Analysis of the mutant selection window and killing of Mycoplasma hyopneumoniae for doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin.

Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating the M. hyopneumoniae type strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent based on microdilution broth and agar dilution methods (bacterial numbers ranged from 105 colony-forming units (CFU)/mL to 109 CFU/mL). The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 µg/mL based on the microdilution broth method, and from 0.008 to 1.0 µg/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 µg/mL. MPC/MIC99 values were ordered tylosin > doxycycline > danofloxacin > tiamulin > valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.

Comparison of the pharmacokinetics of tilmicosin in plasma and lung tissue in healthy chickens and chickens experimentally infected with Mycoplasma gallisepticum.

The objectives of this study were to compare the plasma and lung tissue pharmacokinetics of tilmicosin in healthy and Mycoplasma gallisepticum-infected chickens. Tilmicosin was orally administered at 4, 7.5 and 10 mg/kg body weight (b.w) for the infected and 7.5 mg/kg b.w for the uninfected control group. We found no significant differences in plasma tilmicosin pharmacokinetics between diseased and healthy control chickens. In contrast, the lung tissues in M. gallisepticum-infected chickens displayed a t1/2 (elimination half-life) 1.76 times longer than for healthy chickens. The Cmax (the maximum concentration of drug in samples) of tilmicosin in M. gallisepticum-infected chickens was lower than for controls at 7.5 mg/kg b.w (p < .05), and the AUCinf (the area under the concentration-time curve from time 0 extrapolated to infinity) in infected chickens was higher than for the healthy chickens (p < .05). The mean residence time of tilmicosin in infected chickens was also higher than the healthy chickens. These results indicated that the lungs of healthy chickens had greater absorption of tilmicosin than the infected chickens, and the rate of elimination of tilmicosin from infected lungs was slower.

Pharmacokinetic and Pharmacodynamic Integration and Resistance Analysis of Tilmicosin Against Mycoplasma gallisepticum in an In Vitro Dynamic Model.

Mycoplasma gallisepticum is the major pathogen causing chronic respiratory disease in chickens. In the present study, we successfully established a one-compartment open model with first-order absorption to determine the relationship between tilmicosin pharmacokinetic and pharmacodynamic (PK/PD) indices and M. gallisepticum in in vitro. The aim was to simulate the PK/PD of tilmicosin against M. gallisepticum in lung tissues. The results of static time-killing curves at constant drug concentrations [0-64 minimum inhibitory concentration (MIC)] showed that the amount of M. gallisepticum was reduced to the limit of detection after 36 h when the drug concentration exceeded 1 MIC, with a maximum kill rate of 0.53 h-1. In dynamic time-killing studies, tilmicosin produced a maximum antimycoplasmal effect of 6.38 Log10 CFU/ml reduction over 120 h. The area under the concentration-time curve over 24 h divided by the MIC (AUC24h/MIC) was the best PK/PD parameter to predict the antimicrobial activity of tilmicosin against M. gallisepticum [R2 = 0.87, compared with 0.49 for the cumulative time that the concentration exceeds the MIC (%T > MIC)]. Therefore, tilmicosin showed concentration-dependent activity. Seven M. gallisepticum strains (M1-M7) with decreased susceptibility to tilmicosin were isolated from seven dose groups. These strains of M. gallisepticum had acquired resistance to erythromycin as well as to tylosin. However, no change in susceptibility to amikacin and doxycycline was observed in these strains. Gene mutation analysis was performed on the basis of annotated single nucleotide polymorphisms using the genome of strain S6 as the reference. For strain M5, a G495T mutation occurred in domain II of the 23S rrnA gene. In strain M3, resistance was associated with a T854A mutation in domain II of the 23S rrnB gene and a G2799A mutation in domain V of 23S rrnB. To the best of our knowledge, these tilmicosin resistance-associated mutations in M. gallisepticum have not been reported. In conclusion, tilmicosin shows excellent effectiveness and concentration-dependent characteristics against M. gallisepticum strain S6 in vitro. Additionally, these results will be used to provide a reference to design the optimal dosage regimen for tilmicosin in M. gallisepticum infection and to minimize the emergence of resistant bacteria.

Pharmacokinetic/pharmacodynamic integration of cefquinome against Pasteurella Multocida in a piglet tissue cage model.

To explore the in vivo antimicrobial activity of cefquinome against Pasteurella multocida in piglets, a piglet tissue cage infection model was used in this study. After the population of P. multocida reached 107  CFU/mL in a tissue cage, piglets received an intramuscular administration of cefquinome at 0.2, 0.4, 0.8, 1, 2, and 4 mg/kg once daily for 3 days. To assess the tissue cage pharmacokinetics (PKTCF) of cefquinome, tissue cage fluid was collected for cefquinome analysis at 1, 3, 6, 9, 12, and 24 hr after each of the 3 daily drug administrations. Bacteria were counted every 24 hr after drug administration and at 48 and 72 hr after the last administration. Evaluation of the relationship between pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect showed that the surrogate of %T > minimum inhibitory concentration (MIC) (R2  = 0.981) was the best PK/PD index that correlated with effectiveness of cefquinome against P. multocida. The respective values of %T > MIC required for continuous 1/3-log, 1/2-log, and 1-log reductions were 14.23, 34.45, and 73.44%, respectively, during each 24-hr treatment period. In conclusion, cefquinome exhibited a potent antibacterial effect against P. multocida. When %T > MIC reached 73.44%, cefquinome exhibited a bactericidal effect against P. multocida after three successive daily administrations.

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.

Relationship between danofloxacin PK/PD parameters and emergence and mechanism of resistance of Mycoplasma gallisepticum in In Vitro model.

Mycoplasma gallisepticum is a serious pathogen for poultry that causes chronic respiratory disease in chickens. Increased embryonic mortality, as well as reduced weight gain and egg production have been found in infected chickens, which can lead to considerable economic losses in poultry production. Increased antibiotic resistance compromises the use of tetracyclines, macrolides and quinolones in the farm environment. In the present study, danofloxacin concentrations were simulated below the MIC99, between the MIC99 and MPC (the mutant prevention concentration), and above the MPC in an in vitro dynamic model against M. gallisepticum. The relationship between the simulated danofloxacin pharmacokinetics, pharmacodynamics (PK/PD) parameters and development of resistance for M. gallisepticum was explored based on the available data obtained from various dosing regimens in the in vitro model. Danofloxacin concentration, counts of viable cell and susceptibility were determined during the experiment. The mutations in gyrA, gyrB, parC and parE as well as efflux pumps were examined. The MIC of danofloxacin against M. gallisepticum was increased when drug concentrations were between the lower and upper boundaries of the mutant selection window. The upper boundary of the selection window in vitro was estimated as a Cmax/MPC value of 1. The lower boundary was estimated as Cmax/MPC value of 0.05. Both in terms of the MIC and resistance frequency, M. gallisepticum resistance was developed when danofloxacin concentrations fell inside the mutant selection window (ratios of Cmax to MPC between 0.05 and 1). The single mutation in gyrA (Ser-83→Arg) was found in all mutants, while double mutations in gyrA and parC (Ala-64→Ser) were observed only in the mutant with the highest MIC. In addition, no change of susceptibility in the mutants was observed in the presence of reserpine and carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This suggested that ATP-binding cassette superfamily (ABC transporter) and major facilitator superfamily (MFS transporter) did not play a role in danofloxacin efflux.

Pharmacokinetic/pharmacodynamic assessment of cefquinome against Actinobacillus Pleuropneumoniae in a piglet tissue cage infection model.

To evaluate the relationship between the pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect of cefquinome against Actinobacillus pleuropneumoniae, a tissue cage infection model was established in piglets. In this model, an initial count of A. pleuropneumoniae of approximately 106 CFU/mL was exposed to different concentrations of cefquinome after multiple administration at dosages of 0.2, 0.4, 0.8, 1, 2, 4 mg/kg body weight once a day for 3 days. Concentration of cefquinome and bacterial numbers of A. pleuropneumoniae in the tissue-cage fluid (TCF) were monitered. An inhibitory form of sigmoid maximum effect (Emax) model was used to estimate the relationship between the antibacterial effect and PK/PD indices of cefquinome against A. pleuropneumoniae. The minimum inhibitory concentration of cefquinome against A. pleuropneumoniae was 0.016 µg/mL in TCF. The total maximum antibacterial effect was a 3.96 log10 (CFU/mL) reduction. In addition, the cumulative percentage of time over a 24 h period that the drug concentration exceeds the MIC (%T > MIC) was the pharmacokinetic-pharmacodynamic (PK-PD) index that best correlated with the antibacterial efficacy (R2 = 0.967). The estimated %T > MIC values were 11.59, 27.49, and 59.81% for a 1/3-log10 (CFU/mL) reduction, a 2/3-log10 (CFU/mL) reduction, and a 1-log10 (CFU/mL) reduction, respectively, during the 24h administration period of cefquinome. In conclusion, cefquinome exhibits excellent antibacterial activity and time-dependent characteristics against A. pleuropneumoniae in vivo. Furthermore, these data provide meaningful guidance to optimize regimens of cefquinome to treat respiratory tract infections caused by A. pleuropneumoniae.

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.

Determination of the Mutant Selection Window and Evaluation of the Killing of Mycoplasma gallisepticum by Danofloxacin, Doxycycline, Tilmicosin, Tylvalosin and Valnemulin.

Mycoplasma gallisepticum is a common etiological cause of a chronic respiratory disease in chickens; its increasing antimicrobial resistance compromises the use of tetracyclines, macrolides and quinolones in the farm environment. Mutant selection window (MSW) determination was used to investigate the propensity for future resistance induction by danofloxacin, doxycycline, tilmicosin, tylvalosin and valnemulin. Killing of M. gallisepticum strain S6 by these antimicrobials was also studied by incubating M. gallisepticum into medium containing the compounds at the minimal concentration that inhibits colony formation by 99% (MIC99) and the mutant prevention concentration (MPC). Based on the morphology and colony numbers of M. gallisepticum on agar plates, the four kinds of sera in the order of the applicability for culturing M. gallisepticum were swine serum > horse serum > bovine serum > mixed serum. The MPC/MIC99 values for each agent were as follows: danofloxacin > tilmicosin > tylvalosin > doxycycline > valnemulin. MPC generated more rapid and greater magnitude killing than MIC99 against M. gallisepticum. Under exposure of 105-109 CFU/mL at MPC drug levels, valnemulin had the slowest rate of reduction in viable organisms and danofloxacin had the highest rate of reduction.

[Determination of nine beta-agonist residues in pig tissues by liquid chromatography-tandem mass spectrometry combining with library search].

A new method has been developed using a hybrid triple-quadrupole linear ion trap (QTrap) mass spectrometer for the fast detection and identification of nine beta-agonists, clenbuterol, salbutamol, ractopamine, ritodrine, terbutaline, isoxsuprine, tulobuterol, cimaterol and bambuterol, in one single liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The homogenized tissue samples were purified with liquid-liquid extraction after enzymatic hydrolysis by P-glucuronidase/aryl sulfatase. After gradient elution separation on C(18) LC column using acetonitrile and formic acid aqueous solution as the mobile phases, a multiple reaction monitoring (MRM) scan as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information dependent acquisition (IDA) experiment. Finally, the identification of the drugs was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The analytical method in the present study was well validated and good results were obtained with respect to precision, repeatability and spiked recovery. The limits of detection of residues were 0.1 -0.2 micro g/kg for beta-agonists, and with a linear range from 0.1 to 50.0 micro g/L. Three concentration levels of 0. 5, 1. 0 and 5. 0 pg/kg were spiked in pig tissues, and the overall recoveries were between 72.0% and 95.1% with the relative standard deviations (RSDs) between 3. 1% and 12.1%. The real sample test showed that this method could be used for sensitive and accurate determination of beta-agonist residues in pig tissue

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.

Protocatechuic acid attenuates lipolysaccharide-induced acute lung injury.

Protocatechuic acid (PCA) is a major metabolite of anthocyanins. It has numerous pharmacological effects, including anti-inflammatory, antioxidant, and antitumoral activities. In the present study, we investigated the in vivo protective effect of PCA on acute lung injury (ALI) induced by lipolysaccharide (LPS) in mice. We treated mice with PCA 1 h before the intratracheal (i.n.) administration of LPS. The pulmonary injury severity was evaluated 6 h after LPS administration. We found that pretreatment with a 30 mg/kg of PCA markedly attenuated the LPS-induced histological alterations in the lung. In addition, PCA inhibited the production of several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6, at 6 h in the bronchoalveolar lavage fluid (BALF) after LPS challenge. Furthermore, PCA significantly reduced the number of total cells, neutrophils, and macrophages in the BALF, and it significantly decreased the wet/dry weight (W/D) ratio of lungs and the protein concentration in the BALF. Additionally, Western blotting showed that PCA efficiently blunted nuclear factor-kappa B (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα, as well as the translocation of p65 from cytoplasm to the nucleus. In conclusion, these results indicate that PCA was highly effective in inhibiting acute lung injury (ALI) and may be a promising potential therapeutic reagent for ALI treatment. PCA may utilize the NF-κB pathway to attenuate the nonspecific pulmonary inflammation induced by LPS administration.

[Determination of 12 steroid hormone residues in pig tissues by liquid chromatography-tandem mass spectrometry combining with library search].

A method was developed for the simultaneous determination and identification of 12 steroid hormone residues in pig tissues, including stanolone, aldosterone, boldenone, danazol, metandienone, methyltestosterone, nadrolone, norethindrone, progesterone, stanozolol, testosterone and testosterone propionate, using liquid chromatography-tandem triple-quadrupole linear ion trap mass spectrometry(LC-MS/MS). Homogenized pig tissue samples were purified with a Waters MCX solid phase extraction column after enzymatic hydrolysis by beta-glucuronidase, then separated on a Venusil MP C18 column (100 mm x 2, 1 mm, 3 microm) using gradient elution with the mobile phases of acetonitrile and water with 0.1% (v/v) formic acid. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information dependent acquisition (IDA) experiment. The compound identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The results showed that the limits of detection (LODs, S/N = 3) were in the range of 0.2 - 0.5 microg/kg for the steroid hormones, and with a good linearity (r > 0.99) ranged from 0.5 to 100.0 microg/L. The average recoveries (n = 6) of the 12 steroid hormones spiked in pig tissue samples at 5.0 microg/kg ranged from 72.0% to 98.1% with the relative standard deviations (RSDs) between 3.1% and 12.5%. The method was applied for the qualitative and quantitative determination of steroid hormone residues in pig tissues with sensitive and accurate characteristics.

[Determination of melamine residue in feeds by ultra performance liquid chromatography coupled with electrospray tandem mass spectrometry].

A method for fast determination melamine residue in feeds was established. An ultra performance liquid chromatography coupled with electrospray ionization quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) was used. The residue was quantified with multiple reaction monitoring (MRM) mode. The method was validated and good results were obtained with respect to precision, repeatability and spiked recovery. The limit of detection was 10 microg/kg for melamine, and the linear range was from 10 to 5 000 1g/kg. The average recoveries were between 83% and 94% in the spiked range of 10 - 100 microg/kg, and the relative standard deviations (RSDs) were between 4.2% and 6.5%. The method has good repeatability and high sensitivity, and can be applied for the determination of melamine residue in feeds.