Abstract
Resistance to β-lactam antibiotics in Gram-negative bacteria is mainly mediated by the production of β-lactamases, which are divided into four major Ambler's molecular classes (A, B, C and D). In previous studies, we have demonstrated that CMY-11 from a virulent clinical strain is a plasmid-encoded extended-spectrum class C (AmpC) β-lactamase conferring resistance to cefoxitin and cefotetan, as well as to penicillins and oxyimino-cephalosporins (Lee et al., 2002). We previously obtained 710 Escherichia coli clinical isolates from patients with various infections, 9 of which showed high levels of resistance to amoxicillin, amoxicillin–clavulanic acid, cefalotin, cefoxitin and cefotetan, as well as to cefotaxime, ceftazidime and aztreonam (Kim et al., 2004). All nine E. coli clinical isolates produced CMY-11 (Kim et al., 2004). A blaCMY-11 gene, responsible for this β-lactam resistance, was located on a 130 kb conjugative plasmid harboured by the nine clinical isolates and their transconjugants (Kim et al., 2004). Clonal and plasmid spread contributed to blaCMY-11 dissemination in Korean clinical isolates of E. coli (Kim et al., 2004; Song et al., 2009). This study was carried out to investigate the genetic environment surrounding the blaCMY-11 gene, which can play an essential role in the translocation of the gene from a chromosome (or plasmid) to the conjugative plasmid.
The BLASTN (basic local alignment search tool used against nucleotide sequence databases) program of the National Center for Biotechnology Information () was used for DNA sequence database searches of integron element genes and other genes. The primers for PCR amplification were designed by selecting consensus sequences based on multiple nucleotide alignments (CLUSTAL W program) of searched genes. PCR and DNA sequence analyses (using the primers shown in Table 1) of nine clinical isolates revealed that the blaCMY-11 gene was located in a new complex class 1 integron within the conjugative plasmid (Fig. 1a). To characterize this integron, recombinant plasmids with overlapping fragments were obtained as follows. Long and accurate (LA) PCR amplification was performed with specific primers (Table 1). The LA PCR products were ligated with the pCR2.1-TOPO cloning vector (Invitrogen). The ligation mixture (recombinant plasmids) was introduced into competent E. coli DH5α cells (Invitrogen) by transformation; transformant selection was by kanamycin (50 µg ml–1; Sigma-Aldrich). In Fig. 1(b), the recombinant plasmids are pTO21-33 D (4050 bp, a recombinant plasmid carrying the sul1-type integron), pTO21-33E (3755 bp, a recombinant plasmid carrying a region from within sul1 to within blaCMY-11) and pTO21-33F [5036 bp, a recombinant plasmid carrying a region from within blaCMY-11 to qacEΔ1(140)/sul1]. Consequently, the plasmids covered, together, a 11 839 bp region containing blaCMY-11. To avoid repetition of information about the integron, only the first characterized sequence of this integron was submitted to the GenBank database.