Potential synergism between florfenicol (FF) and thiamphenicol (TAP) was investigated for in vitro efficacy against Actinobacillus pleuropneumoniae and/or Pasteurella multocida as well as in vivo efficacy in swine. Among isolates of A. pleuropneumoniae (n = 58) and P. multocida (n = 79) from pigs in Taiwan that were tested, high percentages showed resistance to FF (52 and 53%, respectively) and TAP (57 and 53%, respectively). Checkerboard microdilution assay indicated that synergism [fractional inhibitory concentration index (FICI) ≤ 0.5] was detected in 17% of A. pleuropneumoniae (all serovar 1) and 24% of P. multocida isolates. After reconfirming the strains showing FICI ≤ 0.625 with time kill assay, the synergism increased to around 32% against both bacteria and the number could further increase to 40% against resistant A. pleuropneumoniae and 65% against susceptible P. multocida isolates. A challenge-treatment trial in pigs with P. multocida showed that the FF + TAP dosage at ratios correspondent to their MIC deduction was equally effective to the recommended dosages. Further on the combination, the resistant mutation frequency is very low when A. pleuropneumoniae is grown with FF + TAP and similar to the exposure to sub-inhibitory concentration of FF or TAP alone. The degree of minimum inhibitory concentration (MIC) reduction in FF could reach 75% (1/4 MIC) or more (up to 1/8 MIC for P. multocida, 1/16 for A. pleuropneumoniae) when combined with 1/4 MIC of TAP (or 1/8 for A. pleuropneumoniae). The synergism or FICI ≤ 0.625 of FF with oxytetracycline (47%), doxycycline (69%), and erythromycin (56%) was also evident, and worth further investigation for FF as a central modulator facilitating synergistic effects with these antimicrobials. Taken together, synergistic FF + TAP combination was effective against swine pulmonary isolates of A. pleuropneumoniae and P. multocida both in vitro and in vivo. Thus, this study may offer a potential alternative for the treatment of A. pleuropneumoniae and P. multocida infections and has the potential to greatly reduce drug residues and withdrawal time.

No abstract

The aim of this study is to investigate whether combinational use of efflux pump inhibitors (EPIs) could improve florfenicol (FF) antimicrobial activity. Five EPIs including Carbonyl Cyanide Chlorophenylhydrazone (CCCP), omeprazole, Phenylalanine-arginine [Formula: see text]-naphthylamide (PA[Formula: see text]N), reserpine and verapamil were individually combined with FF and the minimum inhibitory concentration (MIC) against porcine Actinobacillus pleuropneumoniae and Pasteurella multocida were evaluated by the broth microdilution assay. The results indicated that CCCP demonstrated substantial improvement on the antimicrobial activity (FF MIC reduction [Formula: see text] folds) while PAßN showed minimal effect at high concentrations (80–120[Formula: see text][Formula: see text]g/mL). The MICs of FF were further examined with CCCP at 5[Formula: see text][Formula: see text]g/mL against resistant A. pleuropneumoniae and at 2[Formula: see text][Formula: see text]g/mL against resistant P. multocida. With this combination, a total of 75% (6/8) of A. pleuropneumoniae and 100% (8/8) of P. multocida resistant isolates showed significantly reduced ([Formula: see text] folds) FF MICs. In addition, 100% of the 16 resistant bacterial strains carried the floR gene that regulates the phenicol-specific efflux pump (FloR pump). The time kill experiments were used to verify the effect of CCCP demonstrating a bactericidal effect in resistant A. pleuropneumoniae and synergistic effect in resistant P. multocida. In contrast, the FF MICs were not significantly affected in the FF susceptible strains of these two bacteria. These findings suggested that despite small sample sizes, consistent beneficial effects of CCCP were evident such that the combinational use of selective EPI with FF might be an alternative antibacterial strategy against FF resistant A. pleuropneumoniae and P. multocida. The anti-efflux mechanism was indicated, but more researches to further decipher the mechanisms of CCCP’s effects are warranted.