Inhibitory Effect Of Four Tibetan Medicine Extracts On Clostridium Perfringens From Yak

Clostridium perfringens often causes acute diarrhea,hemorrhagic enteritis and parenchymal organ hemorrhage and necrosis in yaks,which seriously affects the healthy development of local yak breeding industry in Tibet.At present,the control of the disease mainly relies on the use of antibiotics,but the problem of bacterial resistance has become more serious.Some Tibetan medicines and their isolated monomer substances have been confirmed to have certain antibacterial effect on Clostridium perfringens,or can be developed as new antibacterial drugs to replace the use of antibiotics and prevent the generation of bacterial resistance.In this experiment,87 samples of yak diarrhea feces were collected from Nyingchi City,Xizang Province.Clostridium perfringens was isolated and identified,toxin typing,strain resistance and drug resistance genes were detected.The Tibetan medicine with the function of stopping diarrhea,clearing heat and detoxifying was extracted by water,75%ethanol and methanol.The Tibetan medicine extracts with antibacterial effect against Clostridium perfringens from yak were screened out by drug sensitivity test.The minimum inhibitory concentrations of 12 commonly used antibiotics and Tibetan medicine extracts against Clostridium perfringens were detected by microdouble dilution method and checkerboard method,and the antibacterial effects of Tibetan medicine extracts combined with antibiotics against drug-resistant strains were detected.Finally,the film-forming ability of the isolated Clostridium perfringens biofilm and the inhibition effect of antibiotic combined with Tibetan medicine extract on the biofilm were tested.Real-Time PCR was used to investigate the effects of Tibetan medicine extracts on the expression of genes related to biofilm formation and spore formation of Clostridium perfringens.The following research results are obtained.1.Twenty strains of Clostridium perfringens were isolated and identified from 87 samples of yak diarrhea feces,of which 17 were type A and 3 were type D.The results showed that the resistance rates of the isolates to aminoglycosides were gentamicin 85.0%and kanamycin 90.0%,and the resistance rates to polymyxin B and sulfamethoxazole were 80.0%and 85.0%respectively.Sensitive to cefzolin,cefthiophene,doxycycline,minomycin 100.0%.35.0%of the strains were resistant to 4,and even to 6 and 7.Anti-quinolone qnr S,aac（6’）-Ib genes,anti-aminoglycosides aac（6’）/aph（2""）genes,anti-Flfenicol flor genes,anti-β-lactam bla _TEMgenes,tet M,tet A（P）,tet B（P）genes,anti-Tetracycline genes were detected.Sulfanilamide sul2,sul3 genes,a total of10 kinds of drug resistance genes.The results indicated that the prevalence and drug resistance of Clostridium perfringens from yaks existed in Tibet,and there were different degrees of drug resistance.It suggested that we should continuously monitor the prevalence and drug resistance of Clostridium perfringens,pay attention to clinical rational drug use,and consider the cross-application of multiple drugs to avoid the emergence of superbacteria.2.Three methods were used to extract 100 g of four kinds of Tibetan medicine.The results showed that the water extraction amount of pomegranate seeds was the highest,34.20 g.It has 75%ethanol extract of white shore sand and pomegranate seed,29.10 g and 28.33 g,respectively.The extraction amount of Hanshui stone was the lowest,which was less than 3.00 g.Aristolochia showed good bacteriostatic effect among the 24 Tibetan medicines extracted from water,and showed extreme sensitivity（≥20.0 mm）when the concentration of drug-sensitive paper was 102.4 g/L.Among the 24 kinds of Tibetan medicines extracted by 75%ethanol,aristolochia and Chekhoa had the best bacteriostatic effect,followed by polylenia seeds and cardamom.All kinds of Tibetan medicine methanol extracts have better antibacterial effect,Aristolochia still showed extreme sensitivity（≥20.0 mm）when the concentration of drug sensitive paper was 51.2 g/L.The results showed that the extracts of Tibetan medicine had certain bacteriostatic effect,and could be further studied as the prevention and treatment drugs for Clostridium perfringens,in order to find the Tibetan medicine that could replace antibiotics and be economical.3.The MIC detection results of 4 Tibetan medicine extracts against Clostridium perfringens showed that.The minimum inhibitory concentration of the seeds and cardamom extracted from water was more than 25.6 g/L,and the MIC of Chekhoa for 45.0%Clostridium perfringens was less than 0.8 g/L.The inhibitory effect of 75%ethanol extract of C.polyphyllum seed was better,and MIC of all isolates was≤0.4 g/L,followed by that of Aristolochia aristolochia75%ethanol extract on 95.0%strains was≤0.4 g/L.The MIC of aristolochic methanol extract to all isolates was≤0.4 g/L,and the MIC of the methanol extract of potentilla seeds to all isolates was≤0.8 g/L.All 20 strains of Clostridium perfringens were resistant to chloramphenicol,and the resistance rates to gentamicin and polymyxin B were 95.0%.The isolates were sensitive to doxycycline,cephalothiophene,clindamycin and vancomycin.The results of combined drug sensitivity test showed that the combination of 75%ethanol extract and gentamicin had a good effect on 7 strains of Clostridium perfringens,followed by the combination of aristolochia methanol extract and gentamicin or polymyxin B,all of which had a synergistic effect on 6 strains of Clostridium perfringens.The results showed that Tibetan medicine had antibacterial and synergistic effects,but the same drug combination had different effects on different isolates of the same bacteria,which showed synergistic,cumulative,independent and antagonistic effects.Further study on the antibacterial mechanism of Tibetan medicine was needed.4.The biofilm forming ability of 20 strains of Clostridium perfringens was detected.All strains had membrane forming ability,and 55.0%of Clostridium perfringens had strong membrane forming ability.The inhibitory effect of different concentrations of single drug on biofilm showed that polymyxin B had the strongest inhibitory effect on biofilm and still had a strong inhibitory effect at 1/4 MIC.Among the Tibetan medicine extracts,aristolochic methanol extract and Chekhoa chebula 75%ethanol extract had better inhibition effect on biofilm,and had strong inhibition effect at 1/2 MIC.The combined use of antibiotics and Tibetan medicine extract showed that gentamicin combined with 75%ethanol extract of Chekhoa and polymyxin B combined with methanol extract of aristolochia had better inhibition effect on biofilm,and they still had strong inhibition effect when both of them were 1/4 MIC concentration.The four Tibetan medicine extracts had certain effects on the expressions of genes related to biofilm formation of Clostridium perfringens agr B,htr A,abr B,arg G,rib D,rib E,and spore formation genes sig K,cod Y,spo0A and min D.The results showed that the extracts of four Tibetan medicines had an inhibitory effect on the formation of Clostridium perfringens biofilm with strong film forming ability,and combined with antibiotics could enhance the inhibitory effect.The extract of Tibetan medicine affected the expression of genes related to Clostridium perfringens biofilm and spore formation in different degree.