Showing papers by "Zonghui Yuan published in 2012"

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

Abstract: Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

Key genetic elements and regulation systems in methicillin-resistant Staphylococcus aureus

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA), popularly known as a type of superbug, has been a serious challenge for animal and human health. S. aureus has developed methicillin resistance mainly by expression of β-lactamase and PBP2a, which is regulated by the blaZ-blaI-blaR1 and mecA-mecI-mecRI systems. Other genetic elements, including murE and femA, also participate in expression of methicillin resistance, but the mechanism remains unclear. The evolution of the staphylococcal cassette chromosome mec determines the epidemiological risk of MRSA. The plasmid-located gene cfr might contribute to multiresistance and transmission of MRSA. Some virulence factors, including Panton-Valentine leukocidin, phenol-soluble modulin, arginine catabolic mobile element and other toxin elements enhance the pathogenesis and fitness of MRSA. Two-component regulation systems (agr, saeRS and vraRS) are closely associated with pathogenesis and drug resistance of MRSA. The systematic exploration of key genetic elements and regulation systems involved in multidrug resistance/pathogenesis/transmission of MRSA is conclusively integrated into this review, providing fundamental information for the development of new antimicrobial agents and the establishment of reasonable antibiotic stewardship to reduce the risk of this superbug.

Pharmacokinetics of tulathromycin and its metabolite in swine administered with an intravenous bolus injection and a single gavage

Abstract: Tulathromycin is a macrolide antimicrobial agent proposed for therapeutic use in treatment of porcine and bovine respiratory disease. In this study, the absolute bioavailability of tulathromycin solution was investigated in pigs. Eight pigs, with body weight of 20.5 ± 1.6 kg, were given a single dose of tulathromycin at 2.5 mg/kg oral (p.o.) and intravenous (i.v.) in a crossover design. The plasma concentrations of tulathromycin and its metabolite were determined by LC-MS/MS method, and the pharmacokinetic parameters of tulathromycin were calculated by noncompartmental analysis. After p.o. administration, the maximum plasma concentration (C(max) ) was 0.20 ± 0.05 μg/mL at 3.75 ± 0.71 h. The terminal half-life (t(1/2λz) ) in plasma was 78.7 ± 6.75 h, and plasma clearance (Cl/F) was 1.14 ± 0.28 L/h/kg. After i.v. injection, plasma clearance (Cl) was 0.580 ± 0.170 L/h/kg, the volume of distribution (Vz) was 64.3 ± 21.2 L/kg, and the t(1/2λz) was 76.5 ± 13.4 h. In conclusion, an analytical method for the quantification of tulathromycin and its metabolite in plasma in swine was developed and validated. Following p.o. administration to pigs at 2.5 mg/kg b.w., tulathromycin was rapidly absorbed and the systemic bioavailability was 51.1 ± 10.2.

Intestinal metabolism of T-2 toxin in the pig cecum model

Abstract: T-2 toxin, a toxic member of the group A trichothecenes, is produced by various Fusarium species that can potentially affect human health. As the intestine plays an important role in the metabolism of T-2 toxin for animals and humans, the degradation and metabolism of T-2 toxin was studied using the pig cecum in vitro model system developed in the author’s group. In order to study the intestinal degradation of T-2 toxin by pig microbiota, incubation was performed with the cecal chyme from four different pigs in repeat determinations. A large variation in the intestinal degradation of T-2 toxin was observed for individual pigs. T-2 toxin was degraded almost completely in one out of four pigs, in which only 3.0 ± 0.1 % of T-2 toxin was left after 24 h incubation. However, in the other three incubations with pig cecal suspension, 54.1 ± 11.7–68.9 ± 16.1 % of T-2 toxin were still detectable after 24 h incubation time. The amount of HT-2 toxin was increased along with the incubation time, and HT-2 toxin accounted for 85.2 ± 0.7 % after 24 h in the most active cecum. HT-2 toxin was the only detectable metabolite formed by the intestinal bacteria. This study suggests that the toxicity of T-2 toxin for pigs is caused by the combination of T-2 and HT-2 toxins.

In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis.

Abstract: The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10−7, 1.4×10−7, and 1.3×10−8, respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.

Development of quinoxaline 1, 4-dioxides resistance in Escherichia coli and molecular change under resistance selection.

Abstract: Quinoxaline 1, 4-dioxides (QdNOs) has been used in animals as antimicrobial agents and growth promoters for decades. However, the resistance to QdNOs in pathogenic bacteria raises worldwide concern but it is barely known. To explore the molecular mechanism involved in development of QdNOs resistance in Escherichia coli, 6 strains selected by QdNOs in vitro and 21 strains isolated from QdNOs-used swine farm were subjected to MIC determination and PCR amplification of oqxA gene. A conjugative transfer was carried out to evaluate the transfer risk of QdNOs resistant determinant. Furthermore, the transcriptional profile of a QdNOs-resistant E. coli (79O4-2) selected in vitro with its parent strain 79-161 was assayed with a prokaryotic suppression subtractive hybridization (SSH) PCR cDNA subtraction. The result showed that more than 95% (20/21) clinical isolates were oqxA positive, while all the 6 induced QdNOs-resistant strains carried no oqxA gene and exhibited low frequency of conjugation. 44 fragments were identified by SSH PCR subtraction in the QdNOs-resistant strain 79O4-2. 18 cDNAs were involved in biosynthesis of Fe-S cluster (narH), protein (rpoA, trmD, truA, glyS, ileS, rplFCX, rpsH, fusA), lipoate (lipA), lipid A (lpxC), trehalose (otsA), CTP(pyrG) and others molecular. The 11 cDNAs were related to metabolism or degradation of glycolysis (gpmA and pgi) and proteins (clpX, clpA, pepN and fkpB). The atpADG and ubiB genes were associated with ATP biosynthesis and electron transport chain. The pathway of the functional genes revealed that E. coli may adapt the stress generated by QdNOs or develop specific QdNOs-resistance by activation of antioxidative agents biosynthesis (lipoate and trehalose), protein biosynthesis, glycolysis and oxidative phosphorylation. This study initially reveals the possible molecular mechanism involved in the development of QdNOs-resistance in E. coli, providing with novel insights in prediction and assessment of the emergency and horizontal transfer of QdNOs-resistance in E. coli.

Development of an accelerated solvent extraction, ultrasonic derivatisation LC-MS/MS method for the determination of the marker residues of nitrofurans in freshwater fish.

Abstract: A rapid method using accelerated solvent extraction (ASE) and ultrasound enhanced derivatisation has been developed for the quantitative determination of metabolites of nitrofurans, namely 3‐amino‐2‐oxalidinone (AOZ), 5‐morpholinomethyl‐3‐amino‐2‐oxalidinone (AMOZ), 1‐amino‐hydantoin (AHD) and semicarbazide (SEM), in muscle and skin of carp and finless eel. The target analytes were extracted using ASE, ultrasonic derivatisation for 1 h and then purified by solid phase extraction. Averaged decision limits (CCα) and detection capability (CCβ) of the method were in the range of 0.07–0.13 and 0.31–0.49 µg kg−1 in carp and finless eel, respectively. The accuracy in terms of recovery was in the range 77.2–97.4%. The simplified and traditional methods were compared with incurred residue samples. The simplified method reduced the derivatisation time and has been applied to the determination of nitrofurans residues in fish.

Inhibitors targeting on cell wall biosynthesis pathway of MRSA

Abstract: Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA.