| Escherichia coli(E.coli)is one of the Gram-negative bacteria with the highest isolation rate in clinical practice.As a conditioned pathogen,it can cause intestinal or parenteral infection and even result in death.In recent years,due to the irregular use or even abuse of antibiotics in animal husbandry production and veterinary clinical practice,bacterial drug resistance has increased,and the problem of bacterial antimicrobial resistance has become increasingly serious,which hinders the development of animal husbandry and veterinary public health security.The biofilm formation is an important reason for the emergence and increase of antimicrobial resistance in E.coil.Therefore,it is urgent to find new anti-biofilm drugs to control the resistant bacterial infections.There are abundant sources of natural drugs,from which screening antibiofilm drugs is an important way to control resistant bacteria and also is conducive to the development of new veterinary drugs.In this study,ESBLs-producing E.coli from poultry was used as the objects,and the in vitro antibacterial activity of six Tibetan herbs to ESBLs-producing E.coli were determined.Moreover,the intervention effects of Lagotis brachystachya and Swertia mussotii on biofilm of ESBLs-producing E.coli were further studied.The main research contents and results are summarized as follows:1.Isolation and identification of avian E.coli,drug resistance analysis,molecular typing,and biofilm detectionE.coli was isolated and identified from broiler feces samples from 3 broiler farms with different scales in Weinan,Shaanxi using Mac Conkey,Eosin methylene blue identification medium,Gram staining microscopic examination and 16 S r DNA identification.A total of 131 E.coli isolates were isolated.The minimal inhibitory concentration(MIC)of 10 antibacterial drugs against the isolates was determined by microdilution method recommended by CLSI,and the virulence genes were detected by PCR amplification.The results of antimicrobial sensitivity test showed that 131 E.coli isolates showed multi-drug resistance,which was resistant to doxycycline,ceftiofur,ceftriaxone,enrofloxacin,florfenicol and ampicillin,etc.Moreover,the resistance rate of ceftiofur,ceftriaxone,florfenicol,cefotaxime and ampicillin reached 100%.It was sensitive to meropenem,amikacin and polymyxin E.The results of virulence factor detection showed that the detection rate of pap C was the highest prevalence,which was 85.5%.ast A ranked second,accounting for 84.7%.More than 50% of the isolates carried more than one gene of iuc D,iro N and iss.iut A,tsh,irp-2 genes were detected in low rates,ranging from 23.7% to 43.5%.The positive rate of cva was less than 10%,and vat was not detected.Double-disc diffusion method was used to screening the ESBLs-producing bacteria from antimicrobial resistant isolates and multi-locus sequence typing(MLST)was used for molecular typing of ESBLs-producing isolates.Finally,20 ESBLs-producing isolates were screened,which belong to 7 ST types(ST1266,ST162,ST10,ST38,ST1485,ST2847,ST469),all of them were issued ST types.ST162 and ST10 are the main ST types in our study.Crystal violet staining was used to analyze the biofilm formation ability of ESBLs-producing strains,and the strains with strong biofilm production ability were screened out.The results showed that all 20 ESBLs-producing isolates were biofilm positive ones.Among them,8isolates(40%)had medium biofilm production ability and 12 strains(60%)had weak biofilm production ability.2.In vitro antibacterial activities of six Tibetan herbs against E.coliThe disc diffusion test(K-B method)and tube double dilution method were used to determine the antibacterial activity of 6 kinds of Tibetan herbs(Swertia mussotii Franch.,Lagotis brachystachya Maxim.,Uncaria rhynchophylla(Miq.)Jacks(Rubiaceae),Aconitum pendulum Busch.,Gentiana urnula H.Smith.,Dracocephalum tanguticum Maxim.)against the tested strains,and the Tibetan herbs with good antibacterial activity were screened out for subsequent studies.The results of the K-B method showed that the aqueous extracts of all herbs had antibacterial effects on the test isolates except Aconitum pendulum Busch.The ethanol extracts of all herbs had different degrees of antibacterial effects on the tested isolates except Gentiana urnula H.Smith.and Aconitum pendulum Busch..The results of tube double dilution method showed that 6 Tibetan herbs had antibacterial effects on E.coli,but their antibacterial effects were different.Among them,Lagotis brachystachya and Swertia mussotii had the best antibacterial effects on the tested strains(MIC of Lagotis brachystachya was 15.7-125.0 mg/m L,MIC value of Swertia mussotii was 15.7-125.0 mg/m L).In general,the aqueous extracts of 6 kinds of Tibetan herbs had better antibacterial effect on tested strains.3.Interventional effects of Swertia mussotii and Lagotis brachystachya on biofilm of ESBLsproducing E.coliThe effects of aqueous extracts of Lagotis brachystachya and Swertia mussotii on the biofilm of the tested isolates were determined by growth curve measurement,crystal violet staining,scanning electron microscope(SEM)and laser confocal microscope(CLSM).The results of growth curve showed that the aqueous extracts of Lagotis brachystachya and Swertia mussotii could inhibit the growth of the tested strains in a concentration-dependent manner.Crystal violet staining showed that the formation of biofilm was inhibited and the mature biofilm could be removed after adding different concentrations of aqueous extracts of Lagotis brachystachya and Swertia mussotii,and both aqueous extracts had good anti-biofilm activities in a concentration-dependent manner.At the concentrations of MIC,1/2 MIC and1/4 MIC,the Lagotis brachystachya aqueous extracts had the ability to inhibit the formation of biofilm,the inhibition rates were 35.7%-79.9%,20.1%-71.3% and 0%-51.4%,respectively.At the concentrations of MIC,1/2 MIC and 1/4 MIC,the inhibition rates of Swertia mussotii aqueous extracts on biofilm were 28.3%-76.0%,16.0%-71.0% and 0%-58.3%,respectively.At the concentrations of MIC,1/2 MIC and 1/4 MIC,the biofilm eradication rates of the aqueous extracts of Swertia mussotii on biofilm ranged from 15.3%-63.0%,0%-61.7%,0%-56.0%,respectively,and those of Lagotis brachystachya were 27.6%-62.3%,15.6%-57.1%and 0%-41.2%,respectively.The highest inhibition and eradication rates of Swertia mussotii aqueous extracts on biofilm could reach 76.0% and 63.0%,respectively.The highest inhibition and eradication rates of Lagotis brachystachya aqueous extracts on biofilm were 79.9% and62.3%,respectively.The results of SEM showed that after adding Lagotis brachystachya and Swertia mussotii aqueous extracts at the concentrations of MIC,1/2 MIC and 1/4 MIC,the structure of biofilm was dispersed,the aggregation of E.coli was poor,and the structure of biofilm was obviously destroyed,and the anti-biofilm effects became more obvious with the increase of concentration.The results of CLSM further confirmed the anti-biofilm effects of Lagotis brachystachya and Swertia mussotii.After the two Tibetan herbs were treated,the green fluorescence area decreased significantly and the red fluorescence area increased compared with untreated.The results showed that the biofilm of E.coli could be inhibited and removed by Lagotis brachystachya and Swertia mussotii in a concentration-dependent manner. |
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