Emergence of ciprofloxacin heteroresistance in foodborne Salmonella enterica serovar Agona.

BACKGROUND: Bacterial heteroresistance has been increasingly identified as an important phenomenon for many antibiotic/bacterium combinations. OBJECTIVES: To investigate ciprofloxacin heteroresistance in Salmonella and characterize mechanisms contributing to ciprofloxacin heteroresistance. METHODS: Ciprofloxacin-heteroresistant Salmonella were identified by population analysis profiling (PAP). Target mutations and the presence of PMQR genes were detected using PCR and sequencing. Expression of acrB, acrF and qnrS was conducted by quantitative RT-PCR. Competition ability and virulence were also compared using pyrosequencing, blue/white screening, adhesion and invasion assays and a Galleria model. Two subpopulations were whole-genome sequenced using Oxford Nanopore and Illumina platforms. RESULTS: PAP identified one Salmonella from food that yielded a subpopulation demonstrating heteroresistance to ciprofloxacin at a low frequency (10-9 to 10-7). WGS and PFGE analyses confirmed that the two subpopulations were isogenic, with six SNPs and two small deletions distinguishing the resistant from the susceptible. Both subpopulations possessed a T57S substitution in ParC and carried qnrS. The resistant subpopulation was distinguished by overexpression of acrB and acrF, a deletion within rsxC and altered expression of soxS. The resistant population had a competitive advantage against the parental population when grown in the presence of bile salts but was attenuated in the adhesion and invasion of human intestinal cells. CONCLUSIONS: We determined that heteroresistance resulted from a combination of mutations in fluoroquinolone target genes and overexpression of efflux pumps associated with a deletion in rsxC. This study warns that ciprofloxacin heteroresistance exists in Salmonella in the food chain and highlights the necessity for careful interpretation of antibiotic susceptibility.

Pharmacokinetic-pharmacodynamic integration and resistance of tiamulin against Mycoplasma hyopneumoniae in an in vitro dynamic model.

Mycoplasma hyopneumoniae is the major pathogen of enzootic pneumonia in pigs. We established an in vitro dynamic model to investigate the relationship between the pharmacokinetic and pharmacodynamic (PK-PD) parameters of tiamulin against M. hyopneumoniae. Static time-killing curves showed that mycoplasmacidal activity (reduced 3.0 log10 (CFU/mL)) was achieved during 48 h when the drug concentration was 8 MIC, and with a maximum kill rate of 0.072/h. In dynamic time-killing studies, only the dose-fractionated regimen achieved mycoplasmacidal activity when drug concentration was 1.44 and 1.92 mg/L. The duration of post antibiotic effect (PAE) at 1 × MIC was 6.27 ± 0.11 h, and prolonged as the concentration of tiamulin increased. The cumulative percentage of time over a 48-h period that the drug concentration exceeds the MIC (%T > MIC) was the best PK-PD parameter to predict the antimicrobial activity of tiamulin against M. hyopneumoniae (R2 = 0.98). Tiamulin showed time-dependent and prolonged PAE activity. Two strains of M. hyopneumoniae (M1, M2) had acquired resistance to tiamulin as well as to valnemulin, tylosin and amikacin. The genome of strain ATCC 25934 was used as a reference for gene-mutation analysis. For strains M1 and M2, a A2058C mutation occurred in domain V of 23S rRNA. These data showed that tiamulin had excellent efficacy and concentration-dependent characteristics against M. hyopneumoniae in vitro. The lower dose was not safe because it could lead to enrichment of resistant bacteria.

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/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.

In vitro susceptibility of four antimicrobials against Riemerella anatipestifer isolates: a comparison of minimum inhibitory concentrations and mutant prevention concentrations for ceftiofur, cefquinome, florfenicol, and tilmicosin.

BACKGROUND: Mutant prevention concentration (MPC) is an alternative pharmacodynamic parameter that has been used to measure antimicrobial activity and represents the propensities of antimicrobial agents to select resistant mutants. The concentration range between minimum inhibitory concentration (MIC) and MPC is defined as mutant selection window (MSW). The MPC and MSW parameters represent the ability of antimicrobial agents to inhibit the bacterial mutants selected. This study was conducted to determine the MIC and MPC values of four antimicrobials including ceftiofur, cefquinome, florfenicol and tilmicosin against 105 Riemerella anatipestifer isolates. RESULTS: The MIC50/MIC90 values of clinical isolates tested in our study for ceftiofur, cefquinome, florfenicol and tilmicosin were 0.063/0.5、0.031/0.5、1/4、1/4 µg/mL, respectively; MPC50/ MPC90 values were 4/64、8/64、4/32、16/256 µg/mL, respectively. These results provided information on the use of these compounds in treating the R. anatipestifer infection; however, additional studies are needed to demonstrate their therapeutic efficacy. CONCLUSION: Based on the MSW theory, the hierarchy of these tested antimicrobial agents with respect to selecting resistant subpopulations was as follows: cefquinome > ceftiofur > tilmicosin > florfenicol. Cefquinome was the drug that presented the highest risk of selecting resistant mutant among the four antimicrobial agents.

Response of a clinical Escherichia coli strain to repeated cefquinome exposure in a piglet tissue-cage model.

BACKGROUND: In order to provide some basis for effective dosage regimens that optimize efficacy with respect to bacteriological and clinical cures, the in vivo activity of cefquinome against a clinical Escherichia coli (E.coli) strain (the minimum inhibitory concentration value for this strain equals to the MIC90 value of 0.25 µg/ml for 210 E.coli strains isolated from pigs) was investigated by using a piglet tissue-cage infection model. The aim was to elucidate the pharmacokinetic/pharmacodynamics (PK/PD) index associated with cefquinome efficacy, and then to identify the magnitude of the PK/PD parameter required for different degree of efficacy in clinical treatment. RESULTS: Tissue-cage infection model was established in piglets, and then the animals received intramuscular injection of cefquinome twice a day for 3 days to create a range of different drug exposures. The tissue-cage fluid was collected at 1, 3, 6, 9 and 12 h after every drug administration for drug concentrationdetermination and bacteria counting. Different cefquinome regimens produced different percentages of time during that drug concentrations exceeded the MIC (%T > MIC), ranging from 0% to 100%. Cefquinome administration at 0.2, 0.4, 0.6, 0.8, 1, 2 and 4 mg/kg reduced the bacterial count (log10 CFU/mL) in tissue-cage fluid by -1.00 ± 0.32, -1.83 ± 0.08, -2.33 ± 0.04, -2.96 ± 0.16, -2.99 ± 0.16, -2.93 ± 0.11, -3.43 ± 0.18, respectively. The correlation coefficient of the PK/PD index with antibacterial effect of the drug was 0.90 for %T > MIC, 0.62 for AUC0-12/MIC, and 0.61 for Cmax/MIC, suggesting the most important PK/PD parameter was %T > MIC. A inhibitory form of sigmoid maximum effect (Emax) model was used to estimate %T > MIC, and the respective values required for continuous 1/6-log drop, 1/3-log drop and 1/2-log drop of the clinical E.coli count during each 12 h treatment period were 3.97%, 17.08% and 52.68%. CONCLUSIONS: The data derived from this study showed that cefquinome exhibited time-dependent killing profile. And from the results of the present study, it can be assumed that when %T > MIC reached 52.68%, cefquinome could be expected to be effective against a clinical E.coli strain for which the MIC value is below 0.128 µg/ml (3-log drop of bacteria count can be achieved after six successive administrations for 3 days).

Pharmacodynamics of cefquinome in a neutropenic mouse thigh model of Staphylococcus aureus infection.

Cefquinome is a cephalosporin with broad-spectrum antibacterial activity, including activity against Staphylococcus aureus. The objective of our study was to examine the in vivo activity of cefquinome against S. aureus strains by using a neutropenic mouse thigh infection model. Cefquinome kinetics and protein binding in infected neutropenic mice were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo postantibiotic effects (PAEs) were determined after a dose of 100 mg/kg of body weight in mice infected with S. aureus strain ATCC 29213. The animals were treated by subcutaneous injection of cefquinome at doses of 2.5 to 320 mg/kg of body weight per day divided into 1, 2, 3, 6, or 12 doses over 24 h. Cefquinome exhibited time-dependent killing and produced in vivo PAEs at 2.9 h. The percentage of time that serum concentrations were above the MIC (%T>MIC) was the pharmacokinetic-pharmacodynamic (PK-PD) index that best described the efficacy of cefquinome. Subsequently, we employed a similar dosing strategy by using increasing total cefquinome doses that increased 4-fold and were administered every 4 h to treat animals infected with six additional S. aureus isolates. A sigmoid maximum effect (Emax) model was used to estimate the magnitudes of the ratios of the %T that the free-drug serum concentration exceeded the MIC (%T>fMIC) associated with net bacterial stasis, a 0.5-log10 CFU reduction from baseline, and a 1-log10 CFU reduction from baseline; the respective values were 30.28 to 36.84%, 34.38 to 46.70%, and 43.50 to 54.01%. The clear PAEs and potent bactericidal activity make cefquinome an attractive option for the treatment of infections caused by S. aureus.

In vivo evaluation of mutant selection window of cefquinome against Escherichia coli in piglet tissue-cage model.

BACKGROUND: The resistance of cephalosporins is significantly serious in veterinary clinic. In order to inhibit the bacterial resistance production, the mutant selection window (MSW) hypothesis with Escherichia coli (E. coli) ATCC 25922 exposed to cefquinome in an animal tissue-cage model was investigated. RESULTS: Localized infection with E. coli was established in piglets, and the infected animals were administrated intramuscularly with various doses and intervals of cefquinome to provide antibiotic concentrations below the MIC99, between the MIC99 and the mutant prevention concentration (MPC), and above the MPC. E. coli lost susceptibility when drug concentrations fluctuated between the lower and upper boundaries of the window, which defined in vitro as the MIC99 (0.06 µg/mL) and the MPC (0.16 µg/mL) respectively. For PK/PD parameters, there were no mutant selection enrichment when T>MIC99 was ≤ 25% or T>MPC was ≥ 50% of administration interval. When T>MIC99 was > 25% and T>MPC was <50% of administration interval, resistance selection was observed. When AUC24 h/MIC99 and AUC24 h/MPC were considered, the mutant selection window extended from 32.84 h to 125.64 h and from 12.83 h to 49.09 h, respectively. CONCLUSIONS: These findings demonstrate that the MSW exists in vivo for time-dependent antimicrobial agents, and its boundaries fit well with those determined in vitro. Maintenance of antimicrobial concentrations above the MPC for > 50% of administration interval is a straightforward way to restrict the acquisition of resistance in this tissue cage model. This situation was achieved with daily intramuscular doses of 1 mg cefquinome/kg body weight.

Pharmacokinetic/pharmacodynamic integration of amphenmulin: a novel pleuromutilin derivative against Mycoplasma gallisepticum.

Amphenmulin is a novel pleuromutilin derivative with great anti-mycoplasma potential. The present study evaluated the action characteristics of amphenmulin against Mycoplasma gallisepticum using pharmacokinetic/pharmacodynamic (PK/PD) modeling approaches. Following intravenous administration, amphenmulin exhibited an elimination half-life of 2.13 h and an apparent volume of distribution of 3.64 L/kg in healthy broiler chickens, demonstrating PK profiles of extensive distribution and rapid elimination. The minimum inhibitory concentration (MIC) of amphenmulin against M. gallisepticum was determined to be 0.0039 µg/mL using the broth microdilution method, and the analysis of the static time-kill curves through the sigmoid Emax model showed a highly correlated relationship (R ≥ 0.9649) between the kill rate and drug concentrations (1-64 MIC). A one-compartment open model with first-order elimination was implemented to simulate the in vivo anti-mycoplasma effect of amphenmulin, and it was found that bactericidal levels were reached with continuous administration for 3 days at doses exceeding 0.8 µg/mL. Furthermore, the area under the concentration-time curve divided by MIC (AUC/MIC) correlated well with the anti-mycoplasma effect of amphenmulin within 24 h after each administration, with a target value of 904.05 h for predicting a reduction of M. gallisepticum by 1 Log10CFU/mL. These investigations broadened the antibacterial spectrum of amphenmulin and revealed its characteristics of action against M. gallisepticum, providing a theoretical basis for further clinical development.IMPORTANCEMycoplasma has long been recognized as a significant pathogen causing global livestock production losses and public health concerns, and the use of antimicrobial agents is currently one of the mainstream strategies for its prevention and control. Amphenmulin is a promising candidate pleuromutilin derivative that was designed, synthesized, and screened by our laboratory in previous studies. Moreover, this study further confirms the excellent antibacterial activity of amphenmulin against Mycoplasma gallisepticum and reveals its action characteristics and model targets on M. gallisepticum by establishing an in vitro pharmacokinetic/pharmacodynamic synchronization model. These findings can further broaden the pharmacological theoretical basis of amphenmulin and serve as data support for its clinical development, which is of great significance for the discovery of new antimicrobial drugs and the control of bacterial diseases in humans and animals.

Pharmacokinetic/pharmacodynamic relationships of enrofloxacin against Klebsiella pneumoniae in an in vivo infection model in young chicks.

Klebsiella pneumoniae is a serious pathogenic bacterium that poses a significant threat to young poultry and the cause of significant chick mortality and economic loss. We investigated the therapeutic efficacy of enrofloxacin in treating K. pneumoniae infections in chicks and employed an in vivo pharmacokinetic/pharmacodynamic (PK/PD) model. In vivo efficacy was evaluated using 6 multiple-dose groups (oral administration once a day for 3 d) and 2 single-dose groups (oral administration once only). The PK and PD parameters of plasma and lung were analyzed using PK/PD fitting analysis. K. pneumoniae administered intratracheally (108 CFU/mL in 0.4 mL saline) was used to establish a model for pulmonary infection. The plasma protein binding of enrofloxacin was 20.18%. Enrofloxacin displayed T1/2ß values of 4.78 ± 0.69 h and 4.78 ± 1.02 h in plasma and lung of infected chicks, respectively. When the dosage in the multiple-dose group was > 10 mg/kg, bactericidal activity was found and complete eradication was not achieved when the dosage was ≤ 40 mg/kg. When TMSW was set at 20%, the calculated dosage and bacterial reduction (E) based on plasma free drug data were 4.03 mg/kg and -1.982 Log10 CFU/mL, respectively. In the calculation of PK/PD parameters for reducing 3 Log10 CFU/mL and using Cmax/MIC, AUC72h/MIC and TMSW of free drug in plasma values at 9.479, 379.691, and 44.395%, respectively, the value of AUC72h/MIC based on the concentration of drug in lung was 530.800. According to the fitting correlation R2, the PK/PD fitting results of free drug in plasma were better. The corresponding enrofloxacin dosage for AUC72h/MIC of the plasma free drug concentration was 14.16 mg/kg. The administration regimen corresponding to these dosages was once daily for 3 d. This dosage regimen (14.16 mg/kg) was relatively high compared to the clinically recommended dosage in China (7.5 mg/kg) when treating infections caused by K. pneumoniae with MIC ≥ 0.125 µg/mL, so careful consideration is needed.

Rapid Dissemination of blaNDM-5 Gene among Carbapenem-Resistant Escherichia coli Isolates in a Yellow-Feather Broiler Farm via Multiple Plasmid Replicon.

Although carbapenems have not been approved for animal use, carbapenem-resistant Escherichia coli (CREC) strains are increasingly being detected in food-producing animals, posing a significant public health risk. However, the epidemiological characteristics of CREC isolates in yellow-feather broiler farms remain unclear. We comprehensively investigated the genetic features of carbapenem-resistance genes among E. coli isolates recovered from a yellow-feather broiler farm in Guangdong province, China. Among the 172 isolates, 88 (51.2%) were recovered from chicken feces (88.5%, 54/61), the farm environment (51.1%, 24/47), and specimens of dead chickens (15.6%, 41/64). All CREC isolates were positive for the blaNDM-5 gene and negative for other carbapenem-resistance genes. Among 40 randomly selected isolates subjected to whole-genome sequencing, 10 belonged to distinct sequence types (STs), with ST167 (n = 12) being the most prevalent across different sources, suggesting that the dissemination of blaNDM-5 was mainly due to horizontal and clonal transmission. Plasmid analysis indicated that IncX3, IncHI2, and IncR-X1-X3 hybrid plasmids were responsible for the rapid transmission of the blaNDM-5 gene, and the genetic surrounding of blaNDM-5 contained a common mobile element of the genetic fragment designated "IS5-△ISAba125-blaNDM-5-bleMBL-trpF-dsbC". These findings demonstrate a critical role of multiple plasmid replicons in the dissemination of blaNDM-5 and carbapenem resistance.

Lung microdialysis and in vivo PK/PD integration of cefquinome against Actinobacillus pleuropneumoniae in a porcine experimental lung infection model.

This study aim to explore the application of microdialysis in pharmacokinetic (PK) and pharmacodynamic (PD) integration of cefquinome against Actinobacillus pleuropneumoniae in a porcine experimental lung infection model. The model was established via intratracheal inoculation where average bacterial counts (CFU) in the lungs of infected pigs reached 6.57 log10 CFU/g after 3 h. The PK profiles of unbound cefquinome in lung dialysates were determined following intramuscular injection of single doses of 0.125, 0.25, 0.5, 1, 2, and 4 mg/kg. Lung dialysate samples were collected using microdialysis at a flow rate of 1.5 µL/min until 24 h. The PD studies were conducted over 24 h based on 10 intermittent dosing regimens and total daily doses ranged from 0.25 to 4 mg/kg and dosage intervals included 12 and 24 h. The lung tissue was collected after 24 h of treatment and homogenized for bacterial counts. The relationships between PK/PD parameters derived from lung dialysates and drug efficacy were analyzed using an inhibitory sigmoid Emax model. The percentage of time the free drug concentration exceeded the minimum inhibitory concentration (%fT > MIC) was the PK/PD index best describing the antimicrobial activity (R2 = 0.96) in the porcine experimental infection model. The %fT > MIC values required to achieve net bacterial stasis, 1, 2 and 3 log10 CFU/g reductions in the lung were 22.45, 28.86, 37.62, and 56.46%, respectively. Cefquinome exhibited time-dependent characteristics against A. pleuropneumoniae in vivo. These results provide valuable insights into the application of microdialysis in PK/PD integration model studies and optima regimen of cefquinome for the treatment of porcine respiratory diseases caused by A. pleuropneumoniae.

Chlorogenic acid attenuates tet (X)-mediated doxycycline resistance of Riemerella anatipestifer.

Introduction: The increasing resistance of R. anatipestifer has posed a significant threat to the poultry industry in recent years. The tet gene is the primary determinant of tetracycline resistance in numerous bacteria, and the enzyme modification gene tet(X) is predominantly detected in tetracycline-resistant R. anatipestifer strains. Methods: In this study, we evaluated the susceptibility of both the standard strain and clinical isolates of R. anatipestifer to doxycycline. And the expression levels of tet(X), tet(A), and tet(O) genes were detected. To assess drug susceptibility, shuttle plasmids were constructed to transfer the tet(X) gene into the standard strain of R. anatipestifer followed by treatment with chlorogenic acid. Results and discussion: The results revealed that the minimum inhibitory concentration of doxycycline for the standard strain was 0.25µg/mL, whereas it exceeded 8µg/mL for the clinical isolates. Furthermore, there was a significant upregulation observed in expression levels of tet(X), tet(A), and tet(O) genes among induced strains. Interestingly, when transferring the tet(X) gene into the standard strain, its sensitivity to doxycycline decreased; however, MIC values for chlorogenic acid remained consistent between both standard and drug-resistant strains of R. anatipestifer. Moreover, we made a surprising discovery that screening passage with chlorogenic acid resulted in increased sensitivity of R. anatipestifer to doxycycline. Further analysis demonstrated a reversal in expression trends among three differentially expressed genes within induced drug resistance group after intervention with chlorogenic acid. The main objective behind this study is to investigate both killing effect exerted by chlorogenic acid on drug-resistant R. anatipestifer as well as its regulatory impact on drug resistance genes. This will provide novel insights and theoretical basis towards development of chlorogenic acid as a promising drug for treatment and control of drug resistance in R. anatipestifer.

Pharmacokinetic/pharmacodynamic integration of tilmicosin against Pasteurella multocida in a piglet tissue cage model.

Tilmicosin is a semi-synthetic macrolide for veterinary use with strong antibacterial effect on respiratory bacteria. In this study, the pharmacokinetic/pharmacodynamic (PK/PD) integration of tilmicosin against Pasteurella multocida (P. multocida) was evaluated by establishing a piglet tissue cage infection model. Concentration of tilmicosin and bacterial numbers of P. multocida in the tissue-cage fluid were monitered. After the population of P. multocida was equal to or greater than 107 CFU/mL in a tissue cage, piglets received an oral administration of tilmicosin at a dose of 30, 40, 50, and 60 mg/kg b.w., once daily for 3 days, respectively. Bacteria were counted every 24 h after drug administration and at 48 and 72 h after the last administration. A sigmoidal Emax model was used to fit the relationship between PK/PD parameters and the antibacterial effect. AUC24h/MIC was the best PK/PD index that correlated with effectiveness of tilmicosin against P. multocida. The magnitude of AUC24h/MIC required for continuous 1/3-log, 1/2-log, and 3/4-log reductions were 19.65 h, 23.86 h, and 35.77 h, respectively, during each 24 h treatment period. In this study, when the dosage was >50 mg/kg, the AUC24h/MIC was still >35.77 h in the period of 24-48 h after the last administration due to the slow elimination, that is, tilmicosin exhibited a potent antibacterial effect against P. multocida after three successive daily administrations. The data provide meaningful guidance to optimize regimens of tilmicosin to treat respiratory tract infections caused by P. multocida.

PK-PD integration of enrofloxacin and cefquinome alone and in combination against Klebsiella pneumoniae using an in vitro dynamic model.

Introduction: Klebsiella pneumoniae is classified as a critical pathogen in both animals and humans and infections can be fatal in chickens resulting in substantial economic losses. However, the misuse of antibiotics can also lead to drug resistance and a potential transmission chain between animals and humans. Three K. pneumoniae strains with different susceptibility phenotypes were chosen to study the pharmacokinetic/pharmacodynamic (PK/PD) integration of enrofloxacin (ENR) and cefquinome (CEQ) alone and in combination. Results: Checkerboard assay results indicated that the combination treatment for type strain ATCC 700603 was synergistic effect with a fractional inhibitory concentration index (FICI) of ≤0.5. The other two clinical strains demonstrated an additive effect (FICI >0.5 to ≤1). Furthermore, static time-kill curves indicated that enrofloxacin and cefquinome added singly were effective in killing K. pneumoniae at concentrations of >2 MIC and ≥1 MIC, respectively. Additionally, the combination of enrofloxacin and cefquinome led to an enhanced antibacterial activity of cefquinome. The dynamic time-kill curves indicated that enrofloxacin and cefquinome had bactericidal and bacteriostatic activities, respectively at ≥1.5 mg/L (single-dose) and 4 mg/L (8 h split-dose) causing a decrease in bacterial counts of ≥4.45 and >2 log10 CFU/mL. Enrofloxacin possessed no bacteriostatic effects against K. pneumoniae at a constant concentration of 1× MIC. Cefquinome used in combination with 1× MIC enrofloxacin exhibited bactericidal activity at ≥4 mg/L (12 h split-dose) with reductions of ≥3.65 log10 CFU/mL. The PK/PD parameters were also analyzed to determine the concentration and duration of the drugs needed to reduce bacteria by 3 log10 CFU/mL. For enrofloxacin alone, the AUC24h/MIC was 23.29 h and the Cmax/MIC was 3.18. For cefquinome alone, the %T > MIC was 48.66 and when used in combination with enrofloxacin was 18.04. The combined use of cefquinome and enrofloxacin can increase the antibacterial activity of cefquinome against K. pneumoniae under a 12-h split-dose regimen regardless of individual drug susceptibility. Discussion: The static and dynamic time-kill curves indicated that enrofloxacin exhibited concentration-dependent activity, while cefquinome exhibited time-dependent activity. In the in vitro dynamic model, enrofloxacin alone exhibited better antimicrobial effects against K. pneumoniae compared to cefquinome alone. However, the antibacterial effect of cefquinome can be enhanced by combining it with enrofloxacin. These findings suggest a potentially effective approach for combating K. pneumoniae infections.

Withdrawal time of danofloxacin and difloxacin and in vitro binding phenomenon to melanin in black-boned silky fowl.

Fluoroquinolones are commonly used in poultry breeding. Few studies have evaluated the causes of serious drug residues in black-boned silky fowl until enrofloxacin has been banned in black-boned silky fowl breeding in the Chinese Veterinary Commission of Chinese Veterinary Pharmacopoeia (2020). However, similarly structured fluoroquinolones have not been studied in black-boned silky fowl. In this study, the elimination of tissue residues of danofloxacin (DAN) and difloxacin (DIF) was studied in four tissues of black-boned silky fowl. The specific administration methods were 100 mg/L of DIF aqueous solution for free drinking for 5 days and 50 mg/L of DAN aqueous solution for free drinking for 3 days. Based on the experiment, the withdrawal times of 44 days for muscle, 95 days for skin + fat, 3 days for liver, and 44 days for kidney of DAN were acquired, of 43 days for muscle, 61 days for skin + fat, 0 days for liver, and 38 days for kidney of DIF were acquired, which showed that DIF and DAN should be used with caution for application in black-boned silky fowl. In vitro experiments showed that black-boned silky fowl tissues had stronger adsorption capacity to DAN and DIF than yellow chicken tissues (especially in skin + fat), and melanin has a strong adsorption effect on DAN and DIF, which is an important reason for the high residual concentrations of fluoroquinolone in black-boned silky fowl.

Biofilm formation and correlations with drug resistance in Mycoplasma synoviae.

Infectious synovitis in chickens caused by Mycoplasma synoviae infections are characterized by exudative synovial joint membranes and tenosynovitis. We isolated M. synoviae from chickens on farms in Guangdong, China and identifed 29 K-type and 3 A-type strains using vlhA genotyping and all displayed decreased susceptibilities to enrofloxacin, doxycycline, tiamulin and tylosin compared with the type strain WVU1853 (ATCC 25204). M. synoviae biofilms were present after staining as block or continuous dot shape morphologies and these appeared as tower-like and mushroom-like structures in scanning electron micrographs. The optimal temperature for biofilm formation was 33 °C and these biofilms enhanced the resistance of M. synoviae to all 4 antibiotics we tested and minimum biofilm inhibitory concentration for enrofloxacin and biofilm biomass were significantly negatively correlated (r < 0, 0.3 ≤|r|<0.5, P < 0.05). This work is the first study of the biofilm formation ability of M. synoviae and provides the foundation for further investigations.

Liquid chromatography tandem mass spectrometry for the simultaneous determination of mequindox and its metabolites in porcine tissues.

A rapid liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination of mequindox and its five metabolites (2-isoethanol mequindox, 2-isoethanol 1-desoxymequindox, 1-desoxymequindox, 1,4-bisdesoxymequindox, and 2-isoethanol bisdesoxymequindox) in porcine muscle, liver, and kidney, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. The method involved acid hydrolysis, purification by solid-phase extraction, and subsequent analysis with liquid chromatography tandem mass spectrometry using electrospray ionization operated in positive polarity with a total run time of 15 min. The decision limit values of five analytes in porcine tissues ranged from 0.6 to 2.9 µg/kg, and the detection capability values ranged from 1.2 to 5.7 µg/kg. The results of the inter-day study, which was performed by fortifying porcine muscle (2, 4, and 8 µg/kg), liver, and kidney (10, 20, and 40 µg/kg) samples on three separate days, showed that the accuracy of the method for the various analytes ranged between 75.3 and 107.2% with relative standard deviation less than 12% for each analyte.

Emergence and Mechanism of Resistance of Tulathromycin Against Mycoplasma hyopneumoniae in a PK/PD Model and the Fitness Costs of 23S rRNA Mutants.

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.

PK/PD integration of florfenicol alone and in combination with doxycycline against Riemerella anatipestifer.

Riemerella anatipestifer (RA) is an important pathogen found in poultry. RA infection can kill ducks and lead to significant economic losses. Seven RA strains with different susceptibility phenotypes were chosen to study the pharmacokinetic/pharmacodynamic (PK/PD) integration of florfenicol (FF) alone and in combination with doxycycline (DOX). The checkerboard assay indicated that synergy [fractional inhibitory concentration index (FICI) ≤ 0.5] was detected in the CVCC3952 strain of RA and that additivity (FICI >0.5 to ≤ 1) was observed in other strains. Static time-kill curves showed that the bactericidal effect of FF against RA was produced at a FF concentration ≥4 MIC, and the antibacterial activity of FF against RA was enhanced from the aspects of efficacy and efficacy in combination with DOX. Dynamic time-kill curves indicated that FF elicited bactericidal activity against the CVCC3857 strain with a reduction ≥4.88 log10CFU/ml when the dose was ≥8 mg/L. However, a bactericidal effect was not achieved at the maximum administered dose of FF monotherapy (20 mg/L) for isolates with a MIC ≥4 µg/ml. The effect of FF against RA was enhanced upon combination with DOX. The combination of FF with DOX reduced the bacterial burden ≥4.53 log10CFU/ml for all strains with a MIC ≥4 µg/ml. Data were fitted to a sigmoidal Emax model. The PK/PD parameters of AUC24h/MIC (the area under the concentration-time curve over 24 h divided by the MIC) and %T >MIC (the cumulative percentage of time over a 24-h period at which the concentration exceeded the MIC) of FF for eliciting a reduction of 3 log10CFU/ml was 40.10 h and 58.71, respectively. For strains with a MIC ≤ 16 µg/ml, the magnitude of the AUC24h/MIC and Cmax/MIC required for a 3 log10CFU/ml of bacterial killing was 34.84 h and 4.74 in the presence of DOX at 0.5 MIC, respectively. These data suggest that combination of FF with DOX enhanced the activity against RA strains with various susceptibilities to FF and DOX.