PK/PD modelling of enrofloxacin against Glaesserella parasuis infection in pigs.

A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to optimize the dosing regimen of enrofloxacin (EN) against Glaesserella parasuis in pigs. EN (2.5 mg/kg) was administered intramuscularly to eight healthy pigs and eight pigs that were experimentally infected with G. parasuis SW124. Blood samples were collected at predetermined time points. Plasma EN concentrations were determined, and the main PK parameters were estimated. The PD of EN against G. parasuis SW124 was also investigated in vitro and ex vivo. The dynamic behaviour of EN in pigs was consistent with a one-compartment model. Significant differences were observed between healthy and infected pigs in the area under the curve (AUC) (3.58 ± 0.94 and 5.39 ± 1.01 µg h/ml, respectively) and the systemic clearance (CL) (736.32 ± 171.46 and 479.36 ± 96.81 ml/h/kg, respectively), suggesting that the pathogenicity of G. parasuis SW124 to pigs might alter the PK profile of EN, and therefore should be considered in dose optimization. Both the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.125 µg/ml in tryptone soya broth (TSB) medium or plasma. The mutant prevention concentration (MPC) was 0.6 µg/ml. EN inhibited or killed G. parasuis SW124 in a concentration-dependent manner. The targeted endpoints of AUC24 h /MIC for bacteriostasis, bactericidal action, and eradication were 5.10, 7.34, and 8.65 h and 5.91, 9.01, and 10.90 h in healthy and infected pigs, respectively. The optimal doses were 3.58-6.08 mg/kg in healthy pigs and 2.71-4.99 mg/kg in infected pigs from the point of view of preventing drug resistance.

Eutrophication and Related Antibiotic Resistance of Enterococci in the Minjiang River, China.

Antimicrobial resistance (AMR) in the aquatic environment has received increasing attention in recent years, and growing eutrophication problems may contribute to AMR in aquatic ecosystems. To evaluate whether and how eutrophication affects AMR, 40 surface water samples were collected from the Minjiang River, Fujian Province, China. Total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (CODMn) were measured as eutrophication factors. Additionally, enterococci species were isolated and their resistance to six common antibiotics was tested. Eutrophication generally showed a trend of increasing with the flow direction of the Minjiang River, with 25 sites (62.5%) having a TN/TP value over the Redfield value (16:1), which indicated that eutrophication in this region was of phosphorus limitation. High nutrition sites were in or near urban areas. Poor quality water was found in the middle and lower reaches of the Minjiang River system. The resistance frequency of 40 enterococci isolates to the six antibiotics tested was as follows: oxytetracycline > erythromycin > ciprofloxacin > chloramphenicol > ampicillin > vancomycin (70, 50, 17.5, 12.5, 2.5, 0%), and the multi-resistant rate reached 50% with eight resistance phenotypes. AMR also increased along the direction of water flow downstream, and most of the sites with the highest AMR were in or near urban areas, as was true for nutrition levels. Positive correlations between AMR and eutrophication factors (TN, TP, and CODMn) were identified using the Pearson's correlation coefficient, and TN/TP generally was negatively related to AMR. These results indicated that eutrophication may induce or selective for resistance of water-borne pathogens to antibiotics, with a high resistance level and a wide resistance spectrum.

Inhibitory Effect of Two Traditional Chinese Medicine Monomers, Berberine and Matrine, on the Quorum Sensing System of Antimicrobial-Resistant Escherichia coli.

The quorum sensing (QS) system controls bacterial biofilm formation, which is highly related to the virulence and resistance of pathogens. In the present study, the effect of two traditional Chinese medicine (TCM) monomers, berberine and matrine, on biofilm formation and QS-related gene expression of antimicrobial-resistant (AMR) Escherichia coli strains was investigated by laser scanning confocal microscopy (LSCM) observation and real-time PCR. The results indicated a roughly positive relationship between biofilm formation ability and antimicrobial resistance. LSCM observation showed that berberine and matrine inhibited biofilm formation of AMR E. coli strains at 1/2 minimal inhibitory concentration (MIC) (1/2 MIC berberine at OD630: 0.1020; 1/2 MIC matrine: OD630: 0.1045); furthermore, abnormal cell morphology such as rounded and elongated cells was also observed. This finding was consistent with the downregulation of QS-related genes: luxS, pfS, sdiA, hflX, motA, and fliA. At 1/2 MIC and 1/4 MIC concentrations of berberine, a significant downregulation of luxS, pfS, hflX, ftsQ, and ftsE was observed. The results indicate that berberine and matrine can inhibit biofilm formation by inhibiting the QS system and that berberine is more effective than matrine.

PK/PD modeling of Ceftiofur Sodium against Haemophilus parasuis infection in pigs.

BACKGROUND: Ceftiofur Sodium is widely used in China. Our aim was to determine Ceftiofur Sodium activity and optimize dosing regimens against the pathogen Haemophilus parasuis using an in vitro and ex vivo pharmacokinetics/pharmacodynamics modeling approach. By adopting these strategies, we wanted to extend the effective life of Ceftiofur Sodium in reduce drug-resistance in pigs. RESULTS: We established an H. parasuis infection model in pigs, and assessed the pharmacokinetics of Ceftiofur Sodium in both healthy and infected animals. After Ceftiofur Sodium (10 mg/kg, i.m.) administration to healthy and H. parasuis-infected pigs, plasma based desfuroylceftiofur (a metabolite of Ceftiofur Sodium) was measured by High Performance Liquid Chromatography. The pharmacokinetics of Ceftiofur Sodium (desfuroylceftiofur) was consistent with a two-compartment open model, with first-order absorption. We observed no significant differences (P > 0.05) in pharmacokinetic parameters between healthy and infected pigs. Pharmacodynamics data showed that Ceftiofur Sodium was highly inhibitory against H. parasuis, with MIC, MBC, and MPC values of 0.003125, 0.0125 and 0.032 µg/mL, respectively. Desfuroylceftiofur in plasma also had strong bactericidal activity. Almost all H. parasuis cultured in plasma medium of Ceftiofur Sodium-inoculated healthy pigs, at each time point, were killed within 24 h. A weaker antibacterial activity was measured in infected-pig plasma medium at 18, 24, 36, and 48 h, after Ceftiofur Sodium inoculation. Pharmacokinetic parameters were combined with ex vivo pharmacodynamic parameters, and the bacteriostatic effect (36.006 h), bactericidal effect (71.637 h) and clearance (90.619 h) within 24 h, were determined using the Hill equation. Dose-calculation equations revealed the optimal dose of Ceftiofur Sodium to be 0.599-1.507 mg/kg. CONCLUSIONS: There were no significant differences in Ceftiofur Sodium pharmacokinetic parameters between healthy and infected pigs, although pharmacokinetics/pharmacodynamics fitting curves showed obviously differences. The optimal dose of Ceftiofur Sodium was lower than recommended (3 mg/kg), which may provide improved treatments for Glässers disease, with lower drug-resistance possibility.

Rapid Detection of Serratia fonticola by TaqMan Quantitative Real-Time PCR Using Primers Targeting the gyrB Gene.

A gyrB gene is present in the majority of bacterial species, and encodes the ATPase domain of DNA gyraseB-subunit protein, which is essential for transcription and replication of bacteria. The gyrB gene exhibits higher nucleotide sequence variability than the 16S rDNA gene and thus could be more reliable in differentiating Serratia fonticola. A species-specific primer pair and probe were designed for quantitative real-time PCR detection of S. fonticola using gyrB as the target gene. Nine members of the Serratia family (representing nine Serratia species) were chosen to verify the specificity of the primers. Additionally, two species each of Salmonella and Klebsiella, and five other species belonging to five other genera of Enterobacteriaceae, were tested for primer cross-reaction. All the tested strains gave negative results. The limit of detection for S. fonticola using the gyrB gene was 100 copies per PCR reaction. This TaqMan PCR assay provided a specific, rapid, and sensitive method to detect S. fonticola based on its gyrB gene.

Cornmeal-induced resistance to ciprofloxacin and erythromycin in enterococci.

OBJECTIVE: To establish a model ecosystem to study the impact of cornmeal on the appearance and persistence of the erythromycin (ERY)- and ciprofloxacin (CIP)-resistant phenotypes in waterborne enterococci. METHODS: After the model ecosystem was established, the system was divided into six dose groups, with the addition of 8, 4, 1, 0.25, 0.05, and 0 g L(-1) sterilized cornmeal. System mud samples were collected at 0, 1, 3, 7, 14, 30, 40, 61, and 130 d, and enterococci present in the mud samples were evaluated for their sensitivities to CIP and ERY. PCR was employed to detect genes such as gyrA and ermB. The gyrA gene was sequenced, and codons 83 and 87 were analyzed for mutations. RESULTS: (1) The addition of 0.05-8 g L(-1) cornmeal had an impact on CIP resistance. The higher the dose of cornmeal added, the larger the impact it generated. Furthermore, the earlier the emergence of CIP-resistant strains, the greater the incidence of drug resistance. The impact of cornmeal on resistance to ERY was less consistent, and the degree of the impact was not in proportion to the dose of cornmeal added. (2) There were no mutations at codons 83 and 87 in the gyrA genes from 102 strains isolated from the model ecosystem. The incidence of ermB-positive strains of ERY-resistant enterococci (28 strains) was 78.6%, and the incidence of ermB-positive strains of ERY-sensitive enterococci (16 strains) was 0%. CONCLUSIONS: (1) Adding different doses of cornmeal can facilitate resistance to CIP and ERY in waterborne enterococci. In this study, the degree of resistance was related to the cornmeal dose. (2) In the model ecosystem, enterococcal CIP resistance was not caused by a gyrA gene mutation; however, in the vast majority of cases, resistance to ERY was related to the ermB resistance gene.