The Preliminary Sduty On The Effect Of Chinese Traditional Medicines And Antibiotics On The E. Coli Biofilm Formation And Virulence Genes Expression

Escherichia coli is conditional pathogenic bacteria commonly founded in human or animal intestines, most E.coli strains are not pathogenic in normal circumstances, but some serotypes can cause illness. Enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7and E. coli O104:H4, is one type of toxigenic intestinal pathogens that can cause life-threatening hemorrhagic colitis(HC) and renal hemolytic uremic syndrome(HUS). EHEC patients are treated mainly with supportive therapy. The use of antibiotics is not recommended, or even prohibited, because many reports have shown that antibiotics can stimulate E. coli to generate or release Stx, which increase the risks of HC patients becoming HUS patients.Traditional Chinese Medicine (TCM) plays an important role in treating infectious diseases and has been used as an important repository of seeking alternatives to antibiotics drugs, because of its complex composition, broad antibacterial mechanism, low bacterial resistance and it also can weaken the virulence of bacteria. Therefore, the focus of this research is how to extract an effective drug, from TCMs to treat EHEC infections, without inducing the expression of Stx2, eae and the other virulence genes. The main contents and results are as follows:1. Selecting of TCMs which can inhibit the bacterial ability of biofilm formationIn vitro, we finished the biofilm quantitative analysis of Chicken Salmonella enteritidis using micro-plate method and tube method, The Tube method is simple、 convenient、flexible、fast and repeatable. We acquired seven kinds of TCMs which not only have better antibacterial effect but also can inhibit the biofilm formation, they are Corni Fructus、Schisandrae Chinensis Frucus、Fraxini Cortex、Achyranthis Bidentatae Radix、Rehmanniae Radix、Rhizoma seu Radix Notopterygii and Aucklandiae Radix.2. Effects of antibiotics and TCMs on Virulence Genes expression of E.coli0157:H7Real-time RT-PCR was used to quantitatively compared the effects of antibioti c(levofloxacin、streptomycin、chloramphenicol、erythromycin、tetracycline、 cefotaxime sodium) and TCMs(Coptidis Rhizoma、Fraxini Cortex、Schisandrae Chinensis Fructus、Scutellariae Radix、Aucklandiae Radix、Rehmanniae Radix、Radix et Rhizome Rhei、Achyranthis Bidentatae Radix、Corni Fructus、Rhizoma seu Radix Notopterygii) on the expression of stx2, eae and ygaG at the mRNA level. We also finished the quantitative analysis of Stx using a double-antibody (sandwich) ELISA method at the protein level.2.1Effects of antibiotics and TCMs on stx2gene expression of E.coli0157:H7E. coli O157:H7can produce two types of Stx, include Stxl (encoded by stxl gene) and Stx2(encoded by stx2gene), compared with Stxl, Stx2is more toxic and closely related with the occurrence of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The experimental results at mRNA level show that: chloramphenicol、levofloxacin and streptomycin strongly increased stx2gene expression in E. coli O157:H7, the maximum inductive effect was a thousand times higher when compared with control (without antibiotic exposure), while levofloxacin was five hundred times higher, streptomycin is five hundred times higher than that of control. Chloramphenicol had the strongest capacity for inducting the expression of stx2gene, followed by levofloxacin. Compared with the antibiotics, TCMs had no significant inductive effects on stx2expression, Aucklandiae Radix(AR) had the strongest capacity for inducting the expression of stx2gene, was six times higher than that of control, which is far below that of Chloramphenicol, Levofloxacin, and Erythromycin by hundred times and thousand times. In contrast, Coptidis Rhizoma(CR)、Achyranthis Bidentatae Radix(ABR) and Rehmanniae Radix(RR) can inhibit the transcription and expression of the stx2. The results trend at Protein level was basically consistent with the experimental results of the quantitative PCR, chloramphenicol, the Stx releasedwas more than10times than that of control when exposed to streptomycin chloramphenicol and levofloxacin. The results of some TCMs is inconsistent with the experimental results of the quantitative PCR, Coptidis Rhizoma(CR) can inhibit the expression of stx2at mRNA level, but the result of protein level indicate it can induce the expression of stx2, we specurate that CR can increase the release of Stx by increasing the cell permeability, not by promting the transcription and translation of stx2, the other TCMs’ error may be caused by their color.2.2Effects of antibiotics and TCMs on eae gene expression of E.coli0157:H7The protein Intimin encoded by eae gene can induce attaching and effacing lesions (A/E lesions) on human enterocytes, is closely related to the virulence of E. coli O157:H7. The experimental results at mRNA level show that:levofloxacin had no significant effect on the eae expression, cefotaxime sodium can inhibit the eae expression at1/2MIC, the rest of drug concentration had no significant effect on the eae expression, the other four kinds of antibiotics can inhibit the eae expression up to three hundredfold compared with control.TCMs all can have inhibited effects on the expression of eae to some extent.2.3Effects of antibiotics and TCMs on ygaG gene expression of E. coli0157:H7The formation of Biofilm can improve the intestinal pathogen’s virulence, the product of gene ygaG can catalyze S-adenosylmethionin(SAM) to generate AI-2, which is closely related to biofilm formation.The experimental results at mRNA level show that:cefotaxime sodium had no significant effect on the expression of ygaG, chloramphenicol can inhibit its expression, while the other four kinds of antibiotics can induce its expression up to three times compared with control. When the E.coli0157:H7was treated with TCMs at1/2MIC, Schisandrae Chinensis Fructus(SCF)、 Fraxini Cortex(FC)、Fraxini Cortex(FC) and Coptidis Rhizoma(CR) all have inhibitory effect on the expression of ygaG, Corni Fructus(CF)、Achyranthis Bidentatae Radix(ABR)、Rehmanniae Radix (RR)、Rhizoma seu Radix Notopterygii(RsRN) and Aucklandiae Radix(AR) had inductive effect on the expression of ygaG.3. Induction of antibiotics and TCMs to SOS responseWe detected the in vitro mutagenic capacity with the SOS/umu test, ten kinds of TCMs we had selected all cannot induce the SOS response; but some antibiotics such as Chloramphenicol and Erythromycin can induce the SOS response, and some antibiotics such as Cefotaxime, Tetracyclines, and Kanamycin cannot induce the SOS response. Chinese herbal pieces cannot induce the SOS response, while the antibiotics can induce weakly or strongly the SOS response. TCMs cannot induce or can weakly induce the expression of the stx2, while the antibiotics can strongly induce or cannot induce the expression of the stx2. The two trends basically coincide. Wether the drug can induce the release of Stx or not is related with the SOS response, damage DNA can activate the protein RecA, activated RecA can degrade the repressor protein LexA and CI, the degration of CI can activate the expression of Stx encoding genes which were orginal silent, synthetized a large number of phage particles, the bacteria was lysed at last, leading to the release of Stx.E. coli O157:H7is the most commonly encountered member of EHEC, several virulence factors contribute to the pathogencity of E. coli O157:H7. Virulence factors conclude STX(encoded by stx), intimin(encoded by eae),hemolysin(encoded by hly) and bioflim which is related with gene ygaG. Considering the test results above, we conclude:TCMs we selected do not induce the release of the STX, in addition, TCMs can inhibit the expression of eae and formation of biofilm. Compared with antibiotics, TCMs has a comparative advantage in the treatment of infections caused by E. coli O157:H7. TCMs are a good choice for screening new drugs for the treatment of infections caused by E.coli.