Gene floR e a resistência ao florfenicol em isolados de Aeromonas spp. autóctones de organismos aquáticos

Abstract

ABSTRACT.-Silva N.C.S.L., Nogueira J.F., Gouveia J.J.S., Costa M. The floR gene is described in related literature as responsible for resistance to florfenicol, which is a widely used antimicrobial agent in aquaculture. This gene has been reported in many species of bacteria, including the genus Aeromonas. These bacteria cause high mortality in fish farming bringing economic losses. It is important that studies of this gene and possible mutations that can lead to changes in the structure and function of the protein. The aim of this study was to characterize the floR gene in isolates of Aeromonas spp. and check if the presence of this gene is associated with resistance to florfenicol in Aeromonas spp. obtained from the San Francisco Valley. PCR (Polymerase Chain Reaction) were also performed to verify the presence of the floR gene in 27 isolates of Aeromonas spp. Positive samples for the presence of the gene were sequenced and analyzed for the presence of polymorphisms using alignments. Different haplotypes detected were used for analysis with the SIFT and PolyPhen programs for prediction of changes in protein function. The structural modeling of protein encoded by the floR gene was performed using the Modeller software, and the models were evaluated by Procheck, Verify3D and Whatif. The similarity of the dimensional structure of reference protein with the dimensional structures of the proteins encoded by the different haplotypes was compared by TM-align. Bacterial resistance to florfenicol was assessed by the microdilution test, which was also performed in the presence of carbonyl cyanide m-chlorophenyl hydrazone to verify the effect of inhibiting the efflux pump. 14 isolates were positive for the presence of floR gene and 10 were sequenced and allowed the identification of three polymorphisms in the floR gene, which led to construction of three different haplotypes (TAA TTA and CTG). The analyzes carried out with the SIFT and PolyPhen programs showed that the TTA and TAA haplotypes could probably change the protein structure-function. Proteins modeled for the three haplotypes were found to have substantially the same structural conformation with each other. All isolates presenting the gene were resistant to florfenicol and those who did not have were sensitive. The test in the presence of carbonyl cyanide m-chlorophenylhydrazone was conducted for three isolates, representing each single haplotype and was observed inhibition of bacterial growth at all concentrations independent of the haplotype. The results of this study show that resistance to flofenicol in Aeromonas spp. may be explained by the presence of floR gene and that this gene is associated with an efflux pump. Mutations observed in floR gene do not appear to be involved with chenges in structure and function of the protein encoded by gene.