qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin

Abstract

In a previous study, four Salmonella isolates from humans in the Henan province of China showed reduced susceptibility to ciprofloxacin (MIC, 0.125 to 0.25 microg/ml) but were susceptible to nalidixic acid (MIC, 4 to 8 microg/ml). All isolates were negative for known qnr genes (A, B, and S), aac(6')Ib-cr, and mutations in gyrA and parC. Plasmid DNA was extracted from all four isolates and transformed into Escherichia coli TG1 and DH10B cells by electroporation, and transformants were selected on 0.06 microg/ml ciprofloxacin containing brain heart infusion agar plates. Resistance to ciprofloxacin could be transferred by electroporation, and a similar 4,270-bp plasmid was found in all transformants. By sequence analysis, the plasmid was found to carry an open reading frame that had similarities to other qnr genes and that encoded a 214-amino-acid pentapeptide repeat protein. This gene, designated qnrD, showed 48% similarity to qnrA1, 61% similarity to qnrB1, and 41% similarity to qnrS1. Further subcloning of the qnrD coding region into the constitutively expressed tetA gene of vector pBR322 showed that the gene conferred an increase in the MIC of ciprofloxacin by a factor of 32 (from an MIC of 0.002 to an MIC of 0.06 microg/ml). For comparison, qnrA1 and qnrS1 were also subcloned into pBR322 and transformed into DH10B cells, conferring MICs of 0.125 and 0.5 microg/ml, respectively. A phylogenetic analysis of all known qnr sequences was performed and showed that qnrD was more closely related to the qnrB variants but formed an independent cluster. To our knowledge, this is the first description of this qnrD gene.

FIG. 1.

QnrD amino acid sequence. (a) Alignment of amino acid sequences encoded by the qnrA1, qnrB1, qnrS1, and qnrD genes obtained using AlignX with Vector NTI software (InformaxVector NTI Suite 8). (b) Hypothetical structure of the QnrD protein. The amino acid sequence was represented and divided into pentapeptide repeats. The conserved amino acids according to the consensus sequence (A/C/S/T/V)(D/N)(L/F)(S/T/R)(G/R) (22) are in boldface type, and the most characteristic pentapeptide units are underlined.

FIG. 2.

Distance tree between the known qnr genes and variants at the nucleotide level. The evolutionary history was inferred using the neighbor-joining method. Phylogenetic analyses were conducted using MEGA 4 software (23).

FIG. 3.

Graphical map of plasmid p2007057 (GenBank accession number FJ228229). qnrD (positions 554 to 1198) encodes a pentapeptide repeat protein related to previously described qnr genes, ORF2 (positions 1397 to 1597) is a hypothetical protein with 88% similarity to a hypothetical protein described previously (accession number AF448250), ORF3 (positions 1828 to 2091) is a hypothetical protein with no significant matches, ORF4 (positions 2347 to 3309) is a hypothetical protein with 83% similarity to a partial sequence of a mob gene described previously (accession number EU90225), and ORF5 (positions 3312 to 3842) is a hypothetical protein with 73% similarity to a partial sequence of a hypothetical protein described previously (accession number DQ995354). Possible ORFs were searched using Vector NTI software. BLAST searches were performed using the blastn algorithm at the NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi).