New qnr Gene Cassettes Associated with Superintegron Repeats in Vibrio cholerae O1

A novel qnr determinant emerged in ciprofloxacin-resistant Vibrio cholerae O1 from the Amazon region of Brazil. This qnrVC1 was in a typical class 1 integron. Its attC showed 89% identity with V. parahaemolyticus superintegron repeats. Analysis showed V. cholerae O1 carrying qnrVC2 associated with a V. cholerae superintegron repeat.


The Study
V. cholerae O1 isolates from the cholera epidemic in Brazil (1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000) were screened for antimicrobial drug resistance and class 1 integrons. VC627, a clinical strain from the Amazon region isolated in 1998, was resistant to ciprofl oxacin, in contrast to other isolates. This strain was the only one positive for class 1 integrase but not for qacEΔ1 and sulI genes (6). Sequence analysis of the variable region of this class 1 integron showed an aadA2 gene cassette and an ORF similar to qnr determinants. Its deduced amino acid sequence showed 69% sequence identity with a chromosomal protein of Photobacterium profundum, which was recently identifi ed as a quinolone-resistance determinant (QnrPP) (3) and 57% identity with QnrA1, a plasmid-encoded protein found in Enterobacteriaceae (2). This new qnr-like gene in V. cholerae was named qnrVC1 (EU436855).
Qnr proteins belong to the pentapeptide repeat family, which consists of uninterrupted pentapeptide repeats comprising 92% of the sequence and play a role in DNA gyrase protection (7). These repeat features were observed in the QnrVC1 deduced amino acid sequence with the consensus sequence [AC] [DN][LF]XX (amino acids in brackets are found more frequently and XX indicates other amino acids) (8) (online Appendix Figure, available from www.cdc.gov/ EID/content/14/7/1129-appG.htm), with a large proportion of serine residues in the fourth position (fi rst X), as observed by Robicsek et al. (2).
The qnrVC homologs in chromosomes of other bacterial species were investigated by using BLAST analysis (www.ncbi.nlm.nih.gov/blast/Blast.cgi). Similar to other reports of such homologs (3,4), we found Qnr-like proteins in recently published genomes from different families, including other members of Vibrionaceae (Figure). Analysis in conjunction with informatics capabilities (in silico analysis) showed a 657-nt sequence in a class 1 integron of V. cholerae O1 isolated in Vietnam in 2004 (AB200915). This ORF had 75% nt similarity with qnrVC1, and it was designated qnrVC2.
The qnrVC gene cassettes are characterized by attC sites, which are absent in all others qnr genes already identifi ed. Supporting the hypothesis of Vibrionaceae as the source of qnr genes (3), the qnrVC1 attC site has 89% identity with V. parahaemolyticus repeats, and the qnrVC2 putative attC site has 96% identity with a V. cholerae repeat sequence, both of which are characteristic structures of SIs. Rowe-Magnus et al. (5) reported that SI repeat recombination sites are species specifi c and these elements would be the source of gene cassettes found in drug-resistance integrons, such as class 1 integrons. Therefore, we hypothesize that qnrVC1 gene cassette could have originated from a V. parahaemolyticus SI, whereas qnrVC2 originated in V. cholerae. Curiously, no chromosomal qnr genes identifi ed in Vibrionaceae were associated with SIs.
The qnrVC1 gene showed high amino acid divergence compared with all qnr genes described, with similarities ranging from 44% to 69%. The qnrVC1 had a G + C content of 36.8%, which is considerably different from that of the V. cholerae genome (47.5%) and is evidence of horizontal gene transfer.
Promoter Pc, which is found in the 5′ conserved segment of the integron carrying qnrVC1, was identical to the hybrid confi guration described (9,10) and is defi ned by 1 point mutation in the canonical -35 region. The hybrid Pc version has 20-fold lower promoter activity than that of the wild-type (strong) promoter. However, another study verifi ed expression of genes inserted into integrons under the control of a hybrid Pc confi guration (11). Moreover, in silico analysis detected a putative promoter (-35 TTGAGA [17 nt] -10 TAGTCT) in the 5′ untranslated region of the qnrVC1 gene cassette. Therefore, our fi ndings characterize conditions for qnrVC1 expression in the class 1 integron.
Quinolones have excellent antimicrobial activity against V. cholerae (12). Strain VC627 shows a 10-fold increase in resistance to ciprofl oxacin (MIC 0.25 μg/mL) compared with strains from the seventh pandemic lineage in Brazil (MIC 0.02 μg/mL). This increased resistance was also observed in a Shigella fl exneri strain harboring qnrS (13,14) and V. splendidus harboring chromosomal qnrVS1 and qnrVS2 (4). This MIC value resembles that caused by gyrA mutations in V. cholerae ciprofl oxacin-resistant strains (15). Therefore, dissemination of qnr genes by lateral gene transfer may determine, in a 1-step fashion, the same drug-resistance profi le caused by acquired mutations in housekeeping genes.

Conclusions
Our study reports characterization of 2 new qnr genes associated with SI attC sites in typical class 1 integrons and their emergence in V. cholerae. These genetic features have not been observed in either chromosomal-or plasmidborne qnr determinants already described.
Our study suggests that, at least in Vibrionaceae, quinolone-resistance genes were mobilized from SIs by class 1 integrase activity because qnrVC1 and qnrVC2 attC elements are similar to V. parahaemolyticus and V. cholerae SI repeats. These fi ndings corroborate the hypothesis that SIs are a source of cassettes present in drug-resistance integrons (5). These elements are part of a reasonable scenario for a 1-step acquisition of quinolone resistance by bacteria (e.g., Mycobacterium spp. and Neisseria spp.). This acquisition may jeopardize treatment and control and have adverse consequences on infections caused by these organisms.  , EDL69958). Support of the branching order was determined by 1,000 interior branch test replicates. The distancebased tree was generated by using p distance with the neighborjoining method with MEGA version 3.1 (www.megasoftware. net). Values along the horizontal lines are the interior-branch test percentages after testing 1,000 topologies. Scale bar indicates the number of substitutions per alignment site, which is refl ected by branch lengths.