Pharmacokinetic/Pharmacodynamic Integration to Evaluate the Changes in Susceptibility of Actinobacillus pleuropneumoniae After Repeated Administration of Danofloxacin

To evaluate the relationship between pharmacokinetic/pharmacodynamic (PK/PD) parameters and changes in susceptibility and resistance frequency of Actinobacillus pleuropneumoniae CVCC 259, a piglet tissue cage (TC) infection model was established. After A. pleuropneumoniae populations maintained at 108 CFU/mL in TCs, piglets were treated with various doses of danofloxacin once daily for 5 consecutive days by intramuscular injection. Both the concentrations of danofloxacin and the population of vial cells were determined. Changes in susceptibility and resistance frequency were monitored. Polymerase chain reaction (PCR) amplification of quinolone resistance-determining regions (QRDRs) and DNA sequencing were performed to identify point mutations in gyrA, gyrB, parC, and parE genes. Furthermore, the susceptibility of mutants to danofloxacin and enrofloxacin was determined in the presence or absence of reserpine to assess whether the mutants were caused by efflux pumps. The MICs and resistant frequency of A. pleuropneumoniae both increased when danofloxacin concentrations fluctuated between MIC99 (0.05 μg/mL) and MPC (mutant prevention concentration, 0.4 μg/mL). As for PK/PD parameters, the resistant mutants were selected and enriched when AUC24h/MIC99 ranged from 34.68 to 148.65 h or AUC24h/MPC ranged from 4.33 to 18.58 h. Substitutions of Ser-83→Tyr or Ser-83→Phe in gyrA and Lys-53→Glu in parC were observed. The susceptibility of mutants obtained via danofloxacin treatment at 1.25 and 2.5 mg/kg were less affected by reserpine. These results demonstrate that maintaining the value of AUC24h/MPC above 18.58 h may produce a desirable antibacterial effect and protect against A. pleuropneumoniae resistance to danofloxacin

Different Effects of Farrerol on an OVA-Induced Allergic Asthma and LPS-induced Acute Lung Injury

BACKGROUND: Farrerol, isolated from rhododendron, has been shown to have the anti-bacterial activity, but no details on the anti-inflammatory activity. We further evaluated the effects of this compound in two experimental models of lung diseases. METHODOLOGY/PRINCIPAL FINDINGS: For the asthma model, female BALB/c mice were challenged with ovalbumin (OVA), and then treated daily with farrerol (20 and 40 mg/kg, i.p.) as a therapeutic treatment from day 22 to day 26 post immunization. To induce acute lung injury, female BALB/c mice were injected intranasally with LPS and treated with farrerol (20 and 40 mg/kg, i.p.) 1 h prior to LPS stimulation. Inflammation in the two different models was determined using ELISA, histology, real-time PCR and western blot. Farrerol significantly regulated the phenotype challenged by OVA, like cell number, Th1 and Th2 cytokines levels in the BALF, the OVA-specific IgE level in the serum, goblet cell hyperplasia in the airway, airway hyperresponsiveness to inhaled methacholine and mRNA expression of chemokines and their receptors. Furthermore, farrerol markedly attenuated the activation of phosphorylation of Akt and nuclear factor-κB (NF-κB) subunit p65 both in vivo and in vitro. However, farrerol has no effect on the acute lung injury model. CONCLUSION/SIGNIFICANCE: Our finding demonstrates that the distinct anti-inflammatory effect of farrerol in the treatment of asthma acts by inhibiting the PI3K and NF-κB pathway

The PK–PD Relationship and Resistance Development of Danofloxacin against Mycoplasma gallisepticum in An In Vivo Infection Model

Mycoplasma gallisepticum is the causative agent of chronic respiratory disease (CRD), a prevalent disease of poultry, which is responsible for significant economic losses in farms. Although several antimicrobial agents are currently recommended for the treatment and prevention of M. gallisepticum infections, investigations of M. gallisepticum have been hampered by their fastidious growth requirements and slow growth rate. As such, little work has been conducted concerning the PK/PD relationship and mechanisms of antibiotic resistance between antimicrobials against M. gallisepticum. In the present study, danofloxacin was orally administrated to the infected chickens once daily for 3 days by an established in vivo M. gallisepticum infection model. Not only the concentrations of danofloxacin in plasma and lung tissues were analyzed, but also the counting of viable cells and changes in antimicrobial susceptibility in air sac and lung were determined. The PK and PD data were fitted by WinNonlin to evaluate the PK/PD interactions of danofloxacin against M. gallisepticum. PCR amplification of quinolone resistance-determining regions (QRDRs) and DNA sequencing were performed to identify point mutations in gyrA, gyrB, parC, and parE of the selected resistant mutant strains. In addition, susceptibility of enrofloxacin, ofloxacin, levofloxacin, gatifloxacin, and norfloxacin against these mutant strains were also determined. The PK profiles indicated that danofloxacin concentration in the lung tissues was higher than plasma. Mycoplasmacidal activity was achieved when infected chickens were exposed to danofloxacin at the dose group above 2.5 mg/kg. The ratios of AUC24/MIC (the area under the concentration-time curve over 24 h divided by the MIC) for 2 log10 (CFU) and 3 log10 (CFU) reduction were 31.97 and 97.98 L h/kg, respectively. Substitutions of Ser-83→Arg or Glu-87→Gly in gyrA; Glu-84→Lys in parC were observed in the resistant mutant strains that were selected from the dose group of 1 and 2.5 mg/kg. MICs of danofloxacin, enrofloxacin, ofloxacin, levofloxacin, gatifloxacin, and norfloxacin against the resistant mutant strains with a single mutation in position-83 were higher than that with a single mutation in position-87. These findings suggested that danofloxacin may be therapeutically effective to treat M. gallisepticum infection in chickens if administered at a dosage of 5.5 mg/kg once daily for 3 days

Effects of farrerol on OVA-induced inflammatory cell recruitment and mucus hyper-secretion.

<p>Inflammatory cell counts in BALF obtained from sensitized mice 24 h after the last farrerol treatment. Differential cell counts were identified eosinophil (Eos), macrophage (Mac), neutrophil (Neu) and lymphocyte (Lym).</p

Effect of farrerol on Akt, P70S6K, and MAPK activation in vivo.

<p>Immunoblotting of Akt, P70S6K, and MAPK in proteins extracts of lung tissues isolated from mice 24 hours after the last OVA challenge pretreated with 20 or 40 mg/kg farrerol. β-actin was used as an internal control. Experiments were repeated three times and similar results were obtained.</p

Effect of farrerol (20 and 40 mg/kg) on the IL-4, IL-5, and IL-13, Akt, P70S6K, and IκBα phosphorylation and degradation in vitro.

<p>The supernatant of Th2 cells was measured by sandwich ELISA. The values represent the mean±SEM of three independent in vitro experiments. Th2 cells were cultured with anti-CD3 (5 µg/ml) for 1 h (1 mg/L), total cellular proteins were analyzed by western blot with specific antibodies. β-Actin was used as an internal control. Experiments were repeated three times and similar results were obtained.</p

Effects of farrerol on cytokine and chemokine levels in BALF and serum immunoglobulin production in vivo.

<p>BALF and blood were collected and centrifuged 24 hours after the last OVA challenge, and the supernatants and serum were measured by ELISA. Results of IgE in serum (mean±SEM n = 10) are expressed as Optical Density values and are representative of at least three independent in vivo experiments. *p<0.05, **p<0.01 vs. OVA.</p

Effects of farrerol on lung tissue eosinophilia and mucus production.

<p>Histologic examination of lung tissue eosinophilia using HE staining (A) and mucus secretion using AB-PAS staining (B) from: (a) PBS-challenged mice; (b) OVA-challenged mice; (c) OVA-challenged mice treated with farrerol (20 mg/kg); (d) OVA-challenged mice treated with farrerol (40 mg/kg); (e) OVA-challenged mice treated with dexamethasone (2 mg/kg, magnification×400). Quantitative analyses of inflammatory cell infiltration (C) and mucus production (D) in lung sections were performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034634#pone.0034634-Zhou2" target="_blank">[35]</a>. At least 5 different fields for each lung section was performed to score the inflammatory cells and goblet cells. Mean scores were obtained from 5 mice. **P < 0.01. vs. OVA.</p

Chemical structure of farrerol.

<p>Chemical structure of farrerol.</p