Effect of Carbonyl Cyanide Chlorophenylhydrazone on Intrabacterial Concentration and Antimicrobial Activity of Amphenicols against Swine Resistant Actinobacillus pleuropneumoniae and Pasteurella multocida

Abstract

Effects and mechanism of carbonyl cyanide chlorophenylhydrazone (CCCP) on antimicrobial activity of florfenicol (FF) and thiamphenicol (TAP) were investigated against amphenicol-resistant Actinobacillus pleuropneumoniae and Pasteurella multocida isolated from diseased swine. Broth microdilution and time-kill assays indicated that CCCP dose-dependently and substantially (4-32 fold MIC reduction) improved amphenicol antimicrobial activity. When combined with CCCP at the lowest literature reported dose (2-5 μg/mL), 85% FF resistant A. pleuropneumoniae and 92% resistant P. multocida showed significantly reduced FF MICs (≥ 4-fold). In contrast, none or few of the susceptible A. pleuropneumoniae and P. multocida had FF MICs reduction ≥ 4-fold. 90% FF resistant A. pleuropneumoniae and 96% resistant P. multocida carried the floR gene, indicating strong association with the FloR efflux pump. With CCCP, the intracellular FF concentration increased by 71% in floR+ resistant A. pleuropneumoniae and 156% in floR+ resistant P. multocida strains but not the susceptible strains. The degree of reduction in TAP MICs was found consistently in parallel to FF for both bacteria. Taken together, partially attributed to blockage of drug-efflux, the combination of FF or TAP with CCCP at sub-cytotoxic concentrations was demonstrated and showed feasibility to combat amphenicol-resistant A. pleuropneumoniae and P. multocida isolated from diseased swine.