| At present,antibiotics are mainly used for the treatment of E.coli.For a long time,a large number of antibiotics abuse in clinical practice has led to the widespread emergence of E.coli resistance,and the drug resistance spectrum has been increasing,which has a great impact on the prevention and treatment of chicken colibacillosis.In this study,pathogenic bacteria were collected from laying hens in Dangtu area,Anhui Province to isolate and identify the pathogenic bacteria,and their drug resistance was analyzed.From Yujin powder,Rhubarb,Scutellariae Radix and baicalin,which that could eliminate the drug resistance of clinical isolates were screened.The study provides important supports and reliable basis for the effective control of chicken colibacillosis.1.Isolation,identification and drug resistance analysis of E.coli from chickenLiver and heart of sick or dead chickens suspected of having recurrent chicken colibacillosis were collected from an chicken laying farm in Dangtu area,Anhui Province to isolate and identify pathogens.After isolation and culture,morphological observation,purification and amplification,biochemical identification and PCR identification,the results are as follows:wet,smooth,neat,and reddish-colored colonies were formed on the MacConkey agar medium.While on the Eosin-methylene blue agar medium,round,smooth,raised purple-black and typical metallic luster colonies were observed.Colonies picked were Gram stained and a medium-sized,red,obtusely rounded bacterium was observed under a microscope.The two isolates were positive for ferment glucose,lactose,maltose,mannitol,sucrose and MR,negative for VP.The results of PCR identification showed that the phoA gene amplification products of the two isolates had a size of 372 bp,while the uid gene amplification products had a size of 293 bp.Their sequences were similar to 100 E.coli strains with known gene sequences were all ≥99.0%.It was comprehensively determined that the pathogens isolated from the heart and liver were all E.coli and Abbreviated as HE and LE,respectively.The drug resistance test of HE and LE against 20 commonly used antibiotics was analyzed to determine the drug resistance.The results showed that HE and LE were multi-drug resistance,of which HE was resistant to 20 antibiotics and LE was resistant to 12,while sensitive to 8.2.Study on the effect of Yujin Powder and its prescriptions on eliminating the resistance of chicken-derived multidrug-resistant Escherichia coli to gentamicin and ciprofloxacinThe minimum inhibitory concentration(MIC)of the antibiotics,Yujin Powder and its prescriptions on HE and LE was determined by the micro-dilution method.The modified concentration of micro-inoculation colony counting method and the measured growth curve were used to screen the Chinese medicine effective concentration which would eliminate the antibiotics drug resistance.In resistance elimination tests were performed on HE and LE in nutrient broth containing suitable Chinese medicine concentration.Drug sensitivity tests and MIC values were measured to monitor changes in drug resistance of HE and LE.The combined effect of each traditional Chinese medicine with gentamicin and ciprofloxacin was determined by checkerboard dilution method.In the MIC measurement results,the MIC values of HE and LE were the same.The MIC values of Yujin powder,Rhubarb,Scutellariae Radix and baicalin were 250.0 mg/mL,250.0 mg/mL,62.5 mg/mL,2.0 mg/mL.Baicalin had the best antibacterial effect.125.0 mg/mL of Yujin powder,62.5 mg/mL of Scutellariae Radix,15.6 mg/mL of Scutellariae Radix and 1.0 mg/mL of baicalin did not affect the growth of the strains.These concentration Chinese medicines act on multidrug-resistant E.coli to eliminate drug resistance.After 2 generations of each Chinese medicine,all Chinese medicines could eliminate the resistance of HE to gentamicin.The results of HE gentamicin resistance were determined to be sensitive,and the MIC value of gentamicin was from 16.0 μg/mL decreased to 1.0 μg/mL.The sensitivity of HE to gentamicin recovery was stable within 10 generations.After 10 generations of traditional Chinese medicine,Yujin powder,Scutellariae Radix and baicalin had eliminated the drug resistance of HE to gentamicin and quinolones,and Rhubarb could not eliminate the drug resistance to quinolones.All the drug resistance results of HE quinolones were sensitive.The MIC value of ciprofloxacin was 80.0 μg/mL when the HE was not acting with Chinese medicine,and the MIC after each action of Chinese medicine was different,which all decreased to<1.0 μg/mL.The sensitivity of HE to the recovery of quinolone was stable within 10 generations.In the 10 generation of Chinese medicine acting on LE,the resistance of all antibiotics in LE did not change.The combined medication results showed that the combination of Yujin powder,Rhubarb,Scutellariae Radix,baicalin and gentamicin showed no effect;the combination of Yujin powder,Scutellariae Radix,baicalin and ciprofloxacin also showed no effect.3.The effect of Yujin Powder and its prescriptions on anti-infection of multi-drug resistant Escherichia coli.To determine the pathogenicity of HE,ICR mice were injected intraperitoneally with 109 CFU/mL bacterial solution.After inoculation,the clinical symptoms and death of mice were observed and recorded every day.At the end,the hearts were aseptic sampled for bacterial isolation and identification.The amount of E.coli inoculation was determined by measuring the LD50.The bacterial solution was injected intraperitoneally to the mice.The drug was administered 1 h after inoculating the bacterial solution.Test mice were divided into Chinese medicine treatment group,combination medication group,antibiotic treatment,HE control group,and blank control group.When combined medicine,the two drugs were administered simultaneously in different routes.To determine the therapeutic effect,the survival rate,the total number of bacterial colonies,the drug sensitivity of re-isolated bacteria,inflammatory cytokines,endotoxins,and histopathological observations of the spleen were analyzed.The results showed that chicken-derived multidrug-resistant Escherichia coli HE was highly toxicity,the bacterial amount of LD50 was 3.98×108 CFU/mL.The inoculation amount determined for animal tests was 4.49×108 CFU/mL.After the treatment of Yujin powder,Scutellariae Radix and baicalin for 72 h,the resistance of HE to gentamicin can be eliminated,and the sensitivity to ciprofloxacin cannot be restored.After treatment with Yujin Powder,Scutellariae Radix and baicalin,the survival rate of each group was significantly higher than that of the E.coli control group,which could significantly reduce the total number of colonies colonized in the liver,heart and spleen.The inflammatory factors TNF-α,IL-1β and IL-6 can be significantly reduced to normal levels,which can effectively inhibit the inflammation of the spleen;the ciprofloxacin and three groups combined medication had no therapeutic effect. |
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