Journal of Antimicrobial Chemotherapy

JAC Advance Access originally published online on November 16, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):14-23; doi:10.1093/jac/dki398

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Published by Oxford University Press 2005

DNA sequence analysis of the genetic environment of various bla_CTX-M genes

C. Eckert¹, V. Gautier¹ and G. Arlet^1,2,*

¹ Laboratoire de Bactériologie, UPRES EA 2392, Faculté de Médecine Pierre et Marie Curie, Université Paris VI, 27 rue de Chaligny, 75012 Paris, France; ² Service de Bactériologie-Hygiène, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, 4 rue de la Chine, 75970 Paris cedex 20, France

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^* Corresponding author. Tel: +33-1-56-01-70-18; Fax: +33-1-56-01-61-08; E-mail: guillaume.arlet{at}tnn.aphp.fr

Received 7 July 2005; returned 4 September 2005; revised 19 September 2005; accepted 5 October 2005

	Abstract

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Objectives: Over a 3 year period (2000–2003) 21 Escherichia coli, 5 Klebsiella pneumoniae, 1 Serratia marcescens and 1 Proteus mirabilis producing CTX-M-type ß-lactamase were collected from five different hospitals in Paris, France. This study was conducted to analyse the genetic environment of these 28 bla_CTX-M genes.

Methods: Antimicrobial susceptibility testing was performed by the disc diffusion method and MICs of various ß-lactams were determined by an agar dilution method. PCR was used to detect and sequence alleles encoding CTX-M, TEM, SHV and CMY enzymes. The genetic environment was analysed by amplification and direct sequencing using various set of PCR primers or cloning in pBK-CMV.

Results: Sequence analysis revealed that these isolates contained seven different bla_CTX-M genes: bla_CTX-M-1 (4 strains), bla_CTX-M-2 (2 strains), bla_CTX-M-3 (4 strains), bla_CTX-M-9 (1 strain), bla_CTX-M-14 (5 strains), bla_CTX-M-15 (11 strains), bla_CTX-M-24 (1 strain). TEM-1 was associated with CTX-M-type enzymes in 15 isolates. Two strains produced both CTX-M-15 and SHV-2 or CTX-M-14 and CMY-2. In 25 strains the insertion sequence ISEcp1 was located upstream of the 5' end of the bla_CTX-M gene. Among these strains, in five isolates, ISEcp1 was disrupted by insertion sequences such as IS26 (in three of them) or IS1 or IS10. Insertion sequence IS903 was found downstream of bla_CTX-M-14 or bla_CTX-M-24. Examination of the other three bla_CTX-M genes (two bla_CTX-M-2 and one bla_CTX-M-9) by cloning, sequencing and PCR analysis revealed the presence of complex Class 1 integrons, In35, an integron similar to In60 and a novel integron.

Conclusions: This work further confirmed the predominant role of ISEcp1 in the mobilization of bla_CTX-M genes of the CTX-M-1 cluster and the presence of In35, of an integron similar to In60 and a novel complex Class 1 integron.

Keywords: ESBLs , CTX-M ß-lactamases , insertion sequences , integrons

	Introduction

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The CTX-M-type ß-lactamases represent a rapidly emerging group with a typical extended-spectrum ß-lactamase (ESBL)-resistance phenotype but non-TEM/non-SHV derivatives.^1–3 These enzymes, encoded by transferable plasmids, exhibiting extended-spectrum activities, are capable of hydrolysing some broad-spectrum cephalosporins and are inhibited by clavulanic acid and tazobactam. They have a preferential hydrolysis of cefotaxime over ceftazidime. This family of plasmid-mediated ESBLs has been classified in Ambler class A and in group 2be of the Bush, Jacoby and Medeiros classification.^1–3 The first two CTX-M-type enzymes were isolated in Europe and in Argentina in 1989.^4,5 So far, the number of variants described have increased rapidly since 1995, and it is now a major concern in many areas of the world. This group of CTX-M-type ß-lactamases (>30 enzymes) (www.lahey.org/studies/) have been found predominantly in Enterobacteriaceae, most prevalently in Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Salmonella typhimurium.^1–3 In some countries, CTX-M-type enzymes are the ESBLs most frequently isolated from E. coli strains.^6,7 They have been involved in several outbreaks in long-term care facilities and are also becoming a problem in the community.^8–14

These enzymes have been classified into five major groups by amino acid sequence similarities and these are clusters of CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25.³ Two of them, CTX-M-2- and CTX-M-8-types, were shown to be similar (95–100%) to the chromosomally encoded ß-lactamase of Kluyvera ascorbata and Kluyvera georgiana, respectively.^15,16 The natural ß-lactamase KLUC-1 of Kluyvera cryocrescens strain has also been characterized and this enzyme shares only 85–86% identity with the most closely related CTX-M, which belong to the CTX-M-1 group.¹⁷ Recently, two genes identical with bla_CTX-M-3 and bla_CTX-M-14 were detected in the chromosome from a K. ascorbata strain from Argentina and from six strains of K. georgiana isolated in Guyana, respectively.^18,19

Different elements may be involved in the mobilization and expression of bla_CTX-M genes. In clinical isolates, CTX-M-encoding genes have been found on a number of plasmids; some of them are part of transposons or constitute cassettes in integrons.³ Insertion sequences, especially ISEcp1, have repeatedly been found adjacent to genes encoding some of these enzymes.^20–26 Insertion sequences such as IS26 and IS903 have also been found flanking the open reading frame region of the bla_CTX-M genes.^21,24 Unusual Class 1 integrons, similar to In6 and In7, have been reported to carry antibiotic resistance genes such as bla_DHA-1. These complex Class 1 integrons with similar genetic organizations contain the 5' conserved segment (5'-CS) and a partial duplication of the 3' conserved segment (3'-CS); they have an ORF513 between these two 3'-CS. CTX-M-9-encoding genes have been recently characterized as a part of a novel complex sul1-type integron designated In60, including the bla gene and its downstream nucleotide sequence.²⁷ bla_CTX-M-2 and the surrounding DNA, which includes ORF513, have been reported in the complex sul1-type integron, designated InS21 and In35.^28–30 Here we describe the genetic environment of 28 acquired bla_CTX-M genes in strains recovered from the Paris area.

	Materials and methods

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Bacterial strains and plasmids

The 28 strains of Enterobacteriaceae studied are listed in Table 1; they include 21 E. coli, 5 K. pneumoniae, 1 Serratia marcescens and 1 P. mirabilis. They were isolated in five different hospitals in the Paris area between 2000 and 2003. They were identified with API-20 E systems (bioMérieux SA, Marcy l'Étoile, France). E. coli J53-2 rifR (pro met Rif^r) and E. coli DH10B (Invitrogen SARL, Cergy-Pontoise, France) were used for resistance transfer assays (conjugation and electroporation). The plasmid pBK-CMV km^R (Stratagene, La Jolla, CA, USA) was used for cloning experiments.

View this table: [in this window] [in a new window]   Table 1.. Details of cefotaxime-resistant clinical strains isolated from five Parisian hospitals

 
Antibiotic susceptibility

Susceptibility testing was performed using the disc diffusion test on Mueller–Hinton (MH) agar (Bio-Rad, Marnes-la-Coquette, France). The MICs of selected ß-lactams, including penicillins and cephalosporins with and without ß-lactamase inhibitors (clavulanic acid 2 mg/L or tazobactam 4 mg/L), were determined by an agar dilution technique. An inoculum of 10⁴ cfu per spot was delivered with a multipoint inoculator. ESBLs were detected by using the standard double-disc synergy test.

ß-Lactam-resistance transfer assays and epidemiological studies

Mating experiments were performed using E. coli J53-2 (met, pro, Rif^R). Volumes of cultures (1 mL) of each donor and rifampicin-resistant E. coli recipient strain grown in trypticase soy broth (Bio-Rad) were mixed and incubated for 18 h at 37°C. Transconjugants were then selected on Drigalski (Bio-Rad) agar plates containing rifampicin (250 mg/L) and cefotaxime (2.5 mg/L).

Plasmid DNA was isolated by using Takahashi's method,³¹ and 2 µL was transformed into 20 µL of E. coli DH10B cells by electroporation according to the manufacturer's instructions (Bio-Rad). Transformants were incubated for 1.5 h at 37°C and mated on Drigalski medium supplemented with cefotaxime (2.5 mg/L).

Rep-PCR and ERIC-PCR were performed with primers rep-1R and rep-2T and with primer ERIC-2, respectively as previously described.²⁵ The resulting products were run in 1.5% agarose gel in TBE buffer.

Characterization of ß-lactamase-encoding genes

Detection of gene sequences coding for the TEM-, SHV-, CMY- and CTX-M-type enzymes was performed by PCR on DNA extracted by using a commercial genomic DNA purification kit (QIAampDNA Mini Kit, QIAGEN, Courtaboeuf, France). The detection of SHV-type enzymes was performed in all clinical isolates (except for the K. pneumoniae isolates) and for their transconjugants and their transformants. The oligonucleotide primer sets specific for the ß-lactamase genes used in the PCR assays were already described.²⁵

All PCR products obtained from clinical isolates were sequenced twice on both strands with an Applied Biosystems sequencer (model ABI 377), by the dideoxy chain termination method of Sanger et al.³² The nucleotide sequences and deduced protein sequences were analysed with the BLAST and Clustal W programs (multiple sequences alignment, pairwise comparisons of sequences and dendograms).^33,34

Genetic environment of bla_CTX-M genes

The genetic organization of bla_CTX-M was investigated by PCR and by sequencing the regions surrounding these genes. Primers used to investigate the surrounding regions of the bla_CTX-M are described in Table 2.

View this table: [in this window] [in a new window]   Table 2.. Oligonucleotides used for PCR amplification and DNA sequencing the bla genes and their surrounding regions

 
ß-Lactamase gene cloning was performed for four strains with plasmid DNA digested and ligated in the EcoRI, BamHI or HindIII (New England Biolabs Inc. Ozyme, Saint Quentin en Yvelines, France) site of the phagemid pBK-CMV (Stratagene). E. coli DH10B was transformed by electroporation. The transformants harbouring the recombinant CTX-M-encoding plasmids were selected on MH agar supplemented with cefotaxime (2.5 mg/L) and kanamycin (25 mg/L). The molecular sizes of the inserts were estimated from the results of restriction digestion and electrophoresis in 1% agarose gel in TBE buffer. Finally, inserts were investigated by sequencing with M13 universal sequencing primers and then by PCR and sequencing if necessary.

Nucleotide sequence accession numbers

The nucleotide sequences reported in this work have been deposited in EMBL nucleotide sequence database under accession numbers AJ972953 [GenBank] for E. coli TN21, AJ972954 [GenBank] for E. coli TN23, AJ972955 [GenBank] for E. coli TN07, AJ972956 [GenBank] for E. coli TN13, AJ97295 for K. pneumoniae KP-04, AM003902 [GenBank] for S. marcescens SM-01, AM003903 [GenBank] for K. pneumoniae KP-LT, AM003904 [GenBank] for E. coli RB-01, AM003905 [GenBank] for K. pneumoniae KP-RB, AM003906 [GenBank] for P. mirabilis PM-SM, AM040706 [GenBank] for E. coli TN03, AM040707 [GenBank] for E. coli TN17, AM040708 [GenBank] for E. coli TN05, AM040709 [GenBank] for E. coli TN06 and AM040710 [GenBank] for E. coli TN19.

	Results

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Description of clinical isolates

From 2000 to 2003, 28 enterobacterial strains (21 E. coli, 5 K. pneumoniae, 1 S. marcescens and 1 P. mirabilis) were recovered at Tenon Hospital and at four other Parisian hospitals. They were isolated from blood, urinary, rectal, vaginal, wound and pulmonary human specimens (Table 1). Only strains TN03, TN08, TN09, TN14 and TN18 were characterized as the same strains by Rep-PCR or ERIC-PCR as previously described (data not shown).²⁵

ß-Lactam susceptibility profile and associated resistance

Susceptibility testing showed that most of the strains were resistant to ß-lactam antibiotics (including third-generation cephalosporins) (data not shown). One of these strains (E. coli TN13) was also resistant to cefoxitin (MIC, 128 mg/L). Synergy between cefotaxime- and clavulanate-containing discs was present in all strains except for E. coli TN13; for this strain, the synergy was observed only with cefepime. Transconjugants or transformants resistant to cefotaxime were obtained for all strains. Non-ß-lactam antibiotic resistance markers are listed in Table 1. Twenty isolates were resistant to aminoglycosides, and this resistance was transferred in 17 of them.

Characterization of ß-lactamase-encoding genes

PCR experiments were positive for bla_CTX-M with all isolates and their transformants or their transconjugants. Sequence analysis of deduced amino acid sequences showed the presence of various CTX-M-type enzymes: CTX-M-1, -2, -3, -9, -14, -24 and particularly CTX-M-15 (with a glycine at position 240) produced by 11 strains (Table 1). Fifteen isolates were found to carry TEM-1. Only one strain produced SHV-2 enzyme (E. coli TN17). Amplification was obtained with the CMY-specific primers for E. coli TN13, sequencing showed that this ß-lactamase was CMY-2 (Table 1).

Exploration of the regions surrounding bla_CTX-M genes

PCR identified the insertion sequence ISEcp1 in its entirety or partially truncated upstream of the bla_CTX-M gene in 23 strains (Table 3). The sizes of PCR products were 1.7 kb, except for three strains, TN13, PM-SM and KP-RB (Table 3). For two strains, TN13 and KP-RB, the PCR fragments were about >3 and 2.5 kb, respectively, suggesting the insertion of additional DNA. Direct sequencing of PCR products showed that the presence of IS10 and IS1 disrupted ISEcp1 (Figure 1). For the last strain (PM-SM), amplification was obtained with tnpA ISEcp1 primer but not with ISEcp1 5' primer suggesting disruption of this IS.

View this table: [in this window] [in a new window]   Table 3.. Genotypic characterization of blaCTX-M surrounding DNA

 

View larger version (30K): [in this window] [in a new window]   Figure 1.. Schematic representation of the genetic environment of blaCTX-M genes from (a) the 19 clinical Enterobacteriaceae isolates producing CTX-M-1-type ß-lactamases, and from (b) the 8 clinical Enterobacteriaceae isolates producing CTX-M-14 and CTX-M-24.

 
The upstream region of bla_CTX-M in strains TN17, KP-04 and PM-SM, analysed by PCR, contained the transposase gene of the insertion sequence IS26. These strains had IS26 flanking a partially truncated ISEcp1. Length of the disrupted ISEcp1 was 132, 524 and 596 bp in strains TN17, KP-04 and PM-SM, respectively; all these DNA sequences contained the putative promoter region involved in the transcription of bla_CTX-M genes.

ISEcp1 insertion sequences have been observed 42–127 bp upstream of the ORFs encoding the CTX-M enzymes. All of the CTX-M-1 cluster were characterized by a 48 bp region (W sequence) upstream of the bla gene (Figure 1). Different additional fragments (V or X sequence) were present in eight cases. The CTX-M-15 group was characterized by the presence of only the W sequence, whereas the CTX-M-1 group was characterized by the additional X sequence and the CTX-M-3 group was characterized by the V sequence (Figure 1). In all strains belonging to the CTX-M-9 group (CTX-M-9, CTX-M-14 or CTX-M-24), TN05, TN07, TN13, TN21, TN22, TN23 and KP-04, a 42 bp region with an identical sequence (named Y sequence) was found upstream of the start codon of the ß-lactamase gene (Figure 1). An additional sequence Z (52 bp) was found between the right inverted repeat of ISEcp1 and the Y sequence in TN05 (CTX-M-9) (Figure 2).

View larger version (14K): [in this window] [in a new window]   Figure 2.. Schematic representation of sequences surrounding the blaCTX-M genes in the strains TN05, TN06 and TN19, inserted in complex Class 1 integrons.

 
PCR using the tnpA IS903 reverse and IS903 reverse 5' primers produced amplicons in three and five of our strains respectively, suggesting that three strains had the transposase gene of this insertion sequence; in two cases this IS was probably not present in its entirety as confirmed by sequencing. The 19 strains of the CTX-M-1 cluster had the same sequence (between 311 and 395 bp according to the results of the sequencing experiments) downstream of the bla gene corresponding to a truncated part of ORF 477 described downstream of the bla_CTX-M-3 gene of K. ascorbata. Downstream of this common region (CR), the mucA gene was found only in the three strains producing the CTX-M-3 ß-lactamase.

The genetic organization of the bla_CTX-M of E. coli TN03, TN05, TN06 and TN19 was investigated by cloning PCR analysis, and sequencing the regions surrounding these genes.

Upstream of ISEcp1, sequencing the recombinant plasmid of TN03, identified the bla_TEM-1 gene and a resolvase gene tnpR and a transposase gene tnpA of the transposon TN3 (Figure 1). Using primers TnpTN03 and ResTN03 combined with MA-1rev and OT4, respectively, the same organization was found by sequencing for TN08, TN09, TN14 and TN18.

Upstream of the bla_CTX-M gene, TN05, TN06 and TN19 strains harboured a CR to the complex Class 1 integron. This region includes ORF 513, which is present in In6 and In7. Analysis of the genes cassette is showed in Figure 2. For TN05, the gene cassettes were dhfr12 and aadA8 as observed in a previous sequence reported in database (AY852272 [GenBank] ). For TN06, the gene cassettes were aac(6')-Ib, bla_OXA-2 and ORF D (as described in In35, InS21 and In116), and for TN19, dhfrhI and aadA2.

Sequencing analysis of the downstream DNA of the bla_CTX-M-9 gene from E. coli TN05 revealed the presence of ORF 3 and part of ORF 339 from the chromosome of K. georgiana as recently described.¹⁹ In strains TN06 and TN19, we found part of the 3'-CS complex sul1-type integron, downstream of the bla_CTX-M-2 stop codon and between the bla_CTX-M and the second 3'-CS a sequence identical to those found in InS21 and In35 and in K. ascorbata producing CTX-M-2 ß-lactamase.^15,28,29

	Discussion

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In the present study, we analysed the surrounding DNA of 28 bla_CTX-M genes. The genetic organization of these genes was determined by PCR mapping or cloning experiments. We found the presence of ISEcp1 in 23 of our strains. ISEcp1 had been first identified upstream of the plasmid-mediated cephalosporinase gene bla_CMY-4 from an E. coli isolate from the United Kingdom³⁵ (accession number AJ242809 [GenBank] ). This IS was also frequently found associated with several bla_CTX-M.^13,20–26 This ISEcp1 element contained typical –35 and –10 putative promoter regions and could mobilize such genes.^21–23 Interestingly, this IS was disrupted by another IS, IS1 or IS10, in two strains. A similar organization was reported for bla_CTX-M-25 with an IS50 disrupting ISEcp1.³⁶ It is interesting to note that IS10 was identified upstream of the bla_CTX-M-8 (AF189721 [GenBank] ) and may play a role in the mobilization of this bla gene. Another IS, IS26, was found upstream of the bla genes and disrupting ISEcp1. This organization (IS26 and end of ISEcp1) was also found in its entirety upstream of the bla genes encoding the ACC-1 ß-lactamase and upstream of some bla_CTX-M (and accession number AY462238 [GenBank] ).^19,24,25,37 Interestingly, the insertion site of this IS is different from strain to strain. Upstream of the ISEcp1, in strains TN03, TN08, TN09, TN14 and TN18, we found the transposon Tn3 (truncated in the 5' end of the tnpA by ISEcp1) and the bla_TEM-1 gene. This organization of our DNA sequence shows 100% identity with the corresponding region of the plasmid pC15-Ia found associated with an outbreak involving an E. coli producing CTX-M-15 in Canada.⁹ It will be very interesting to compare our plasmid with pC15-Ia.

Analysis of the DNA downstream of the bla genes showed a different organization. In the CTX-M-1 cluster, sequence ORF 477 was present for all the strains and in three cases additional fragment mucA was found as described for the plasmid pCTX-M-3 (accession number NC 00464). Another insertion sequence, IS903, has been already described in the literature.^21–23 This structure was found downstream of the bla_CTX-M-24 and bla_CTX-M-14 of three of our strains; this IS was truncated in two cases (TN07 and TN13).

It is worth mentioning that PCR amplification with primers specific for the tnpA genes of ISEcp1 and IS26 was negative for three strains. Analysis of the surrounding regions after cloning and end-sequencing allowed us to see that these strains (two bla_CTX-M-2 and one bla_CTX-M-9 gene) had a different construction and their bla genes were located in unusual Class 1 integrons. This genetic organization was already described in the literature for two bla_CTX-M-2 sequences and one bla_CTX-M-9 sequence inserted in novel complex Class 1 integrons InS21, In35, In116 and In60, respectively.^13,27–30 This unusual integron contains a partial duplication of the 3'-conserved segment, and a region that includes bla_CTX-M and the ORF 513 between both 3' conserved segments. In addition, integrons bearing bla_CTX-M-2 have resistance cassettes including aac(6')-Ib-oxa-2-orfD (In35, InS21 and In116).^28–30 Sequencing analysis of the entire gene cassettes on TN06 carrying bla_CTX-M-2 revealed a similar organization to In35, InS21 and In116. Interestingly, the bla_CTX-M-2 gene and the bla_CTX-M-9 of the strains TN19 and TN05 were located in an unusual Class 1 integron having, respectively, dhfrhI-aadA2 or dhfrXII-orfF-aadA8 genes cassettes within the variable region.

Among the ESBLs, the cefotaximases (CTX-M) constitute a rapidly growing cluster of enzymes. Analysis of the promoter regions in bacteria producing plasmid-mediated ß-lactamases revealed mobile IS such as IS26, IS903 and ISEcp1. These elements could play an important role in the spread of such ESBLs (it has been demonstrated for ISEcp1).^21,22 These sequences ensure the transfer of the resistance genes from the bacterial chromosome to the plasmids. Other constructions may be implicated in the transfer of these genes, like integrons. Integrons are very sophisticated site-specific recombination systems that capture various genes cassettes, between their 5' and 3' conserved segment. Thus ISEcp1 and integrons may be efficient tools for mobilization and expression of ß-lactamase genes. These results showed the diversity and the complexity of the surrounding DNA of bla_CTX-M genes carried by enterobacterial strains.

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No declarations were made by the authors of this paper.

	Acknowledgements

 
We are grateful to Nadia Hidri, Thierry Lambert, Claire Poyart and Michèle Saladin-Allard for providing strains. This work was supported by grants from Faculté de Médecine Saint-Antoine, Université Pierre et Marie Curie, and from European Community (6th PCRD Contract : LSHM-CT 2003-503335).

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