Ultra-performance liquid chromatography-tandem mass spectrometry determination and depletion profile of flunixin residues in tissues after single oral administration in rabbits.

An ultra-performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS) method was developed for the detection of flunixin residues in rabbit tissues. The samples were extracted with acidic acetonitrile, defatted with n-hexane, and then purified by HLB solid-phase extraction cartridge. Analysis was carried out on UPLC-ESI-MS/MS working with multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the method were 0.3-0.8µgkg(-1) and limits of quantification (LOQs) were 1.0-3.0µgkg(-1) in rabbit tissues, respectively. In all fortified samples at a concentration range of 1.0-300.0µgkg(-1), mean recoveries were 61.7-115.7% with relative standard deviations (RSDs) below 16%. Residue depletion of flunixin in rabbit was conducted after oral administration at a dose of 5mgkg(-1) of body weight. The average concentrations for flunixin measured 2h post-administration in kidney and intestine were significantly higher than in liver, heart and muscle. The concentrations for flunixin in all rabbit tissues were below the LOD or not detected in all tissues after 96h administration of drug. A minimum withdrawal time of 21h was indicated for residue levels in heart, liver, kidney, intestine and muscle below the maximum residue limits (MRLs).

A survey of ß-lactamase and 16S rRNA methylase genes among fluoroquinolone-resistant Escherichia coli isolates and their horizontal transmission in Shandong, China.

The prevalence of ß-lactamase, 16S rRNA methylase genes, and plasmid-mediated fluoroquinolone-resistance (PMQR) determinants (qnrC and qnrD) was determined by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli isolated from a chicken farm, a pig farm, and a hospital in Shandong, China in 2007. The bla(TEM) and bla(CTX-M) were the most prevalent ß-lactamase genes in isolates from chickens (88.4%, 175/198 and 81.3%, 161/198) and hospitalized patients (87.8%, 122/139 and 69.1%, 96/139). The bla(TEM) was the most prevalent ß-lactamase gene observed in isolates from pigs (98.5%, 135/137). The gene bla(CMY-2) was also predominant among isolates from chickens (20.2%, 40/198). The bla(LAP-1) gene was first detected in one strain from chickens and humans (pig farm workers) in China. Only one strain from hospitalized patients was found to possess bla(SHV). The rmtB was the most prevalent 16S rRNA methylase gene detected in isolates from chickens (19.7%, 39/198) and hospitalized patients (15.8%, 22/139). To our knowledge, this is the first report of the detection of the qnrD gene in E. coli from chickens and pigs in China. The qnrC and bla(KPC) genes were not detected in any of the isolates. Results of southern hybridization revealed that PMQR determinants, ß-lactamases, and 16S rRNA methylase genes were located on the same plasmid in E. coli strains derived from patients. Also, PMQR determinants and ß-lactamase genes were localized on the same plasmid in an E. coli strain of animal origin. Results of conjugation experiments revealed that all of these plasmid-based resistance genes can be transferred by conjugation through horizontal transmission.

A survey of plasmid-mediated fluoroquinolone resistance genes from Escherichia coli isolates and their dissemination in Shandong, China.

Bacterial resistance to fluoroquinolones result from mutations in the quinolone resistance-determining regions of the drug targets, overexpression of efflux pumps, and/or the more recently identified plasmid-mediated low-level resistance mechanisms. We investigated the prevalence of and characterized plasmid-mediated fluoroquinolone resistance genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli (n = 530) isolated from a chicken farm, a pig farm, and hospitalized patients in Shandong, China, in 2007. The aac(6')-Ib-cr gene was the most prevalent resistance gene that was detected in bacteria isolated from all sources. Next was the qnrS gene, which was predominantly present in isolates from the pig farm. Only eight (5.8%) isolates from hospital patients were found to possess the qepA gene, and these isolates were first reported in qepA-carrying E. coli from humans in China. The qnrA and qnrB genes were not detected in any of the isolates. Further, most of the isolates were also resistant to beta-lactams and aminoglycosides as determined by the broth microdilution method. Pulsed-field gel electrophoresis analysis of the E. coli isolates with similar resistance patterns that also carried resistance genes showed great genomic diversity among these bacteria, suggesting that the multiresistant E. coli isolates carrying the qnr, aac(6')-Ib-cr, or qepA genes were not derived from a specific clone, but represented a wide variety of different genotypes. The results of Southern hybridization revealed that qepA, qnrS, and parts of aac(6')-Ib-cr genes were localized on plasmids and/or chromosome. qepA and aac(6')-Ib-cr genes were colocalized with aac(6')-Ib-cr and qnrS genes, respectively, on the same plasmids. Our study demonstrated that two different genes (qepA and aac(6')-Ib-cr) were identified on the same plasmid in E. coli strains derived from patients and qnrS and aac(6')-lb-cr genes on the same plasmid in an E. coli strain of animal origin.