Study On The Isolation And Identification Of E.coli And Mongolian Medicine Antibacterial Components Selection And The Function

Calf diarrhea is a common disease in calves. Currently, large cattle farms often use antibiotics therapy to treat the disease. However, long-term use of antibiotics therapy on calf diarrhea can cause a number of side effects including drug residue animal and drug resistance Mongolian medicine is pure natural. According to previous research, Mongolian medicine Babu-7 has strong bacterioustasis effect and curative efficative is significant. Our aim is to select the effective inhibitory ingredients in this compound in vitro and vivo, and to explore related mechanism. The result of our experiments are as follows:l.To understand the serological distribution of pathogenic E.coli around Hohhot, We collected 127 fecal samples(37 samples of calf diarrhea;40 samples of dairy cattle and 40 samples of breed cows.) from a cow farm around Hohhot. We obtained 97 strains of suspected E.coli after separation and cultivation. By morphological observation and biochemical identification, we confirmed that 65 of them were E.coli. We isolated three serotypes through "O" serotype identification, and they wrer O145, O152, and O167,accounting for 1.96%,1.96%, and 7.84% in these 65 strains of E.coli, respectively. Then we used PCR to further identify these 65 strains. After sequencing the PCR products, we obtained two new serotypes of O26 and O83. These two serotypes were not detected in the previous serotype experiments, indicating that PCR was more sensitive. We also tasted pathogenicity in animals of these 65 stauns, of which 51 (40%) were highly pathogenic, suggesting a wide distribution of highly-pathogenic E.coli.2. Using Oxford cup method, we found that five out of six active inhibitory ingredients in Mongolian medication Babu-7 were effective on calf diarrhea. After testing MIC and FIC indices, we confirmed that these five ingredients were not antagonized and suitable for compatibility. The MIC of these five ingredients on E.coli were:Alizarin 5mg/mL, Rhein 20mg/mL, Emodin 20mg/mL, Quercetin 7.5mg/mL, and Galic acid 7.5mg/mL.3. We used orthogenal design to test bacterioustasis of above-mentioned five ingredients to obtain the most effective one. We also used orthogenal design to determine dosage proportion between medicines. The optimal dosage combination for inhibiting E.coli was A2B1C3D2E2.4. We used microdilution method to measure MIC of the inhibitory composition and the result was 0.675mg/mL. The mice protection ratio in vivo of this composition was 50%, close to the ratio in CPFX (66.7%).5.To provide theoretical evidence for the optimized composition, we used the ONPG method to determine its effect on permeability of cytomembrane in E.coli. We further tested its effects on electrical conductivity and cell wall to better understand the impact of this composition on cellular membrane of E.coli. The results showed that the permeability of cytomembrane, the electrical conductivity of solution and the AKP amount all increased after the composition was given. These results proved that this composition functioned through destroying cytomembrane to increase membrane permeability.6. To determine the antibacterial spectrum of this Mongolian inhibitory composition, we measured the inhibitory diameter of bacteriostasis in vitro and MIC. The results showed that the composition had a broad spectrum and could inhibit with E.coli and Staphylococcus aureus.