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Antimicrobial Agents and Chemotherapy, August 2007, p. 3017-3019, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00279-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Complex Class 1 Integron Bearing an ISCR1 Element in an Escherichia coli Isolate Carrying the blaCTX-M-14 Gene{triangledown}

Il Kwon Bae,1 You-Nae Lee,1 Wee Gyo Lee,2 Sang Hee Lee,3 and Seok Hoon Jeong1*

Research Institute for Antimicrobial Resistance and Department of Laboratory Medicine, Kosin University College of Medicine, 602-030, 34 Amnam-Dong, Suh-Gu, Busan,1 Department of Laboratory Medicine, Ajou University School of Medicine, 442-749, San 5 Wonchun-Dong, Youngtong-Gu, Suwon,2 Department of Biological Sciences, Myongji University, 449-728, San 38-2 Nam-Dong, Yongin, Gyeonggido, Korea3

Received 24 February 2007/ Returned for modification 20 April 2007/ Accepted 8 May 2007


   ABSTRACT
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This work identifies an ISCR1-related blaCTX-M-14 gene, which has never been reported before, from a clinical isolate of Escherichia coli. The blaCTX-M-14 gene was preceded by an ISCR1 element that was followed by a class 1 integron containing three different insert gene cassettes, i.e., dfrA12, orfF, and aadA2.


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The CTX-M enzymes comprise more than 60 variants (http://www.lahey.org/studies/webt.asp) belonging to five different clusters (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) on the basis of their amino acid sequence similarities (3). CTX-M enzymes have a wide substrate range, including penicillins and narrow- and expanded-spectrum cephalosporins, and as the designation CTX indicates, these enzymes preferentially hydrolyze cefotaxime but not ceftazidime, although some CTX-M enzymes, including CTX-M-15, CTX-M-19, and CTX-M-54 enzymes, have been associated with expansion of activity towards ceftazidime (2, 11, 13).

CTX-M-14 is a member of CTX-M-9 cluster and differs from CTX-M-9 only by the substitution Ala231Val (9). The amino acid sequences of CTX-M-14 and CTX-M-18 are identical (http://www.lahey.org/studies/webt.asp). CTX-M-14, one of the most widespread CTX-M enzymes, has been reported repeatedly in a very wide geographic area, including Europe, North America, South Asia, and East Asia (7, 10, 16). This enzyme has been found predominantly in Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Shigella sonnei, Salmonella spp., and Proteus mirabilis (7, 9, 15, 16, 19).

The rapid dissemination of CTX-M enzymes involves plasmid or strain epidemics, but it also involves mobile elements, including ISEcp1-like insertion sequences and the ISCR1 element (previously called orf513) (6). Most blaCTX-M genes belonging to the CTX-M-1, CTX-M-2, and CTX-M-9 clusters are associated with ISEcp1-like insertion sequences, while ISCR1 elements were identified upstream of the blaCTX-M-2 and blaCTX-M-9 genes (1, 6, 17). Interestingly, the blaCTX-M-14 gene, although closely related to the blaCTX-M-9 gene, has been reported to be mainly associated with ISEcp1 (6). This report identifies an ISCR1-related blaCTX-M-14 gene, which has never been reported before, from a clinical isolate of E. coli.

E. coli AJE0508 was isolated from a sputum specimen from a 26-year-old male patient hospitalized at a tertiary care hospital in Suwon, Korea, in July 2005 for subarachnoid hemorrhage and pulmonary pneumonia. Strain AJE0508 exhibited resistance to ampicillin, ampicillin-sulbactam, ceftazidime, cefotaxime, aztreonam, tobramycin, and trimethoprim-sulfamethoxazole and susceptibility to cefoxitin, cefepime, imipenem, amikacin, gentamicin, and ciprofloxacin by disk diffusion assay. Agar dilution MIC testing on Mueller-Hinton agar (Difco Laboratories, Detroit, MI) with an inoculum of 104 CFU per spot confirmed that the strain was resistant to ampicillin (MIC, >256 µg/ml), ceftazidime (MIC, 32 µg/ml), cefotaxime (MIC, 64 µg/ml), and aztreonam (MIC, 64 µg/ml) but susceptible to cefoxitin (MIC, 8 µg/ml) and imipenem (MIC, 0.1 µg/ml) (5). Clavulanic acid (at a fixed concentration of 4 µg/ml) restored the activities of ceftazidime (MIC, 2 µg/ml) and cefotaxime (MIC, 1 µg/ml). The strain exhibited a positive double-disk synergy test, thus indicating the production of extended-spectrum beta-lactamases (16).

Plasmid analysis, which was carried with a commercial kit (QIAGEN, Valencia, CA) according to the instruction manual, showed that E. coli AJE0508 contained two plasmids with molecular sizes of ca. 82 kbp (pAJE0508) and 25 kbp. The strain transferred pAJE0508 to the E. coli J53 azide-resistant recipient in mating experiments in which transconjugants were selected on Mueller-Hinton agar plates supplemented with cefotaxime (2 µg/ml) and sodium azide (100 µg/ml) (2). PCR amplifications using primers (16) specific for extended-spectrum beta-lactamase-encoding genes revealed that the transconjugant (E. coli trcAJE0508) possessed blaSHV, blaTEM, and blaCTX-M-9 cluster genes. Sequences of the PCR amplicons for three type genes were 100% identical to the blaSHV-12, blaTEM-1, and blaCTX-M-14 sequences, respectively. The blaSHV-12 and blaCTX-M-14 genes might be attributed to high-level resistance of strain AJE0508 to cefotaxime and cetazidime.

The internal ISCR1 (ISCR1-F and ISCR1-mF) or ISEcp1 (TN1-F and BTN1-F) forward primers and blaCTX-M-9 cluster reverse primers (CTX-M-9-R and CTX-M-9-mR) were used to investigate the upstream region of the blaCTX-M-14 gene. While a sequence having homology with the ISCR1 element was detected upstream of the blaCTX-M-14 gene on pAJE0508, ISEcp1-like insertion sequences were not. These results suggested that the blaCTX-M-14 gene is associated with a complex class 1 integron containing ISCR1, as the blaCTX-M-9 gene on plasmid pMSP071 is associated with the complex In60 (17). To analyze the complex class 1 integron, sequencing of several overlapping PCR fragments obtained from pAJE0508 with primers corresponding to internal regions of In60 and the blaCTX-M-14 gene was performed (Table 1).


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  		TABLE 1. Primer sequences used in this study

 
The blaCTX-M-14 gene was preceded by an ISCR1 element that was followed by a typical class 1 integron containing two conserved elements, 5'-CS and 3'-CS (Fig. 1). The integron contained three different insert gene cassettes. The first contained a dihydrofolate reductase type A12 gene, dfrA12 (previously called dhfrXII), which confers resistance to trimethoprim. The second contained an open reading frame, orfF, of unknown function, and the third contained an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin (8). Each gene cassette contained a 59-base element recombination site. Although the dfrA12-orfF-aadA2 array has been globally disseminated since it was first described in Finland in 1969 (8), this is the first report of the array being associated with the blaCTX-M-14 gene.


Figure 1
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  		FIG. 1. Schematic map of the complex class 1 integron carrying the blaCTX-M-14 gene on plasmid pAJE0508. Open arrows, open reading frames; white ovals, 59-be; black oval, attI1 recombination site. The +1 transcription initiation site and the ATG start codon of the blaCTX-M-14 gene are in boldface type. The –35 and –10 motifs of the putative promoter and the recombination crossover site are boxed.

 
An ISCR1 element was found downstream of the 3'-CS element and upstream of the blaCTX-M-14 gene. Recently it has been suggested that ISCR elements are members of an extended family of IS91-like elements that can transpose adjacent DNA sequences by a mechanism termed rolling-circle transposition and are responsible for the mobilization of virtually every class of antibiotic resistance genes, including the blaCTX-M genes (18). A recombination crossover site (RCS) (33-bp DNA sequence) at which insertion of resistance genes into the complex class 1 integron containing ISCR1 takes place (14) was observed within the 3' noncoding sequence of ISCR1. A 94-bp region which is identical to that of In60 was identified between the RCS and the start codon of the blaCTX-M-14 gene. A putative promoter consisting of the –35 (TAAACG) and –10 (TAAGAT) regions, which drives blaCTX-M-14 transcription, was observed just upstream of the RCS. The –35 and –10 sequences were separated by 17 bp.

Sequence analysis of the downstream DNA of the blaCTX-M-14 gene in the complex class 1 integron on the pAJE0508 revealed the presence of an IS903-like element, the role of which in the mobilization process of the blaCTX-M genes has not yet been demonstrated (12). This insertion sequence has been found downstream of the blaCTX-M-17, blaCTX-M-19, blaCTX-M-24, and blaCTX-M-54 genes (2, 4, 6, 12) and also has been identified downstream of the blaCTX-M-14 genes in three E. coli strains and one K. pneumoniae strain recently isolated in Paris (6).

This work describes a complex class 1 integron bearing an ISCR1 element and shows that mobilization and expression of the blaCTX-M-14 gene may be associated with ISCR1 elements. The ISCR1 element is a powerful genetic element that can mobilize antibiotic resistance genes, so further spread of the blaCTX-M-14 gene can be anticipated.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper are available in the GenBank nucleotide database under accession number EF450247.


   ACKNOWLEDGMENTS
 
This work was supported by a Korea Research Foundation grant (KRF-2006-331-E00455).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Laboratory Medicine, Kosin University College of Medicine, 602-030, 34 Amnam-Dong, Suh-Gu, Busan, Republic of Korea. Phone: 82-51-990-6373. Fax: 82-51-990-3034. E-mail: kscpjsh{at}ns.kosinmed.or.kr Back

{triangledown} Published ahead of print on 21 May 2007. Back


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Antimicrobial Agents and Chemotherapy, August 2007, p. 3017-3019, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00279-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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