Study On Drug Resistance Mechanism Of Aeromonas Hydrophila Isolated From Freshwater Fish Species

Aeromonas hydrophila(Ah), belonging to family Aeromonadaceae and genus Aeromonas, is a kind of typical and conditional pathogenic bacteria which could infect human, poultry and aquatic animals. There have been lots of reports about the outbreak death of bacterial septicemia from aquatic farming animals caused by Aeromonas hydrophila. Because of the irrational use of antibiotics, the resistance of Ah isolates to fluoroquinolons and tetracyclines gradually increased recently. Therefore, this study performed the following experiments to explore the drug-resistance mechanism of Ah to fluoroquinolons and tetracyclines.Firstly, 10 strains were selected from Ah clinical isolates which were identified from farming carp and crucian in the lab. Combining the gram staining, physiological and biochemical identification, and molecular characteristics, these strains were identified and the results showed that 10 strains were all gram-negative and oxidizing enzyme positive. They could liquefy gelatin, ferment glucose and produce acid and gas, and so on. All of the biochemical and physiological characteristics were consistent with the national standard of Ah. Blast analysis indicated that 16 S r RNA sequences of the strains were highly identical to Aeromonas hydrophila in Gen Bank database with 99% identity. According to the results above, the selected 10 strains of bacteria were all identified to be Aeromonas hydrophila.The antibiotic susceptibility of 10 Ah strains to many kinds of antibiotics were detected by K-B disk diffusion and broth microdilution method. 9 Ah strains sensitive to fluoroquinolones and 7 Ah strains sensitive to tetracyclines were then selected to be the objectives of this study. In order to investigate the relationship between antibiotic susceptibility and gene mutations, efflux pump activities of selected strains, the strains were induced in vitro to obtain the drug resistance to fluoroquinolons and tetracyclines through adding norfloxacin(NF), enrofloxacin(EN) and doxycycline(DO)to the solid medium. The minimal inhibitory concentrations(MICs) of induced strains to NF, EN, DO and other 16 kinds of non-selected antibiotics were determined. Meanwhile, MIC values were also determined after adding efflux pump inhibitor 1-Methyl-2-pyrrolidinone(NMP) to the medium in order to evaluate the effects of efflux function on drug-resistance of the strains. The cross-resistance of induced strains to other antibiotics was also detected. Additionally, drug-resistance related genes in drug-induced strains were determined. In NF and EN induced strains, gry A and par C were amplified and the sequences were analyzed and compared. In DO induced strains, five tet genes were amplified and sequenced.The results showed that the MICs of the 9 strains induced by EN and NF increased 409.6- and 4096-fold after induction, respectively, while the MICs of strains to non-selected fluoroquinolones and other antimicrobials also changed greatly. Gene mutations were found in the quinolone resistance-determining region(QRDR) encoded by gyr A and par C genes after induction, which were Ser 83 to Ile in Gyr A and Ser 87 to Ile/Arg in Par C. After NMP was added, MICs of all selected strains to EN and NF decreased to different degrees. Cross-resistance of induced strains was closely related to the strains, among of which, E3, N3, E8 and N8 had no cross-resistance to 16 kinds of non-selected antibiotics. The strains induced by EN and NF didn’t produce the cross-resistance to aminoglycosides and rifamycins. All induced strains had great cross-resistance to tetracyclines and chloramphenicols.The MICs of the 7 DO-induced strains to DO increased significantly after induction, while the MICs of strains to other antibiotics also increased to different degrees. The MICs of induced strains to fluoroquinolone increased dozens to thousands of times than that of control. The induced strains exhibited a little higher sensitivity to aminoglycoside and rifampicin. However, the MICs of all induced strains to DO decreased to different degrees after adding NMP to the medium. The detection of tet genes indicated that the tet A and tet E were positive in D7 after induction and the tet C gene was positive in T2, D2 before and after induction. The tet E genes were detected in strains T1, 3, 4, 5, 6, 7 and D1, 3, 4, 5, 6, 7, no matter whether it was induced or not.The research suggested that more attention should be paid to the cross-resisance when applying antibiotics to prevent and control diseases clinically. The drug-resistance mechanism of Ah to fluoroquinolone might include the target gene mutations and an active efflux system. The active efflux system might also exsit in resistance mechanisms of Ah to tetracyclines. tet E gene may be the predominant gene mediating tetracyclines-resistance of Ah, which provided theoretical basis to clarify the drug-resistance mechanism of Aeromonas hydrophila to tetracyclines.