The intrinsic macrolide resistome of Escherichia coli.

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.

Pharmacokinetics of tilmicosin in plasma, urine and feces after a single intragastric administration in donkey (Equus asinus).

Tilmicosin, a macrolide antibiotic, has the potential to treat bacterial infections in donkeys. However, the pharmacokinetics of tilmicosin in donkeys have not been reported. The aim of this study was to investigate the pharmacokinetics of tilmicosin in donkey plasma, urine, and feces after a single intragastric administration to determine the suitability of tilmicosin for donkeys. A total of 5 healthy male donkeys with similar body weights were selected. The donkeys were administered a single dose of 10 mg · kg-1 body weight (BW) tilmicosin by gavage. The concentrations of tilmicosin in plasma, urine, and feces were determined. The results showed that after a single intragastric administration of 10 mg · kg-1 body weight, tilmicosin in donkey plasma reached a maximum concentration of 11.23 ± 5.37 mg · L-1 at 0.80 ± 0.10 h, with a half-life of 14.49 ± 7.13 h, a mean residence time of 28.05 ± 3.05 h, a Cl/F of 0.48 ± 0.18 L · kg-1 · h-1, and a Vd/F of 9.28 ± 2.63 Lkg-1. The percentage of tilmicosin excreted through the urine of donkeys is 2.47%, and the percentage excreted through the feces is 66.43%. Our study provides data to inform the use of tilmicosin in donkeys.

Depletion of tilmicosin residue in Gushi chickens following oral administration via drinking water.

This study aimed to examine the depletion of tilmicosin residues in Gushi chickens following the administration at a concentration of 75 mg/L in their drinking water for three consecutive days. Plasma, liver, kidney, lung, muscle, and skin + fat samples were collected from 6 chickens at 6 h, 1, 3, 5, and 7 days after the treatment. Tilmicosin concentrations in the samples were determined using a high-performance liquid chromatography (HPLC) method. The findings revealed that the highest tilmicosin residues were detected in the liver, followed by the kidney, lung, skin + fat, muscle, and plasma. Notably, at 7 days post-treatment, no drug residue was detected in all samples except for the liver and kidney. The non-compartmental model was employed to calculate relevant pharmacokinetic parameters. The elimination half-lives (t1/2λz ) of tilmicosin were as follows, ranked from long to short: skin + fat (45.42 h), liver (44.17 h), kidney (40.06 h), plasma (37.64 h), lung (31.39 h), and muscle (30.05 h). Considering the current residue depletion and the maximum residue limits (MRLs) set by Chinese regulatory authorities, the withdrawal times for tilmicosin were estimated as 18.91, 10.81, and 8.58 days in the kidney, liver, and skin + fat, respectively. A rounded-up value of 19 days was selected as the conclusive withdrawal time. Furthermore, based on the observed tilmicosin concentrations in plasma and lung, combined with previously reported minimum inhibitory concentration (MIC) values against Mycoplasma gallisepticum, the current dosing regimen was deemed adequate for treating Mycoplasma gallisepticum infections in Gushi chickens.

Disposition kinetics and tissue residues of tilmicosin following intravenous, subcutaneous, single and multiple oral dosing in geese (Anser anser domesticus).

Tilmicosin (TMC), a semi-synthetic macrolide antibiotic, is widely used in veterinary medicine due to its broad-spectrum, bacteriostatic properties. Frequently administered in various birds species, it is likely used off-label in geese as well. The study sought to investigate TMC's pharmacokinetics, tissue residues, in geese through in vivo experiments. The study involved longitudinal open studies on 15 healthy adult males, with three phases separated by one-month washout periods. Geese were administered TMC through intravenous (IV, 5 mg/kg), subcutaneous (SC, 10 mg/kg), and oral (PO, 25 mg/kg for five consecutive days) routes, with blood samples drawn at specific intervals. Tissue samples were also collected for subsequent analysis at pre-assigned times. TMC in goose plasma was quantified by a fully validated HPLC method. Plasma concentrations were quantified up to 4 hr for the PO and IV routes, and up to 10 hr in the SC route. Significant variations in bioavailability were observed between SC (87%) and PO (4%) routes. The body extraction ratio was low at 0.03, suggesting minimal ability of the liver and kidneys to eliminate TMC. Multiple oral doses showed no plasma accumulation, but tissue data revealed extensive distribution and prolonged residence, up to 120 h, suggesting a sustained therapeutic effect despite the brief plasma half-life. Regarding the multiple PO doses, provisional withdrawal times of 6, 7.5, and 8 days were suggested for the liver, muscles, and kidneys, respectively, according to the MRL set for these matrices in chickens by EMA. In conclusion, while the practical oral administration is discouraged at the population level, SC administration of TMC may be appropriate for geese, albeit impractical for flock therapy.

Glutamicibacter nicotianae AT6: A new strain for the efficient biodegradation of tilmicosin.

The degradation of tilmicosin (TLM), a semi-synthetic 16-membered macrolide antibiotic, has been receiving increasing attention. Conventionally, there are three tilmicosin degradation methods, and among them microbial degradation is considered the best due to its high efficiency, eco-friendliness, and low cost. Coincidently, we found a new strain, Glutamicibacter nicotianae sp. AT6, capable of degrading high-concentration TLM at 100 mg/L with a 97% removal efficiency. The role of tryptone was as well investigated, and the results revealed that the loading of tryptone had a significant influence on TLM removals. The toxicity assessment indicated that strain AT6 could efficiently convert TLM into less-toxic substances. Based on the identified intermediates, the degradation of TLM by AT6 processing through two distinct pathways was then proposed.

Production of monoclonal antibody against tylosin and tilmicosin with homogeneous cross-reactivity and its application in lateral flow immunoassay.

To avoid false negative results due to the low cross-reactivity rate (CR) in rapid immunoassay, a group-specific antibody with homogeneous CR toward target compounds is needed for accuracy. In this study, tylosin (TYL) and tilmicosin (TM) were selected as model molecules. Firstly, two-dimensional similarity, electrostatic potential energy, spatial conformation and charge distribution of the haptens TYL-CMO, TYL-6-ACA, TYL-4-APA, TYL-CHO and DES-CMO and target compounds of TYL and TM were obtained using Gaussian 09W and Discovery Studio. The optimal hapten was DES-CMO because it is the most similar to TYL and TM. Subsequently, the mAb 14D5 cell line was obtained with IC50 values of 1.59 and 1.72 ng/mL for TYL and TM, respectively, and a CR of 92.44%. Finally, amorphous carbon nanoparticles (ACNPs) were conjugated with mAb 14D5 to develop an accurate lateral flow immunoassay (LFA) for detection of TYL and TM by the reflectance value under natural light. The recoveries of TYL and TM ranged from 77.18 to 112.04% with coefficient of variation < 13.43%. The cut-off value in milk samples was 8 ng/mL, and the limits of detection were 11.44, 15.96, 22.29 and 25.53 µg/kg for chicken muscle, bovine muscle, porcine muscle and porcine liver samples, respectively, and the results being consistent with HPLC-UV. The results suggest that the developed LFA is accurate and potentially useful for on-site screening of TYL and TM in milk and animal tissue samples.

Pharmacokinetics of tilmicosin in chicken joints based on a novel microdialysis sampling method.

Microdialysis is a continuous direct sampling technique used in live animals to study pharmacokinetic (PK) characteristics of drugs directly in target organs. The antibiotic tilmicosin used to treat arthritis in chickens caused by Mycoplasma synoviae. However, the PK study of tilmicosin in chicken joint has not been reported. The aim of this study was to explore the PK characteristics and penetration of tilmicosin by microdialysis incorporated with High-Performance Liquid Chromatography Mass Spectrometry (HPLC-MS/MS). An articular cavity microdialysis sampling model was established by determining in vivo and in vitro recovery results. Tilmicosin was orally administered to chickens and flow rate testing combined with retro-dialysis were used to determine tilmicosin concentration in the target synovial space. HPLC-MS/MS quantification of tilmicosin from plasma and joint dialysate indicated that recovery was negatively correlated with flow rate and the optimal perfusion rate was determined to be 1.0 µL/min. The AUC, Cmax , MRT and t1/2 in plasma were 4.6, 3.0, 2.2 and 1.6 times higher than in the joint dialysate, respectively, but Tmax did not significantly differ. The penetration of tilmicosin from plasma to joint (AUCdialysate /AUCplasma ) was 0.24 and indicated tilmicosin concentration in joints was much lower than that of plasma. Microdialysis technology provides a novel technique to study pharmacokinetics directly in target tissues and our study provides a reference for the clinical use of tilmicosin for treatment of M. synoviae infections in articular cavities.

In Vitro and In Vivo Activities of Tilmicosin and Acetylisovaleryltylosin Tartrate against Toxoplasma gondii.

Toxoplasma gondii is a widespread intracellular pathogen that infects humans and a variety of animals. The current therapeutic strategy for human toxoplasmosis is a combination of sulphadiazine and pyrimethamine. However, this combination still has a high failure rate and is ineffective against chronic infections. Therefore, it is important to discover a new anti-T. gondii drug that is safer and more effective in both humans and animals. In this study, we describe the anti-T. gondii activities of the 16-membered macrolide tilmicosin and acetylisovaleryltylosin tartrate (ATLL). Both tilmicosin and ATLL potently inhibited T. gondii with a half-maximal effective concentration (EC50) of 17.96 µM and 10.67 µM, respectively. Interestingly, tilmicosin and ATLL had different effects on the parasites. ATLL exhibited a potent inhibitory effect on intracellular parasite growth, while tilmicosin suppressed parasites extracellularly. By studying the lytic cycle of T. gondii after treatment, we found that ATLL potently inhibited the intracellular proliferation of tachyzoites, while tilmicosin affected the invasion of tachyzoites. Immunofluorescence analysis using ATLL-treated T. gondii showed morphologically abnormal parasites, which may be due to the inhibition of tachyzoite proliferation and division. In addition, tilmicosin and ATLL significantly delayed the death of mice caused by acute toxoplasmosis. Our results suggest that ATLL has potent anti-Toxoplasma activity both in vitro and in vivo and may be an alternative to toxoplasmosis in the future.

Immunochromatographic assays based on three kinds of nanoparticles for the rapid and highly sensitive detection of tylosin and tilmicosin in eggs.

Three kinds of immunochromatographic assays (ICAs) are proposed for the highly sensitive and rapid determination  of tylosin (TYL) and tilmicosin (TIM) in eggs based on colloidal gold (CG), latex microsphere (LM), and time-resolved fluorescent microsphere (TRFM). Three types of ICAs could tolerate the egg matrix via simple sample pretreatment and demonstrated high sensitivity for TYL and TIM with cut-off values of 6/6/3 µg/kg and 14/14/6 µg/kg, respectively. Furthermore, in a single-blind parallel study 20 egg samples were analyzed  by the three developed ICAs and confirmed by LC-MS/MS. The  results showed good consistency, and there were no false positive and false negative results in our three ICAs. Consequently, the proposed three ICAs offered rapid, highly sensitive, reliable, and selectable testing platforms for screening veterinary medicine or other small molecule contaminants.

Pharmacokinetics and bioavailability of solid dispersion formulation of tilmicosin in pigs.

Tilmicosin (TMS) is a semisynthetic macrolide antibiotic restricted to veterinary use but is only partially soluble in aqueous solutions, which limits its administration in treatments. We developed a strategy to enhance the supersaturated solubility of TMS using amorphous solid dispersion (SD). The dissolution profile shown that the dissolution rate of TMS-SD was obviously faster than TMS. The pharmacokinetics of tilmicosin (TMS) and tilmicosin solid dispersion (TMS-SD) in pigs after oral administration at a single dose of 50 mg/kg b.w were investigated. The tmax of TMS-SD (2.50 hr) was 1.80 times faster than TMS (4.50 hr) (p < .05). There were no significant differences in the other PK parameters (Cmax , t1/2ß , V/F, CL/F, MRT, and AUC0-inf ) (p > .05). The mean relative bioavailability of TMS-SD compared with TMS was 140.39%, according to the AUC0-inf values. These results demonstrated that the solid dispersion technique enhanced the bioavailability of TMS and the new formulation administered to animals via drinking water may be used as a therapeutic alternative for clinical treatments.

The dose regimen formulation of tilmicosin against Lawsonia intracellularis in pigs by pharmacokinetic-pharmacodynamic (PK-PD) model.

In this study, the rational dose regimens of tilmicosin against Lawsonia intracellularis (L. intracellularis) were studied using pharmacokinetic-pharmacodynamic (PK-PD) model approach to provide a maximal efficacy. The healthy and infected pigs were orally administrated the tilmicosin premix at a single dose of 10 mg/kg, and then the plasma and ileum content were collected at different time points. The time to peak (Tmax), the peak concentration (Cmax), the area under concentration time curve (AUC0-24h), the apparent volume of distribution by bioavailability (V/F), the body clearance rate by bioavailability (CL/F) and the mean residence time (MRT) of tilmicosin premix for plasma were 2.00 h, 1.08 ± 0.04 µg/mL, 9.61 ± 1.47 µg h/mL, 34.43 ± 1.02 L/kg, 0.71 ± 0.03 L/h/kg and 15.03 ± 0.04 h in healthy pigs, and 2.00 h, 0.99 ± 0.03 µg/mL, 9.30 ± 1.43 µg h/mL, 58.59 ± 1.81 L/kg, 0.44 ± 0.02 L/h/kg and 15.75 ± 0.03 h in infected pigs, respectively. The Tmax, Cmax, AUC0-24h, V/F, CL/F and MRT of tilmicosin premix for ileum content were 2.00 h, 3.78 ± 0.03 µg/mL, 20.41 ± 1.64 µg h/mL, 11.29 ± 0.97 L/kg, 0.44 ± 0.02 L/h/kg and 11.29 ± 0.09 h in healthy pigs, and 2.00 h, 3.41 ± 0.06 µg/mL, 22.65 ± 1.32 µg h/mL, 8.16 ± 1.51 L/kg, 0.41 ± 0.01 L/h/kg and 11.44 ± 0.05 h in infected pigs, respectively. Based on the intracellular minimum inhibitory concentration (MIC) of L. intracellularis isolate was 2 µg/mL, the results of the mutant prevention concentration (MPC), the post-antibiotic effect (PAE) and time-killing curves all showed strong concentration-dependenttendencies. Integrating the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data using the sigmoid Emax (Hill) equation to obtain the ileum content AUC0-24h/MIC values of 6.87, 26.80, and 36.02 h to achieve the bacteriostatic activity, bactericidal activity, and virtual eradication of bacteria, respectively. Based on these results, a dosage regimen of daily 14.39 mg/kg for 3 d could be sufficient in the treatment of L. intracellularis. This study will provide a guidance of dosage regimen formulation for drug against animal intracellular bacterial infections.

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.

Synthesis of Tilmicosin Nanostructured Lipid Carriers for Improved Oral Delivery in Broilers: Physiochemical Characterization and Cellular Permeation.

This study aimed to develop nanostructured lipid carriers (NLCs) for improved oral absorption of tilmicosin (TMS) in broilers. Thus, palmitic acid, lauric acid, and stearic acid were selected as solid lipids to formulate TMS-pNLCs, TMS-lNLCs, and TMS-sNLCs, respectively. They showed similar physicochemical properties and meanwhile possessed excellent storage and gastrointestinal stability. The TMS interacted with the lipid matrix and was encapsulated efficiently in NLCs in an amorphous structure. NLCs could enhance oral absorption of TMS compared to 10% tilmicosin phosphate solution in broilers, among which the TMS-sNLCs were the most efficient drug delivery carriers, with a relative oral bioavailability of 203.55%. NLCs could inhibit the efflux of P-glycoprotein (P-pg) toward TMS, which may be involved with improved oral absorption. Taken together, these types of solid lipids influenced the enhanced level of NLCs toward oral bioavailability of TMS, and the sNLCs proved to be the most promising oral delivery carriers of TMS.

Protective effect of ghrelin against tilmicosin-induced left ventricular dysfunction in rats.

This study was conducted to investigate the possible protective effects of ghrelin against tilmicosin-induced acute ventricular dysfunction in rats. Forty adult male Sprague Dawley rats were randomly divided into 4 equal groups: control, ghrelin, tilmicosin, and ghrelin + tilmicosin. The left ventricular structural and functional parameters together with cardiac biomarker levels were evaluated. The results showed that tilmicosin treatment alone significantly decreased the left ventricular fractional shortening, left ventricular ejection fraction, left ventricular stroke volume, and cardiac output when compared with control group. In addition, tilmicosin led to a significant increase in left ventricular internal dimension in systole and left ventricular fractional end-systolic volume. At the same time, serum lactate dehydrogenase, creatine kinase, and creatine kinase-myocardial B fraction levels were significantly increased in tilmicosin-treated group when compared with control group. However, ghrelin pretreatment significantly prevented the left ventricular internal dimension in systole, left ventricular fractional end-systolic volume, left ventricular stroke volume, left ventricular ejection fraction, left ventricular fractional shortening, and cardiac output changes caused by tilmicosin. Moreover, ghrelin pretreatment could reduce significantly serum lactate dehydrogenase, creatine kinase, and creatine kinase-myocardial B fraction levels. These data indicated that ghrelin treatment may provide a protective effect against tilmicosin-induced left ventricular systolic dysfunction.

Tilmicosin enteric granules and premix to pigs: Antimicrobial susceptibility testing and comparative pharmacokinetics.

The purpose of this study was to compare the pharmacokinetics and relative bioavailability of tilmicosin enteric granules and premix after oral administration at a dose of 40 mg/kg in pigs. Three kinds of different respiratory pathogens were selected for determination of minimal inhibitory concentration (MIC) to tilmicosin. Eight healthy pigs were assigned to a two-period, randomized crossover design. A modified rapid, sensitive HPLC method was used for determining the concentrations of tilmicosin in plasma. Pharmacokinetic parameters were calculated by using WinNonlin 5.2 software. The MIC90 of tilmicosin against Haemophilus parasuis, Actinbacillus pleuropneumoniae, and Pasteurella multocida were all 8 µg/ml. These results indicated that these common pig respiratory bacteria are sensitive to tilmicosin. The main parameters of time to reach maximum plasma concentration (Tmax ), elimination half-life (t1/2ß ), mean residence time (MRT), and apparent volume of distribution (VF ) were 2.03 ± 0.37 hr, 29.31 ± 5.56 hr, 25.22 ± 2.57 hr, 4.06 ± 1.04 L/kg, and 3.05 ± 0.08 hr, 17.06 ± 1.77 hr, 15.55 ± 1.37 hr, 2.95 ± 0.62 L/kg after the orally administrated tilmicosin enteric granules and premix. The relative bioavailability of tilmicosin enteric granules to premix was 114.97 ± 7.19%, according to the AUC0-t values. These results demonstrated that tilmicosin enteric granules produced faster tilmicosin absorption, slower elimination, larger tissue distribution, and higher bioavailability compared to the tilmicosin premix. The present study results manifest that tilmicosin enteric granules can be used as a therapeutic alternative to premix in clinical treatment.

Comparison of pharmacokinetics of tilmicosin in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae.

Aim: To compare the pharmacokinetic profiles of tilmicosin, administered orally at a single dose of 20 mg/kg bodyweight, in healthy pigs and in pigs experimentally infected with Actinobacillus pleuropneumoniae. Methods: Twelve healthy crossbred pigs, aged approximately 8 weeks, were randomly assigned to uninfected and infected groups, with six pigs per group. Pigs in the infected group were inoculated intranasally with a bacterial suspension of A. pleuropneumoniae containing approximately 108 cfu. Each pig received a single oral dose of 20 mg/kg bodyweight of tilmicosin, given 3-4 hours after inoculation in infected pigs. Blood samples were collected before drug administration and up to 48 hours after tilmicosin administration. Concentrations of tilmicosin in plasma samples were determined by HPLC. Throughout the experimental period pigs were observed for signs of inappetence and clinical abnormalities. After sampling was complete pigs were subject to euthanasia and samples collected for gross and histopathology as well as microbiology. Results: Infected pigs showed signs of bradykinesia, nasal discharge dyspnoea, and coughing 1 hours after inoculation and A. pleuropneumoniae was cultured from the lungs of all infected pigs postmortem. Comparing pharmacokinetic parameters in uninfected and infected pigs, the maximum plasma concentration of tilmicosin was higher in uninfected pigs (1.17 (SD 0.17) vs. 0.96 (SD 0.17) µg/mL), the time to reach maximum concentration was shorter (1.53 (SD 0.23) vs. 2.40 (SD 0.37) hours), and the half-life of the absorption phase and half-life of the elimination phase were both shorter (0.66 (SD 0.08) vs. 1.00 (SD 0.27) hours) and (12.93 (SD 0.96) vs. 16.53 (SD 0.55) hours), respectively. The apparent volume of distribution was smaller in uninfected than infected pigs (1.91 (SD 0.22) vs. 2.16 (SD 0.21) L/kg). The relative bioavailability of tilmicosin in infected relative to uninfected pigs was 108.6 (SD 9.71)%. Conclusions and clinical relevance: The results of this study indicate that A. pleuropneumoniae infection significantly changed certain pharmacokinetic parameters of tilmicosin in pigs. In infected pigs tilmicosin exhibited a longer drug persistence and a better extent of absorption. These results indicate that it is necessary to monitor and adjust the dose of tilmicosin administration during the presence of pleuropneumonia. It is expected that this can optimise clinical efficacy and help avoid the development of resistance.

Impact of intramammary tilmicosin infusion as a dry cow therapy.

Three hundred subclinically infected quarters of 259 Holstein cows infected with gram-positive bacteria were selected via quota sampling based on the California Mastitis Test (CMT) result and were divided randomly and equally into treatment and test groups. Quarters of test group (n = 150 in 128 cows) were treated with an intramammary infusion of tilmicosin, and quarters of the control group (n = 150 in 131 cows) were treated with cloxacillin as a traditional intramammary infusion of dry cow (DC) ointment. Cows with more than one infected quarter were randomly assigned to the same group, and adjacent quarters were treated the same. The milk samples of all quarters were obtained, and bacterial cultures and somatic cell count (SCC) were tested before dry cow therapy (DCT) (50 ± 15 days before parturition), and finally on day 2 of the next lactation. Results have shown that total bacteriological cure rates on day 2 of the next lactation were 45% and 78%, (p = .01), new infection rates were 43.3% and 56.6%, and SCC was (6.732 × 105  ± 3.124 × 105 ) and (5.025 × 105  ± 2.935 × 105 ), (p > .05) in test and control groups, respectively. Tilmicosin had less effect on reducing IMI due to Corynebacterium bovis, and had no effect on Streptococcus agalactiae, but had a potent effect against Staphylococcus aureus. It was concluded that tilmicosin alone should not be infused as an alternative to conventional dry cow therapy. However, it had a significant effect against S. aureus, and the potential of tilmicosin to treat S. aureus IMI should be confirmed in further studies.

Tilmicosin- and florfenicol-loaded hydrogenated castor oil-solid lipid nanoparticles to pigs: Combined antibacterial activities and pharmacokinetics.

The combined antibacterial effects of tilmicosin (TIL) and florfenicol (FF) against Actinobacillus pleuropneumoniae (APP) (n = 2), Streptococcus suis (S. suis) (n = 2), and Haemophilus parasuis (HPS) (n = 2) were evaluated by chekerboard test and time-kill assays. The pharmacokinetics (PKs) of TIL- and FF-loaded hydrogenated castor oil (HCO)-solid lipid nanoparticles (SLN) were performed in healthy pigs. The results indicated that TIL and FF showed synergistic or additive antibacterial activities against APP, S. suis and HPS with the fractional inhibitory concentration (FIC) ranging from 0.375 to 0.75. The time-kill assays showed that 1/2 minimum inhibitory concentration (MIC) TIL combined with 1/2 MIC FF had a stronger ability to inhibit the growth of APP, S. suis, and HPS than 1 MIC TIL or 1 MIC FF, respectively. After oral administration, plasma TIL and FF concentrations could maintain about 0.1 µg/ml for 192 and 176 hr. The SLN prolonged the last time point with detectable concentrations (Tlast ), area under the concentration-time curve (AUC0-t ), elimination half-life (T½ke ), and mean residence time (MRT) by 3.1, 5.6, 12.7, 3.4-fold of the active pharmaceutical ingredient (API) of TIL and 11.8, 16.5, 18.1, 12.1-fold of the API of FF, respectively. This study suggests that the TIL-FF-SLN could be a useful oral formulation for the treatment of APP, S. suis, and HPS infection in pigs.

Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column (2.1 × 30 mm, 3.5 µm) using acetonitrile-water (90:10, v/v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r2 = 0.9998). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC-MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability.

The anti-nociceptive potential of tilmicosin against chemical-induced but not thermal-induced pain in mice.

The aim of the present study was to assess the analgesic activity of the macrolide antibiotic tilmicosin at dose levels of 20 and 40 mg/kg of body weight, subcutaneously, against chemical- and thermal-induced acute pains, using acetic acid-induced writhing, formalin-induced pain, hot-plate, and tail-flick models in mice. Tilmicosin showed a dose-dependent significant decrease in the number of writhes in the acetic acid-induced writhing test and significant decrease in hind paw-licking time in the late phase of the formalin test. However, it did not cause any significant changes in the reaction times to heat stimuli in the hot-plate and tail-flick models. In chemically-induced pains, both dose levels of tilmicosin showed significant effects compared to those of the corresponding standard peripheral analgesic, acetylsalicylic acid (200 mg/kg of body weight, subcutaneously) being 26.37±2.88 and 43.64±3.85% vs. 73.35±1.44% in acetic acid test; and 19.23±3.85 and 44.90±1.80% vs. 73.63±2.39% in the late phase of formalin test, respectively. These results may indicate that tilmicosin possesses a significant peripheral but not central analgesic potential that may be beneficial in symptomatic relief of pain when it is used in therapy, in addition to its well-established antibacterial effect.