Research On Characterization Of Brucella Mutations In The Pressure Of Antimicrobial Drug Selection

Brucellosis is on the rise in many regions in China. Antibiotic therapy is the first choice totreat the Brucella-infected patients. Since improper use of antibiotics leads to resistancemutation, correctly choosing the antibiotics for the treatment of Brucella infection is veryimportant. To assist doctors to dose the antibiotics, a theory of mutant prevention has beenproposed stating that a dosage of an antibiotic above the minimal inhibitory concentration(MIC) but below the mutant prevention concentration (MPC) kills sensitive bacteria butenriches the resistance mutants; therefore, dosage between MIC and MPC is called mutantselection window (MSW). In order to avoid resistance, antibiotics with lower MPC should beused so dosage above MPC can be easily achieved.In the present project, MSW and selection index (SI=MPC/MIC) for four antibiotics(rifampin, rifabutin, cefdinir, cephalosporin cefepime) to Brucella strains S2were determinedby broth dilution method and agar dilution method; MSWs were4-460μg/mL,0.703-512μg/mL,0.2361-0.5764, and0.0083μg/mL-0.2951μg/mL and SIs were115,728,2.44,and for the4antibiotics respectively. Our results indicated that cefdinir and cephalosporincefepime have favorable MSW and SI for preventing the antibiotic resistance; rifampin andrifabutin were prone to generate drug resistance.To further understanding the resistance, mutations in the drug target coding genes werecharacterized by sequencing the DNA extracted from the selected colonies under differentdrug concentrations. Rifampin and rifabutin resistant mutations were found at H536Y, S532L,H536R, R539H amino acid residues in the rpoB gene. In addition, rifampicin selected Q523Lwhile rifabutin selected the521~523LSQ (1561~1569bp) deletion, Q523K and S541Lmutation. The penA gene that encodes the target for cephalosporin was found mutated withinthe amino acid sequence385to560with N477S, S484L, and A559D point mutation weremost common followed by F519I, L387F, A559T, G485V, T499S mutations; with increase ofthe drug concentration, eventually only G485V T499S remained. In the mrcA gene that encodes the target for cefdinir no mutations were found, in consistence with its low MPC andsmall SI.Our studies should provide useful information for doctors in choosing the right antibioticsto treat brucellosis.