In vivo Pharmacokinetic/pharmacodynamic relationship of florfenicol in combination with doxycycline against Riemerella anatipestifer in ducks and the effect upon resistance development.

Abstract

Antimicrobial chemotherapy is necessary to control Riemerella anatipestifer (RA), among which florfenicol (FF) is regarded as one of the preferred options. Based on the consideration of drug combination to improve efficacy, the pharmacokinetics and pharmacodynamics of FF combined with doxycycline (DOX) against RA were studied. FF was administered at doses of 20 or 40 mg/kg in combination with DOX (1, 2.5, 5, 10, or 20 mg/kg) via a single intramuscular injection (i.m.). DOX showed slow elimination in ducks with elimination half-life (T) in plasma, lung, and liver of 11.21, 11.53, and 13.01 h, respectively. A single dose of DOX (≥10 mg/kg) combined with FF (20 mg/kg) could exert a bactericidal effect on some tissues (heart, liver, spleen, lungs) in a model of RA strain CVCC3857 (minimum inhibitory concentration (MIC) of FF = 1 µg/mL, MIC of DOX = 2 µg/mL) infection within 24 h, and bactericidal effects (3.01-4.36 logCFU/mL) were achieved in various tissues at a FF dose of 40 mg/kg. The AUC/MIC of DOX combined with FF at 20 mg/kg required to produce a drop of 3 LogCFU/mL was 39.19 h (predicted dose of 25.03 mg/kg) and the value was 19.98 h (predicted dose of 12.76 mg/kg) when the dose of FF was 40 mg/kg. Combination of these two drugs could be used against insensitive strains (RA38 infection model with MIC of FF = 4 µg/mL, MIC of DOX = 2 µg/mL) by administering them twice for 24 h. Continuous passage under antibiotic pressure for 30 days suggested that resistance to FF was delayed in the presence of DOX. Genome resequencing and analyses of single-nucleotide polymorphisms revealed seven mutated genes (fahA, pfam, TonB-dependent receptor gene, proS, porU, RpiB). TonB-dependent receptor genes play a role in bacterial susceptibility. Additionally, both TonB-dependent receptor genes and fahA are involved in bacterial virulence and biofilm formation capabilities. Antimicrobial-treated strains were different from ancestor strains in terms of growth and virulence. Our study provides a data basis for the clinical use of FF and DOX against RA.