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 Log 10 CFU/ml reduction over 120 h. The area under the concentration–time curve over 24 h divided by the MIC (AUC 24h /MIC) was the best PK/PD parameter to predict the antimicrobial activity of tilmicosin against M. gallisepticum [R 2 = 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.
Mycoplasma hyopneumoniae is the major pathogen causing enzootic pneumonia in pigs. M. hyopneumoniae infection can lead to considerable economic losses in the pig-breeding industry. Here, this study established a first-order absorption, one-compartment model to study the relationship between the pharmacokinetics/pharmacodynamics (PK/PD) index of tilmicosin against M. hyopneumoniae in vitro. We simulated different drug concentrations of timicosin in the fluid lining the lung epithelia of pigs. The minimum inhibitory concentration (MIC) of tilmicosin against M. hyopneumoniae with an inoculum of 106 CFU/mL was 1.6 μg/mL using the microdilution method. Static time–kill curves showed that if the drug concentration >1 MIC, the antibacterial effect showed different degrees of inhibition. At 32 MIC, the amount of bacteria decreased by 3.16 log10 CFU/mL, thereby achieving a mycoplasmacidal effect. The M. hyopneumoniae count was reduced from 3.61 to 5.11 log10 CFU/mL upon incubation for 96 h in a dynamic model with a dose of 40–200 mg, thereby achieving mycoplasmacidal activity. The area under the concentration-time curve over 96 h divided by the MIC (AUC0–96 h/MIC) was the best-fit PK/PD parameters for predicting the antibacterial activity of tilmicosin against M. hyopneumoniae (R2 = 0.99), suggesting that tilmicosin had concentration-dependent activity. The estimated value for AUC0–96 h/MIC for 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction from baseline was 70.55 h and 96.72 h. Four M. hyopneumoniae strains (M1–M4) with reduced sensitivity to tilmicosin were isolated from the four dose groups. The susceptibility of these strains to tylosin, erythromycin and lincomycin was also reduced significantly. For sequencing analyses of 23S rRNA, an acquired A2058G transition in region V was found only in resistant M. hyopneumoniae strains (M3, M4). In conclusion, in an in vitro model, the effect of tilmicosin against M. hyopneumoniae was concentration-dependent and had a therapeutic effect. These results will help to design the optimal dosing regimen for tilmicosin in M. hyopneumoniae infection, and minimize the emergence of resistant bacteria.
Macrolides are widely used in diseases caused by Mycoplasma spp. The new semi-synthetic macrolide antibiotic tulathromycin is currently in wide use for the treatment of respiratory diseases of livestock. The objective of this study was to evaluate the antibacterial effect of tulathromycin against Mycoplasma hyopneumoniae using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model to reveal mechanisms of antibiotic resistance and to evaluate the fitness of drug-resistant strains. In this study, high performance liquid chromatography-tandem mass spectrometry was used to determine drug concentrations for the in vitro model after dosing. The peak concentrations were in the range 0.3125–20 μg/mL (1 × MIC-64 × MIC). The ratio of the area under the concentration-time curve (AUC) over 72 h divided by the MIC (AUC72h/MIC) had the highest correlation with the antibacterial effect of tulathromycin against M. hyopneumoniae. Tulathromycin also showed concentration-dependent antimicrobial effects and promoted the emergence of drug-resistant bacteria after being cultured for 168 h and most were mutations in 23S rRNA at site A2058G (E.coli numbering) and only a single isolate was an A2058T (E.coli numbering) mutant. In the presence of reserpine, we determined the MIC of tulathromycin, tilmicosin, tiamulin and tylosin against these drug-resistant bacteria and the strains with efflux pump mechanisms were found among the strains resistant to tilmicosin. Gene expression analysis indicated that the ABC and MATE transporter efflux pump genes RS01935, RS02670, RS01115, RS01970, RS02395 and RS03540 (MATE family efflux transporter) were up-regulated in the three strains (P < 0.05 or P < 0.01). These investigations provide guidance for clinical administration of tulathromycin and elucidate the mechanism and fitness cost of drug resistance in M. hyopneumoniae.
= 333 21 Text = 3434 22 Figure counts: 5 2 23 Abstract 24 Mycoplasma hyopneumoniae is the major pathogen causing enzootic pneumonia in 25 pigs. M. hyopneumoniae infection can lead to considerable economic losses in the 26 pig-breeding industry. Here, this study established a first-order absorption, 27 one-compartment model to study the relationship between the 28 pharmacokinetics/pharmacodynamics (PK/PD) index of tilmicosin against M. 29 hyopneumoniae in vitro. We simulated the drug concentration of timicosin in the fluid 30 lining the lung epithelia of pigs. The minimum inhibitory concentration (MIC) of 31tilmicosin against M. hyopneumoniae with an inoculum of 10 6 CFU/mL was 1.6 32 μg/mL using the microdilution method. Static time-kill curves showed that, if the 33 drug concentration >1 MIC, the antibacterial effect showed different degrees of 34 inhibition. At 32 MIC, the amount of bacteria decreased by 3.16 log 10 CFU/mL, 35 thereby achieving a mycoplasmacidal effect. The M. hyopneumoniae count was 36 reduced from 3.61 to 5.11 log 10 CFU/mL upon incubation for 96 h in a dynamic 37 model with a dose of 40-200 mg, thereby achieving mycoplasmacidal activity. The 38 peak concentration by MIC (C max /MIC) and the area under the concentration-time 39 curve over 96 h divided by the MIC (AUC 0-96 h /MIC) were the best-fit PK/PD 40 parameters for predicting the antibacterial activity of tilmicosin against M. 41 hyopneumoniae (R 2 = 0.99), suggesting that tilmicosin had concentration-dependent 42 activity. The estimated value for C max /MIC and AUC 0-96 h /MIC for 2log 10 (CFU/mL) 43 reduction and 3log 10 (CFU/mL) reduction from baseline was 1.44 and 1.91, and 70.55 44 h and 96.72 h, respectively. Four M. hyopneumoniae strains (M1-M4) with reduced 3 45 sensitivity to tilmicosin were isolated from the four dose groups. The susceptibility of 46 these strains to tylosin, erythromycin and lincomycin was also reduced significantly. 47 For sequencing analyses of 23S rRNA, an acquired A2058G transition in region V 48 was found only in resistant M. hyopneumoniae strains (M3, M4). In conclusion, in an 49 in vitro model, the effect of tilmicosin against M. hyopneumoniae was 50 concentration-dependent and had a therapeutic effect. These results will help to design 51 the optimal dosing regimen for tilmicosin in M. hyopneumoniae infection, and 52 minimize the emergence of resistant bacteria. 53 54 Keywords: Mycoplasma hyopneumoniae, tilmicosin, in vitro dynamic model, 55 Pharmacokinetic, Pharmacodynamic, enzootic pneumonia, resistance. 56 57 1 Introduction 58 Mycoplasma hyopneumoniae is the primary pathogen of mycoplasmal pneumonia in 59 pigs. M. hyopneumoniae is widespread in various regions, and can cause huge 60 economic losses to the pig industry [1]. Infected pigs are the main source of infection. 61 The pathogen can be transmitted directly through air and contact, so the infection rate 62 is extremely high [2]. Once flocks of pigs are infected with M. hyopneumoniae, 63 eradication is difficult because M. hyopneumoniae can tr...
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