Effect Of Cyadox On Human Colonic Microflora In Chemostat Models

Cyadox is a new antimicrobial growth promoter of quinoxalines. It can promote the growth of animals, enhance productive performance of livestock, poultry and aquatic animals, reduce rates of infection incidence, therefore, it has a good prospect for application because of its property of good safety, highly effectivety and low toxicity. Laboratory preliminary systematic researches demonstrated that cyadox metabolism and the law of residues and elimination, toxicology research showed its safety was good, however, there is no related microbiological safety research whether cyadox has adversed effects on human colonic microflora or not, in order to guarantee food safety, it is necessary to evaluate microbiological effects, including the composition and population of the intestinal microbiota, the activities of microbiota, ratesof resistance and colonization barrier effect. At the same time, microbial effect of antimicrobial drugs was an important part of food security assessment, so chemostat models were applied to systematically assess the effect of cyadox on human colonic microflora in order to provide microbiological safe data more entirely and systematically and offer scientific and comprehensive data for cyadox declaration and a further step application.1Effect of cyadox on human colonic microfloraChemostat models which FDA recommended are adopted to study the effect of cyadox on human intestinal flora. at first, The cyadox and metabolites susceptibility test was analysted to four dominant bacteria(Escherichia coli, Enterococci, Bifidobacteria and B.fragilis), Cyl,Cy2,Cy4,Cy6,Cy10,Cy12metabolites had no antibacterial activity compared with cyadox, therefore, it was only observed microbiological effect of cyadox on colonic microflora. The result of cyadox sensitivity test to four clinical isolate dominant bacteria was1-16μg/mL. Based on this range,0.5-128μg/mL series concentrations wered designed to incubate with stool samples in anaerobie glove box, changes of population of Escherichia coli, Enterococci, Bifidobacteria and B.fragilis were detected, proportion of resistant Escherichia coli and Enterococci,4μg/mL,16μg/mL,32μg/mL,128μg/mL final concentrations were set to have a pretest in chemostat models. Chemostat models ran steady state after inoculating faecal samples in7d, the models were exposed to cyadox4,16,32and128μg/mLfroml3to20d, population of dominant bacteria(Escherichia coli, Enterococci, Bifidobacteria, B.fragilis), change of SCFA(AC, PC, BC) and propotion of resistant facultative anaerobes(Escherichia coli and Enterococci) were observed during administration. Each models were challenged with108CFU/mL Salmonella typhimurium inoculated in1mL/d at21,22and23d, samples collected daily were observed the population of Salmonella typhimurium, wich as a judement index of colonization resistance.Populations of four dominant bacteria, concentrations of SCFA, propotions of resistant facultative anaerobes and populations of Salmonella typhimurium were in95%confidence intervals at the groups of4μg/mL and16μg/mL, and there was no effect on human intestinal microbiota in this two drug groups. The variance of populations of Escherichia coil and Enterococci were retained106and104magnitude order, the variances of Bifidobacteria and B.fragilis were maintained in107magnitude order, the populatins of four dominant bacteria not only were in95%confidence intervals, but also in the range of normal physiological data during administration; the populaion of Salmonella typhimurium was numbered, results showed that there was no signifinant difference in4,16μg.mL compared with control group, Salmonella typhimurium did not grow in this two drug chemostat models exerted effective colonization resistance.Conversely, at32,128μg/mL there were different degrees effect on four observation indexes, populations of four dominant bacteria were reduced at32and128μg/mL groups, especially for anaerobes(Bifidobacteria and B.fragilis); the susceptibility of Escherichia coli and Enterococci were increased in two groups and the population of Salmonella typhimurium which was significant difference compared with no-drug group presented a trend of increase at32,128μg/mL group, and the colonization barrier of colonic flora was disrupted after32,128μg/mL cyadox administration. However, there was no effect on concentrations of SCFA, concentrations of acetic acid; propionic acid and butyric acid were in95%confidence intervals during32,128μg/mL cyadox administrations. It may be related to chemostat models not to simulate intestinal absorption function, which makes changes of concentration of SCFA unobvious. By studying the influences of4,16,32,128μg/mL cyadox on human colonic microflora in models, it showed that16μg/mL cyadox had no effect on observation indexes, and this concentration does not disrupt microflora balance.2Analysis of resistance manner of Escherichia coil and EnterococciResistant Escherichia coli and Enterococci were isolated from32,128μg/mLcyadox chemostat models samples before administration(the8-13d) and during administration(the14-20d),10strains resistant Escherichia coil and Enterococci were isolated from32μg/mL concentration in chemostat model before administration, respectively; and in128μg/mL model9srains resistant Escherichia coil and9srains Enterococci were isolated. During administration14strains resitant bacteria(Escherichia coil and Enterococci) were isolated from32μg/mL model, respectively, and13strains E.coil and Enterococci resistant isolates from128μg/mL cyadox chemostat model. Then we analysed resistant phenotype of Escherichia coil and Enterococci isolated from models before and during administration, it was showed there was high level resistance for quinoxalines (MIC>64μg/mL), at the same time, bacteria were also resistant to β-lactam antibiotics (MIC>128μg/mL). Then primers were designed to amplify target fragments of quinoxalines resistant genes oqxAB in isolated resistant bacteria, the target fragments of oqxA and oqxB were670bp and520bp. oqxAB genes were in3of10strains and1in9strains Escherichia coil from32,128μg/mL modes before administration, respectively. There had oqxAB genes in5of14strains Escherichia coli from32μg/mL concentration and3of13strains in128μg/mL concentration during administration. Escherichia coil srains isolated from the two models before and during administration were cyadox-resistance, Escherichia coil isolated from models before administration was intrinsic resistant, resistance rate rising of Escherichia coil at32,128ug/mL groups was the action of intrinsic resistant Escherichia coil selection, resistance rate rising was caused by overgrowing of intrinsic resistant E.coil, a study showed MBC of cyadox against Escherichia coil under anaerobic condition was <32μg/mL, therefore it was impossible to induce cyadox-resistance Escherichia coilat32,128μg/mL concentration groups. however, oqxAB genes were not amplified in Enterococci before and during adminisration, may be it had other resistance genes, but what resistance genes were needs further study. Plasmids were extracted from oqxAB-positive Escherichia coil, extraction plasmids were used as amplified template to amplify oqxAB genes and the lengthes of oqxAB were correct, at the same time, sequencing results further confirmed that resistance genes were oqxAB, The oqxA and oqxB sequencing results were aligned with the sequence (Genbank, GI167613948), indicating that the homology was99%and100%between two sequences, respectively. Therefore, oqxAB genes of our isolated resistant Escherichia coli were located in plasmid; the resistance manner was plasmid-mediated quinoxalines resistance for Escherichia coil, oqxAB genes located in plasmid was correspond to reported in literatures.In conclusion, different concentration cyadox were designed to determine the effect of durg on human colonic microflora, there was no impact of16μg/mL cyadox on intestinal microflora, including population of four dominant bacteria, resistance rates of Escherichia coiland Enterococci, concentration of short chain fatty acid(acetic acid, propionic acid and butyric acid), and colonization barrier; human intestinal microflora was affected at32,128μg/mL concentration groups by different degrees,16μg/mL concentration had no effect on intestinal microflora, cyadox was an safer antimicrobial growth promoter. On the other side, resistance rate rising of Escherichia coildat32,128μg/mL groups was the action of intrinsic resistant Escherichia coil selection exposed to cydox, resistance rate rising was caused by overgrowing of intrinsic resistant Escherichia coil, at the same time, killing sensitive Escherichia coil, these resistant Escherichia coil isolates could be amplified oqxAB genes by PCR, plasmid confered resistance to Quinoxalines which was correspound to correlational study, it was showed there was some epidemic of oqxAB genes, therefore reinforce market supervision and ues antibacterial agents more safer and reasonability, and this can assure safty of animal derived food and human health.