (1994), from E. coli and S. enterica serovar Virchow in Spain (1996), from E. coli, Klebsiella pneumoniae and Enterobacter cloacae in China (1997) and from E. coli in Brazil (1998).31

Hybridization experiments with the CTX-M-9 probe on the 30 strains, previously digested with XbaI, showed a high variability. Several hybridization bands of different molecular weights were obtained. The blaCTX-M-9 gene, therefore, seemed to be located either at different positions within the same plasmid or in different plasmids. This blaCTX-M-9-carrying plasmid variability was confirmed by S1 digestion, PFGE and hybridization with the CTX-M-9 probe (Figure 1 and Table 2). The size of the plasmids carrying the blaCTX-M-9 gene ranged from ~80 to 430 kb, in contrast with data obtained with other blaCTX-M-carrying smaller plasmids (7–160 kb).3133 Some of these blaCTX-M-9-carrying plasmids also hybridized with the TEM probe (Table 2). The presence of these two ß-lactamases on the same plasmid has also been reported by other authors.32

The presence of the blaCTX-M-9 gene in two different weight plasmids in the same strains (858-D and 1213-D) and the only chromosomal location of the blaCTX-M-9 gene in the 1277-D strain (confirmed by DNA I-CeuI restriction, PFGE and hybridization with the 16S rRNA and CTX-M-9 probes) may suggest that a transposon mobilizes this gene. However, we were unable to detect the In60 transposition in 785-D and MSP492 strains using killer-trap vectors according to a previously reported strategy34 (data not shown). Some CTX-M and Toho ß-lactamases were also observed in the chromosome.3537 The presence of several plasmids within a single isolated E. coli strain suggests that they belong to several incompatibility groups.

Several mobile elements may contribute to disseminating resistance genes among DNA replicons.29,38,39 Genetic arguments show the mobilization of natural blaCTX-M genes from the Kluyvera ascorbata and Kluyvera georgiana chromosomes to plasmids. Insertion sequences such as ISEcp1 and ISEcp1-like may play an important role in the mobilization of blaCTX-M genes, such as blaCTX-M-2,-3,-5,-10,-15,-17,-18,-19,-25,-26,-27.31,40,41 IS26, ISEc8, IS10 and IS6100 have been associated with other resistance genes.31,4246 In the present study, the IS26 was inserted within the IS3000 in the 1334-D strain, and the insertion sequence ISEc8 was located upstream of the IS3000 in the 1104-D strain.

Other important genetic elements in the mobility of resistance genes are the integrons. The blaCTX-M-9 and blaCTX-M-2 genes are situated in an orf513-bearing class 1 integron also called complex or unusual class 1 integron (In60, In35 and InS21).17,47,48 The blaCTX-M-9 gene, in the MSP492 strain, was located in In60. When this structure was analysed from integrase to IS3000 in the 30 strains of this study, 36% showed changes in this environment. Eight of these strains presented deletions upstream of the blaCTX-M-9, whereas downstream of this gene, near or within IS3000, one strain presented a deletion and three strains presented insertions. Other deletion and recombination events in orf513-bearing class 1 integrons have been reported by Arduino et al.47 and Boyd et al.49 These authors described the deletion of the orf513 gene together with the resistance gene dfrA10. Moreover, the putative transposase Orf513 may be involved in its mobility and in the acquisition of different genes as in In6 and In7.9

The In60 structure described for pMSP071 (accession number AF174129), 785-D transconjugant, turned out to be different from the structure shown for its donor strain. Downstream of IS3000, qacE{Delta}1 and sul1 genes were present in pMSP071, but when these two genes were screened by PCR mapping for the donor strain, the PCR fragments obtained were smaller than expected. These amplified fragments did not correspond to the qacE{Delta}1 and sul1 gene, suggesting reorganization in this region. The donor strain, 785-D, presented two plasmids: one, of ~270 kb, hybridized with the CTX-M-9 probe whereas the other, of ~110 kb, did not hybridize either with CTX-M-9 or with TEM probes (data not shown). Nevertheless, its transconjugant presented only one plasmid (pMSP071) of ~370 kb that hybridized with the CTX-M-9 probe. The size of this plasmid suggested a possible co-integration phenomenon in which the second copy of the 3'-CS region could be added by recombination. The In60 structure in pMSP071 could thus be the result of an in vitro reorganization. Moreover, the In60 structure as previously described has only been partially detected in some of the studied strains.

The apparent genetic divergence between plasmids carrying the blaCTX-M-9 gene and the variability in the genetic surrounding of this gene may perhaps be due to DNA acquisition and/or deletion events (plasmid evolution). Such events have been extensively demonstrated in various studies. It has also been observed that the environmental closely-related plasmids may recombine in the plasmid backbone.50,51 Moreover, the high variability observed in the region surrounding the blaCTX-M-9 gene suggests that structures such as IS3000, or orf513 and its hot spot, may play an important role in the evolution of this region. These events may enhance plasmid diversity and bacterial adaptability.

In conclusion, a high variability has been observed within the regions surrounding the blaCTX-M-9 gene and also between the sizes of the plasmids that carry it. Although the evolution of plasmids could partially explain these differences, the fact that the blaCTX-M-9 gene has been observed within the chromosome of one strain suggests that a mobile element may be involved. The fast diffusion of CTX-M-9 enzyme in our city could therefore be due to both diffusion of plasmids and mobile elements.


    Acknowledgements
 
We wish to thank Professor J. Barbé for his helpful advice and critical review of the manuscript prior to submission. This study was partially supported by grants PI020368, 97/0623 and the network ‘Red Española de Investigación en Patología Infecciosa’ (REIPI; C03/14) from the ‘Fondo de Investigaciones Sanitarias de la Seguridad Social de España’.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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