The Effect Of Glucose On The Formation Of Antibiotics Resistance Of Water Source Enterococcus

Objective: In this study, model ecosystems were set up to study the effect ofGlucose on the resistant phenotype of water source Enterococcus to ciprofloxacin(CIP), erythromycin (ERY), oxytetracycline (OTC), chloramphenicol (CHL),ampicillin (AMP) and vancomycin (VAN). We also explored the resistant mechanismof Enterococcus to ciprofloxacin and erythromycin.Methods: Soil samples were collected and Enterococcus strains were isolated, thesensitivity of these strains to six kinds of antibiotics were detected, the sample showedthe sensitive results were used as the sediment in model ecosystems. After theestablishment of model ecosystems, six groups were set up, sterilization Glucose wereadded at the doses of0.02g/L,0.1g/L,0.4g/L,1.6g/L,3.2g/L and0g/L respectively,each group had two replications. Enterococcus strains were isolated from the modelecosystems at the time of0d,1d,8d,16d,28d,40d,84d and140d, sensitivity test ofthese Enterococcus strains to those antibiotics were detected; meanwhile, thedissolved oxygen(DO) and pH value of the sediment-water interface in differentstages were detected to set up the relationship between them to resistance. PCRmethods were used to detect the ermB, mefA, gyrA and Esp gene of Enterococcusstrains, gyrA gene sequence was determined and the83site and87site were analyzedto detect of mutation.Results:(1) The effect of different dose of Glucose to the isolation rate of Enterococcus wasdifferent. In the range of0.02g/L-0.4g/L, the addition of Glucose could increase theisolation rate remarkably. Adding0.02g/L Glucose increased the isolation rate from20%to90%; and0.1g/L and0.4g/L dose groups have same effect;1.6g/L and3.2g/Ldose groups have same isolation rate to control group.(2) At day1after adding of Glucose, CIP resistant Enterococcus were detected at the0.4,1.6,3.2g/L groups. General speaking, the more the addition dosage, the bigger the effect was, with the earlier appearance of resistant Enterococcus and the higherresistant rate. At day1after the adding of Glucose, ERY resistant Enterococcus weredetected at the0.1and3.2g/L groups. There was no correlation between the additiondosage and the resistant rate to ERY. At day8after the adding of Glucose, OTCresistant Enterococcus were detected at the0.02,0.1,0.4g/L groups and these threegroups have bigger and lasting effect on the resistant rate to OTC. The addition ofGlucose could induced the resistant phenotype to CHL, but the appearance of it waslater.(3) The rate of ermB gene was63.6%in44strains of erythromycin resistantEnterococcus while the rate was0%in the16strains of erythromycin sensitiveEnterococcus; Among28strains of ermB gene positive Enterococcus,24strains ofthem have the resistant level at256μg/mL and128μg/mL. Meanwhile, there was only1strain of Enterococcus could detect the mefA gene in all detected strains. In the72strains of Enterococcus which were isolated from model ecosystems, the rate of gyrAgene was83.3%; but there were no mutation detected at the site of83and87in thegyrA gene. Detection of drug-resistant strains did not find the Esp gene.Conclusion:(1) The Addition of Glucose in model ecosystems could easily induced theappearance of resistant Enterococcus to CIP and ERY, flowed by resistantEnterococcus to OTC, later was resistant Enterococcus to CHL. There were nodetectable resistant phenotype changes of Enterococcus to vancomycin andampicillin.(2) There was correlation between the dosage of Glucose and the appearance ofresistant Enterococcs to CIP and the resistant rate, but there was no correlationbetween the dosage of Glucose and the resistant rate of Enterococcs to ERY. In0.02,0.1,0.4g/L dosages, Glucose could easily induced the appearance of OTC resistantphenotype.(3) In the model ecosystem, the appearance of erythromycin resistant Enterococcus has strong correlation to the appearance of ermB gene. The addition of Glucose couldinduce the appearance of ermB gene and led to the resistance of some Enterococcus toERY. The appearance of ciprofloxacin resistant Enterococcus was not caused by themutation of gyrA gene; and the resistant phenotype has no correlation to Esp and mefAgenes changes.