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Antimicrobial Agents and Chemotherapy, July 2006, p. 2544-2546, Vol. 50, No. 7
0066-4804/06/$08.00+0     doi:10.1128/AAC.00609-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Common Region CR1 for Expression of Antibiotic Resistance Genes

Jose-Manuel Rodriguez-Martinez,1,2 Laurent Poirel,1 Rafael Canton,3 and Patrice Nordmann1*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris Sud, K.-Bicêtre, France,1 University Hospital Virgen Macarena, University of Sevilla, Sevilla,2 Servicio de Microbiologia, Hospital Universitario Ramon y Cajal, Madrid, Spain3

Received 16 May 2005/ Returned for modification 29 July 2005/ Accepted 10 April 2006


   ABSTRACT
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The CR1 element defined by an orf513 gene encoding a putative recombinase and a recombination crossover site has been identified upstream of several antibiotic resistance genes in Enterobacteriaceae. This CR1 element was shown to bring promoter sequences that play a role in the expression of unrelated antibiotic resistance genes.


   TEXT
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The role of class 1 integrons in acquisition, dissemination, and expression of antibiotic resistance genes is now well established in Enterobacteriaceae (1, 3, 8). Class 1 integrons possess two conserved features consisting of an integrase gene, intI1, in a so-called 5' conserved sequence (5'-CS) and qacE{Delta}1 and sul1 genes in a so-called 3' conserved sequence (3'-CS) (1). Promoter sequences that drive the expression of downstream-located antibiotic resistance genes have been identified in the 5' end of the integrase gene (1). The complex class 1 integrons contain the 5'-CS and part of the 3'-CS flanking one or more gene cassettes (6). Following the 3'-CS is a region known as the common region (CR) consisting of orf513 and a recombination crossover site followed by genes that do not resemble a gene cassette and flanked by another copy of the qacE{Delta}1/sul1 complex (6). The encoded Orf513 may play a role in the integration of genes located nearby (mostly antibiotic resistance genes) (6). The orf513 gene and the 33-bp DNA sequence located at its right-hand boundary that may correspond to a recombination crossover site (RCS) are the common region CR1. We have recently shown that CR1 plays a role in the expression of the plasmid-mediated quinolone resistance qnrA gene by providing a promoter structure (5). The aim of the study was to analyze CR1 elements and their putative role in the expression of other antibiotic resistance determinants from nonrelated enterobacterial isolates. Six strains that encoded resistance determinants including QnrA and emerging extended-spectrum ß-lactamases of the CTX-M type were included in this study (Table 1). Since the nucleotide sequence separating CR1 from the antibiotic resistance gene may vary, strains with different structures located upstream of identical qnrA and blaCTX-M-9 genes were studied (Table 1). Primers used for PCR identification and sequencing of CR1 elements and associated resistance genes are shown in Table 2.


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  		TABLE 1. Characteristics of the clinical strains included in this study

 

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  		TABLE 2. Primers used in this study

 
Mapping of the transcription start sites was performed by 5' rapid amplification of cDNA ends (5'-RACE). Total RNA was isolated from the different strains studied using the RNeasy Midi kit (QIAGEN, Courtaboeuf, France). 5'-RACE reactions were performed using 5 µg of total RNA of each strain and the 5'-RACE System kit (version 2.0; Invitrogen Life Technologies, Cergy Pontoise, France) according the manufacturer's recommendations. After a reverse transcription step with gene-specific primer GSP1 and reverse transcriptase, the cDNA was tailed with terminal deoxynucleotidyl transferase and was subsequently amplified with another gene-specific primer, GSP2, combined with an oligo(dT) adapter primer provided with the kit (Table 2). This PCR product was used as a template for a nested PCR with another adapter primer and primer GSP3. The PCR product obtained was cloned into pCR-BluntII-Topo (Invitrogen), and the corresponding clones possessing the larger insert were sequenced. Analysis of the cloned sequence allowed the determination of the transcription initiation site(s), defined as the first nucleotide following the sequence of the adapter primer. Promoter sequences were determined subsequently. For each transcription assay, at least 10 clones were analyzed, and the entire experiment was repeated twice for all strains.

Identical CR1 elements were identified in all cases, and their 3' ends are shown in Fig. 1. The CR1 element was associated with the qnrA gene in two cases, with blaCTX-M-9 in two cases, with blaCTX-M-2 in one case, and with the dfrA10 gene encoding trimethoprim resistance in one case (Table 1 and Fig. 1). The distance separating the RCS of CR1 from the start codon of the antibiotic resistance genes varied. This distance upstream of the qnrA gene was 98 and 31 bp for Escherichia coli Lo and Klebsiella pneumoniae K149, respectively (Table 1). An identical 94-bp region was identified between the RCS of CR1 and the start codon of the blaCTX-M-9 gene in E. coli B36 and K. pneumoniae KP40. However, a 95-bp duplication was identified at the right-hand boundary of CR1 in E. coli B36 (Fig. 1). A 149-bp sequence was identified between CR1 and the dfrA10 gene in Acinetobacter baumannii AYE, whereas a 266-bp region was identified upstream of the blaCTX-M-2 gene in E. coli JAB.


Figure 1
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  		FIG. 1. Promoter structures for the expression of antibiotic resistance genes as determined by 5'-RACE experiments for the qnrA gene from E. coli Lo and K. pneumoniae K149 (A), bla CTX-M-9 from E. coli B36 and K. pneumoniae KP40C (B), dfrA10 from A. baumannii AYE (C), and blaCTX-M-2 from E. coli JAB (D). The +1 initiation sites of transcription and the active promoter sequences are in boldface type. The –35 and –10 motifs of other promoters are boxed. Promoter PCR1-2, which was identified previously for the qnrA gene in E. coli Lo, is indicated for the sake of consistency (5). The ATG start codons are capitalized. The right-hand boundary of the CR1 element is shaded in gray.

 
Analysis of the 5'-RACE PCR products obtained from QnrA-positive E. coli Lo revealed a single type of transcription product that was different from that previously reported (5). Although promoter PCR1-2 has been identified previously (5), a +1 transcription site located 136 bp upstream of the qnrA gene led to the identification of another promoter, termed PCR1-1 (Fig. 1). In the previous study, identification of the PCR1-2 promoter was likely the result of selection of truncated transcripts during the 5'-RACE experiment. The same promoter, PCR1-1, controlled the expression of the qnrA gene in K. pneumoniae K149, the blaCTX-M-9 gene in E. coli strain B36 and in K. pneumoniae KP40C, and the dfr10 gene in A. baumannii AYE (Fig. 1). These results also indicated that promoter PCR1-1, provided by the CR1 element, was active in Enterobacteriaceae and A. baumannii. The expression of the blaCTX-M-2 gene in E. coli JAB depended on another promoter, PORI, located outside the CR1 element between the CR1 and the initiation codon of the resistance gene (Fig. 1).

Our results showed that CR1-mediated promoter sequences were involved in the expression of the qnrA, dfrA10, and blaCTX-M-9 genes (Fig. 1) and that PCR1-1 plays a major role in antibiotic resistance gene expression when CR1 is present. This study emphasizes that CR1 plays a significant role by providing promoter sequences for the expression of unrelated antibiotic resistance genes.


   ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France, and by a grant from the European Community (6th PCRD, LSHM-CT-2003-503335). L.P. is a researcher from the INSERM (Paris, France), and J.-M.R.-M. was a recipient of a travel grant from the Spanish Society for Clinical Microbiology and Infectious Diseases in 2004.

We thank A. Pascual for constant support of J.-M.R.-M.


   FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre78, rue du General Leclerc, 94275 Le Kremlin-Bicêtre, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr. Back


   REFERENCES
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  6. Partridge, S. R., and R. M. Hall. 2003. In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob. Agents Chemother. 47:342-349.[Abstract/Free Full Text]
  7. Poirel, L., O. Menuteau, N. Agoli, C. Cattoen, and P. Nordmann. 2003. Outbreak of extended-spectrum ß-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J. Clin. Microbiol. 41:3542-3547.[Abstract/Free Full Text]
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Antimicrobial Agents and Chemotherapy, July 2006, p. 2544-2546, Vol. 50, No. 7
0066-4804/06/$08.00+0     doi:10.1128/AAC.00609-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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