| As a common intestinal microflora of livestock and poultry,Escherichia coli can form bacterial biofilms under the extensive and persistent pressures of antibiotics to resist the changes of the external living environment.With the emergence of super-resistant bacteria and the increase of multi-drug resistant bacteria,many researchers at home and abroad have turned their attention to the research and development of new antibacterial drugs to deal with the increasingly serious problem of bacterial resistance,and the plant drugs and probiotics are of the more active new areas.Therefore,four Tibetan veterinary drugs(Spenceria ramalana Trimen,Thalictrum delavayi Franch.,Gentiana sino-ornata Balf.f.and Lonicera rupicola Hook.f.& Thomson)and two strains of Enterococcus faecium(S16 and S17)were selected for combined susceptibility testing to explore the antibacterial activity to Escherichia coliisolated from Tibetan pigs and Tibetan chickens in this study,and the results could lay the foundation for the developmentand utilization of new antibacterial drugs.Methods:(1)203 samples of feces,urine,eggs,surrounding soil and water samples of Tibetan pigs and Tibetan chickens were collected from for Escherichia coli isolation and identification using the conventional methods.(2)Semi-quantitative method of modified crystal violet staining method and the broth microdilution method were used respectively to analyze the biofilm formation ability of and drug susceptibility tests of 12 commonly used antibiotics for Escherichia coli isolates.(3)The selected 10 strains with strong biofilm formation ability and multi-drugre sistant Escherichia coli were chosen for study,and the combined chemosensitivity test method was used to evaluate the combined bactericidal activity of the differen textracted ingredients of the four Tibetan veterinary medicines(Spenceria ramalana Trimen,Thalictrum delavayi Franch.,Gentiana sino-ornata Balf.f.,Lonicera rupicola Hook.f.& Thomson),probiotics of two Enterococcus faecium isolations and high resistant antibiotics.(4)To explore the biofilm scavenging effect of the different extracted ingredients of the four Tibetan veterinary medicines(Spenceria ramalana Trimen,Thalictrum delavayi Franch.,Gentiana sino-ornata Balf.f.,Lonicera rupicola Hook.f.&Thomso n),probiotics of two Enterococcus faecium isolations and high-resistant antibiotics,E6 strain was chosen for the research object and the rapid silver staining method wasused for relative amount of extracellular polysaccharides to verify the biofilm formation ability.Results:(1)A total of 152 Escherichia coli were isolated from 203 samples,including 61 strains from Tibetan pigs,82 strains from Tibetan chickens,a strain from environmental soil,two strains from pig urine and two strains of from eggs,the total isolation rate of the strains was 74.88%.(2)Most of the 152 Escherichia coli isolates had the biofilm-forming ability and the ratio value of OD570 was between one and three;The analysis results using WHONET 5.6 software showed that the drug resistant rates of the 152 isolates to Chloramphenicol,Sulfamethazine,Oxytetracycline,Ampicillin and Sulfamethoxazole were all higher than 90%,of which the resistance rate of ampicillin was as high as 100%;The drug resistant rates of the 152 isolates to difloxacin,ciprofloxacin,amikacin,cefotaxime,tobramycin and ceftriaxone were all lower than 50%,of which the sensitiverate of amikacin was the highest and the drug resistance rate was only 19.90%;the dominant drug resistant pattern of the 152 isolates was chloramphenicol/difloxacin/ sulfamethazine/oxytetracycline/doxycycline/ampicillin/sulfamethoxazole.(3)The results of combined chemosensitivity test of the extracted ingredients of the four Tibetan veterinary medicines(Spenceria ramalana Trimen,Thalictrum delavayi Franch.,Gentiana sino-ornata Balf.f.and Lonicera rupicola Hook.f.& Thomson)with strong antibacterial effect against Escherichia coli,probiotic ethyl acetate extract and four antibiotics(chloramphenicol,oxytetracycline,ampicillin and sulfamethoxazole)with higher drug resistant rates showed thatthe MIC range of the methanol extract from Spenceria ramalana Trimen to Escherichia coli ranged from 3.93 to 15.63 mg/m L,revealing additive effect with chloramphenicol and oxytetracycline and indifferent effect with ampicillin and sulfamethoxazole;The MIC range of the ethyl acetate extract from Thalictrum delavayi Franch.to Escherichia coli ranged from 3.93 to 31.52 mg/m L,revealing synergistic effect with chloramphenicol and additive effect with oxytetracycline,ampicillin and sulfamethoxazole;The MIC range of the methanol extract from Gentiana sino-ornata Balf.f.to Escherichia coli ranged from 7.81 to 15.63 mg/m L,revealing indifferent effect with chloramphenicol,oxytetracycline and ampicillin and antagonistic effect with sulfamethoxazole;The MIC range of the ethyl acetate extract from Lonicera rupicola Hook.f.& Thomson to Escherichia coli ranged from 3.93 to 31.52 mg/m L,revealing additive effect with chloramphenicol,synergistic effect with oxytetracycline and ampicillin and indifferent effect with sulfamethoxazole.The MIC range of the ethyl acetate extract of Enterococcus faecium S16 to Escherichia coli ranged from 0.45 to 3.63 mg/m L,revealing indifferent effect with chloramphenicol and sulfamethoxazole,and synergistic effect with oxytetracycline and ampicillin;The MIC range of the ethyl acetate extract of Enterococcus faecium from S17 to Escherichia coli ranged from 0.42 to 13.38 mg/m L,revealing indifferent effect with chloramphenicol,synergistic effect with oxytetracycline and additive effect with sulfamethoxazole and ampicillin.The methanol extract from Gentiana sino-ornata Balf.f.and the ethyl acetate extractof the two strains of Enterococcus faecium showed additive effect;The ethyl acetate extract from Lonicera rupicola Hook.f.& Thomson and the ethyl acetate of the Enterococcus faecium S17 showed synergistic effect and the ethyl acetate of the Enterococcus faecium S16 showed additiveeffect;The ethyl acetate extract from Thalictrum delavayi Franch.and the ethyl acetate extract of the two strains of Enterococcusfaecium showed additive effect;The methanol extract from Spenceriaramalana Trimen and the ethyl acetate extract of the Enterococcus faecium S16 showed indifferent effect and the ethyl acetate extract of the Enterococcus faecium S17 showed additive effect.(4)The result of rapid silver staining of E6 strain showed more amount of the secretion of extracellular polysaccharides and had the strong biofilm-forming ability;The biofilm inhibition rate on E6 strain of the different ingredients extracted from the four Tibetan veterinary drugs(Spenceriaramalana Trimen,Thalictrum delavayi Franch.,Gentiana sino-ornata Balf.f.and Lonicera rupicola Hook.f.& Thomson)showed was positively correlated with the drug concentration.Under the concentration of MIC,the biofilm inhibitory rate of combined application of the ethyl acetate extract from Lonicera rupicola Hook.f.& Thomson with oxytetracycline and ampicillin were 48.92% and 48.58%,respectively;the biofilm inhibitory rate of combined application of the ethyl acetate extract from Thalictrum delavayi Franch.with oxytetracycline and ampicillin were 42.58% and 47.84%,respectively.The result is higher than the inhibition rate of single drug,which proves the synergy between the two drugs.Under the concentration of MIC,the biofilm inhibitory rate of combined application of the ethyl acetate extract from Enterococcus faecium S16 with oxytetracycline and ampicillin were 41.53% and 33.88%,respectively;the biofilm inhibitory rate of combined application of the ethyl acetate extract from Enterococcus faecium S17 with oxytetracycline and ampicillin were 37.37% and 34.26%,respectively.The result was higher than the inhibition rate of single drug and also proved the synergy between the two drugs.Under the concentration of MIC,the biofilm inhibitory rate of combined application of the ethyl acetate extract from Lonicera rupicola Hook.f.& Thomson with the ethyl acetate extract from Enterococcus faecium S16 and S17 were 37.33% and 34.51%,respectively;and the biofilm inhibitory rate of combined application of the ethyl acetate extract from Thalictrum delavayi Franch.with the ethyl acetate extract from Enterococcus faecium S16 and S17 were 33.50% and 35.61%,respectively.The inhibition rate is higher than that of the single drug,which also proves the synergistic effect of the probiotics and antibiotics. |
PDF Full Text Request