| Antibiotics are widely used and even abused in many fields,resulting in serious environmental pollution.Antibiotics in the environment may enter the organism and even the human body through the food chain,then change the structure and quantity of flora in the body,and even lead to the death of the body infected with some superbacteria,which not only brings difficulties to the recycling of water resources,but also poses a potential threat to human health.In recent years,traditional Chinese medicine has been widely used in the medical industry because of its outstanding curative effect.Then,will antibiotics and traditional Chinese medicine have a joint impact on the flora in the body? However,there are few related studies and the mechanism is unclear.Therefore,eight commonly used medical drugs,seven antibiotics including penicillin G potassium(PEN),penicillin V potassium salt(PHE),piperacillin sodium(PIP),streptomycin sulfate(STR),tobramycin(TOB),ampicillin sodium(AMP),tetracycline hydrochloride(TET)and one Chinese medicine berberine chloride hydrate(BCH),were selected as the research object in this paper.A common strain Escherichia coli(E.coli)in humans and animals,was selected as the test organism and 96-well transparent microplate as the experimental carrier,the time-dependent toxicity microplate assay(t-MTA)based on E.coli was established.The binary and multi-component mixture systems(a total of 162rays)were designed by direct equipartition ray method(Equray)and uniform design ray method(UD-ray),respectively.Their bacteriostatic effects on E.coli were systematically investigated at exposure time of 0.25,2,4,8 and 12 h.The combined bacteriostatic interactions within mixtures were analyzed by concentration addition model(CA).The absolute residual model(d CA)was used to quantitatively evaluate the intensity of the interaction.The bacteriostatic mechanism was revealed by measuring the changes of conductivity parameters,relative nucleic acid content,bacteriostatic circle diameter and cell morphology of 8 drugs and their multi-component mixtures before and after exposed.The main conclusions of this paper are as follows:(1)Taking five antibiotics as the research object,the t-MTA based on E.coli was established by adjusting experimental factors such as medium composition,exposure time,data processing method and mixture system design.And the bacteriostatic data of eight drugs and their mixture systems against E.coli under different exposure time and concentration were systematically determined by t-MTA.The results show that,compared with the traditional method,it not only has simple operation and accurate data,but also can detect a wider range of concentrations and study more influencing factors.In addition,the antibacterial dose-response relationship can be effectively characterized by Weibull function,which is "S" type,and has time-dependent antibacterial activity.The antibacterial intensity increases with the extension of exposure time.Most of the mixture systems have significant component-and concentration-ratio dependent antimicrobial activity.(2)The interaction of 26 binary to five representative mixture systems of five antibiotics(PEN,PHE,PIP,STR and TOB)is antagonistic or synergistic,and the interaction intensity of different systems shows different laws with the change of exposure time and concentration.The maximum synergistic or antagonistic intensity is mainly located in the high concentration area of long exposure time(8?12 h),and the maximum synergistic and antagonistic intensity can reach 0.50 and 0.40.(3)Simple mixtures ystems including four binary and ternary mixture systems of two antibiotics and one traditional Chinese medicine(AMP,TET and BCH)have strong synergistic effects.The synergistic intensity is positively correlated with exposure time and concentration,and the maximum synergistic intensity is more than 0.85.(4)The interaction of an complex eight-component mixture system composed of antibiotics and traditional Chinese medicine changes with exposure time and concentration,from the addition of medium and low concentration areas and the antagonism of high concentration areas to the overall addition,and then to the synergy of medium and high concentration areas.The antagonistic intensity decreased with the extension of exposure time and increased with the increase of mixture concentration,and the maximum antagonistic intensity was 0.33.The synergistic intensity increased with the extension of exposure time,increased first and then decreased with the increase of drug concentration,and the maximum synergistic intensity was 0.83.(5)Antibiotics and traditional Chinese medicine have no obvious inhibition at low dose,but the inhibition is significantly enhanced after their combination,that is,the combined inhibition of antibiotics and traditional Chinese medicine are significantly clearer than that of single drugs.The interaction of the mixture system composed of antibiotics is synergy or antagonism,while the interaction of the mixture system composed of antibiotics and traditional Chinese medicine is strong synergy.(6)Most of the mixture systems do not produce obvious drug resistance.After the exposure to the eight drugs and their representative mixture systems,the permeability of E.coli cell membrane gradually increases,the relative nucleic acid content continues to rise,and the cell morphology is seriously damaged.This results in long strip,round ball,concave convex shrinkage,swelling and coarsening,rupture and adhesion,dry holes and other phenomena,which promotes the failure and death of the cell. |
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