Vibrio splendidus as the Source of Plasmid-Mediated QnrS-Like Quinolone Resistance Determinants

Abstract

Plasmid-mediated quinolone resistance due to Qnr-like proteins is increasingly reported worldwide (5, 9) since the first report from the United States in 1998 (4). Six variants of both QnrA and QnrB determinants and two of QnrS have been described so far (9). QnrS1 was firstly identified in a Shigella flexneri isolate from Japan (3) and then from several other countries (2, 6), whereas the QnrS2 variant (92% identity with QnrS1) was only identified from a mobilizable IncQ-related plasmid from Germany (1) and also reported from a single non-Typhi Salmonella isolate from the United States (2). Whereas the origin of the qnrA gene was identified as being Shewanella algae (7), the reservoirs of the qnrB and qnrS genes remain unknown. However, we have already considered that they might be close to some species of the Vibrionaceae family which possess chromosome-encoded Qnr-like determinants (40% to 67% identity with QnrA, QnrB, and QnrS determinants) conferring resistance to quinolones (8). By in silico analysis of the recently released genome sequence of Vibrio splendidus strain 12B01 (accession no. {type:entrez-nucleotide,attrs:{text:NZ_AAMR01000029,term_id:84391733,term_text:NZ_AAMR01000029}}NZ_AAMR01000029, we identified an open reading frame (V12B01_17906) coding for a 218-amino-acid protein sharing 84% and 88% amino acid identity with QnrS1 and QnrS2, respectively. Therefore, DNA fragments corresponding to the entire sequences of the qnrVS1 and qnrVS2 genes were PCR amplified from V. splendidus strains 12B01 (seawater, United States) and LGP32 (oyster, France) (D. Mazel, personal collection), respectively, were cloned into the kanamycin resistance pCR-BluntII-TOPO plasmid (Invitrogen, Life Technologies, Cergy-Pontoise, France) as previously described (8), together with the qnrS1 and qnrS2 genes as controls. When overexpressed in Escherichia coli TOP10 (Invitrogen), recombinant plasmids with the qnrVS1 (pVS1) and qnrVS2 (pVS2) genes conferred an 8-fold increase in the MIC of nalidixic acid and 4- to 16-fold increases in the MICs of fluoroquinolones compared to those for E. coli TOP10 (Table ​(Table1),1), as previously reported for other species of the Vibrionaceae family (8). In addition, MICs of quinolones for E. coli TOP10 harboring recombinant plasmid pVS1 or pVS2 were very similar to those obtained with E. coli with recombinant plasmids harboring the qnrS1 (pS1) and qnrS2 (pS2) genes (Table ​(Table11). TABLE 1. MICs of quinolones and fluoroquinolones for V. splendidus isolates and E. coli recombinant clones The QnrVS1 and QnrVS2 protein sequences were almost identical, with only four amino acid substitutions, corresponding to 98.1% amino acid identity. In addition, the QnrVS1 and QnrVS2 determinants shared 83.9% and 83.1% amino acid identity, respectively, with the plasmid-mediated QnrS1 determinant and 87.6% and 87.1% identity, respectively, with plasmid-mediated QnrS2. In silico analysis showed that chromosome-encoded Qnr-like proteins were identified in other species of the Vibrionaceae family (V. angustrum, V. alginolyticus, V. fischeri, V. vulnificus, V. parahaemolyticus, V. salmonicida, Photobacterium profundum) and shared no more than 64% amino acid identity with any Qnr determinants, including the QnrS2 determinant. In addition, the G+C contents of the qnrVS1 (45.2%) and qnrVS2 (45.1%) genes are close to those of qnrS1 and qnrS2 (both are 43.8%). These results underline that gram-negative bacterial species of the aquatic environment may be the reservoir of plasmid-mediated Qnr-like determinants. Although the exact progenitor of the plasmid-encoded QnrS determinant remains unknown, it should be closely related to V. splendidus.