IS Ecp1 -Mediated Transposition of qnrB -Like Gene in Escherichia coli

Abstract

A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene.Resistance to quinolones in Enterobacteriaceae most commonly arises stepwise as a result of chromosomal mutations responsible for the modification of target enzymes (DNA gyrase and topoisomerase IV) or decreased intracellular drug accumulation by the upregulation of efflux pumps and/or modified outer-membrane porins (27). Since the discovery of the first plasmid-mediated quinolone resistance (PMQR) determinant, QnrA, in 1998 (15), four other PMQR determinants have been identified to date: the QnrB (12) and QnrS (10) proteins, the aminoglycoside acetyltransferase AAC(6Ј)-Ib-cr (25), and the efflux pump QepA (19,30). Resistance due to Qnr determinants is increasingly reported worldwide in enterobacterial isolates (18,26) and has been recently identified outside Enterobacteriaceae in environmental Aeromonas isolates from France (3). The three types of Qnr determinants, QnrA, QnrB, and QnrS, belong to the pentapeptide repeat family of proteins (18,26). By protecting DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones, Qnr proteins confer resistance to quinolones (e.g., nalidixic acid) and decreased susceptibility to fluoroquinolones, therefore facilitating the recovery of chromosome-encoded target mutants with a higher level of resistance to fluoroquinolones (18, 26). Whereas qnrAlike genes have only been identified as part of the complex sul1-type integrons in association with the orf513 transposase gene (18,26), which is part of a region redefined as ISCR1 (28), qnrB-like genes have been found to be associated with either the orf1005 gene encoding a putative transposase (12) or the ISCR1 element (8,24).The aim of this study was (i) to investigate the genetic environment of a qnrB-like gene from an Escherichia coli clinical isolate, (ii) to evaluate experimentally the mobility of that putative transposon in E. coli, and (iii) to determine the promoter sequences of the qnrB-like gene responsible for the expression of that gene.E. coli R4525 expressing an extended-spectrum ␤-lactamase (ESBL) phenotype had been isolated in 2002 from a wound culture of a patient hospitalized at the Hospital San Jeronimo in Monteria, Colombia. In the course of studying the genetic support of the ESBL determinant in E. coli R4525, conjugation experiments followed by selection with sodium azide (100 g/ ml) and amoxicillin (50 g/ml) as previously described (3) gave E. coli J53 transconjugants displaying an ESBL phenotype and decreased susceptibility to fluoroquinolones. This result prompted us to search for the presence of PMQR determinants. The screening of qnr genes using a multiplex strategy (5) identified a qnrB-like gene in both E. coli R4525 and it...

Plasmid-Mediated Quinolone Resistance: a Multifaceted Threat

“…The impact of some qnr allele variants on quinolone MICs has additionally been examined. Overall, the patterns of resistance have been similar, with 2-to 8-fold and 8-to 32-fold increases in MICs of nalidixic acid and ciprofloxacin, respectively (17,23,70,91,126,161,166).…”

“…These genes differ from qnrB1 by 4 and 11 amino acids, respectively (GenBank accession numbers EU043311 and EU043312, respectively). qnrB19, which differs from qnrB1 by six amino acids, was also found in an E. coli isolate from Colombia by the same group (GenBank accession number EU432277) (23). qnrB10 (161), discovered in C. freundii, differs from qnrB1 by five amino acids, and qnrB12 (104), identified in Citrobacter werkmanii, differs from qnrB1 by eight amino acids in addition to the five differences that characterize qnrB10.…”

“…More recently, using the same multiplex PCR method, qnrB13, qnrB14, and qnrB15 (GenBank accession numbers ABX72042, ABX72044, and ABX72227, respectively), differing from qnrB1 in four, five, and five amino acids, respectively, were identified among Citrobacter freundii strains from South Korea (190). Using degenerate primers for the detection of qnrB in a collection of isolates of Enterobacteriaceae from Kuwait City, Kuwait, Cattoir et al identified qnrB7 in Enterobacter cloacae and qnrB8 in C. freundii (23). These genes differ from qnrB1 by 4 and 11 amino acids, respectively (GenBank accession numbers EU043311 and EU043312, respectively).…”

“…The PMQR gene responsible for this phenotype coded for a 214-or 226-amino-acid protein (depending on which potential initiation codon was taken as the start) and was termed qnrB1 (GenBank accession number DQ351241). A recent determination of the transcription start site supports a peptide length of 214 amino acids, and homology between different qnrB variants bears this out (23,209). The QnrB1 protein shares 43% and 44% amino acid identities with QnrA and QnrS, respectively (91).…”

Plasmid-Mediated Quinolone Resistance

“…Alleles qnrB2, qnrB4, qnrB6, and qnrB10 are associated with ISCR1, usually as a single copy (73)(74)(75), but in some plasmids two copies of ISCR1surround qnrB2 (76,77). qnrB19 has been found in three genetic environments: within large plasmids associated with ISEcp1C-based transposons, in large plasmids bracketed by IS26, and in small ColE1-type plasmids (~3-kb) lacking insertion sequences in which a flanking oriT locus and Xer recombination site have been proposed to be involved in site-specific recombination (73,(78)(79)(80)(81)(82). In all three settings qnrB19 is linked to a fragment of pspF implying that the putative mobilization pathways may be related.…”