Mechanisms of Quinolone Resistance in Vibrio parahemolyticus strains Isolated from Imported Shrimp

Sixty-one quinolone-resistant strains of Vibrio parahaemolyticus were isolated from 247 shrimp samples. These isolates had MICs of 256 and 4-8 µg/mL for nalidixic acid and ciprofloxacin respectively. Template DNA of the 61 isolates were screened by PCR for the quinolone resistance genes. Purified PCR amplicons of gyrA, gyrB and parC from these isolates were sequenced and analyzed for point mutations that confer resistance to these antibiotics. Point mutations in the quinolone resistance determining region (QRDR) of GyrA at positions 68, 83, 85 and 89 and in ParC at positions 85 as well as in the non QRDR of GyrA at positions 48 along with 4 different point mutations in GyrB at positions 311, 354, 360 and 374 conferred resistances to these antibiotics.  Structural analysis were undertaken to determine the role of these four novel point mutations in V. parahaemolyticus at codons 48, 68, 83 and 89 on GyrA in enhanced MICs to quinolone antibiotics. Homology analysis indicated that all four mutations are localized in the vicinity of quinolone binding and DNA binding major grooves. These four mutations are known to play pivotal roles in readjusting the position of α-helices, stabilization of GyrA, loss of electrical charges or the formation of A2 dimers resulting in enhanced MICs to the quinolone antibiotics. Ethidium bromide uptake experiments indicated higher efflux pump activities in drug resistant V. parahaemolyticus than their sensitive counterparts. Our results indicate that imported shrimp is a reservoir of quinolone-resistant V. parahaemolyticus.