| Sheep colibacillosis is an acute,septicemic infectious disease caused by pathogenic Escherichia coli that can cause significant economic losses to the sheep farming industry.Antibiotics are the main means of treating the disease,but the prevention and control of the disease are seriously affected by factors such as the irrational use of antibiotics and bacterial biofilm formation.Sheep breeding is one of the essential industries in Ta’er District,Xinjiang.There still needs to be more relevant data on the situation of sheep E.coli carriers in Ta’er District Xinjiang.In this study,415 lamb feces with diarrhea were collected from eight large-scale sheep farms in Ta’er Reclamation Area,Xinjiang,from January to March 2022,and E.coli was isolated and cultured,and the biofilm-forming ability,drug resistance gene,virulence gene,and infection ability to mice of the isolated strains were tested,and the effects of D-amino acid on biofilm formation of E.coli and the combined use of D-amino acid and antibiotics on The study on the inhibitory effect of D-amino acid and antibiotics on E.coli.1.Molecular epidemiological investigation of pathogenic Escherichia coli of sheep originIn this study,73 E.coli strains were isolated and identified from 415 samples,and the results of phylogenetic groups and E.coli LPS core types showed that group D(30.13%)and B1(42.46%)were the dominant populations among the 73 E.coli strains,and R1 core type(57.53%)was the dominant E.coli LPS core type.The virulence genes of 73 E.coli strains were detected by PCR,and 13 virulence factors were seen,among which the detection rate(100%)of type I gross fim H was the highest.The detection rates of 7 virulence factors,including mat,she,sod A,yijp,ibe B,csg A,and omp A,were over 80%.To evaluate the pathogenicity of the isolated strains,mouse pathogenicity tests were performed on the strains carrying multiple virulence factors.The results showed that 7 of the 9 E.coli strains carrying numerous virulence factors were highly pathogenic.The results of drug sensitivity tests on 73 strains of E.coli of sheep origin showed that except for polymyxin,imipenem,meropenem,and tigecycline,which showed sensitivity,the other 12 drugs showed resistance rates of 4%-67%.Among them,35 strains were multi-drug resistant(47.94%).The drug resistance gene testing results showed that the detection rate of quinolone resistance genes exceeded 90%.bla-TEM detected 75% of β-lactam resistance genes,OXA-1,CXT-MU,tet A,tet M,Aph,aac’(6)-Ib,ant’(4),oqx A,oqx B,and mar A,sul1,sul2 were detected.The biofilm formation of 73 E.coli strains was detected by crystalline violet staining,53 strains could form a biofilm(72.6%),23 songs with solid biofilm formation ability(31.5%),8 themes with a moderate ability(10.95%),22 strains with a weaker ability(30.1%)and 20 strains without biofilm formation(27.39%).2.Inhibitory effect of D-amino acids on E.coli biofilmThis study investigated the effects of 19 D-amino acids on the biofilm formation of EC344-1 and EC125-1 strains by the 96-well crystal violet microplate method.The results showed that 15 mmol/m L of D-histidine,D-methionine,D-asparagine-aqueous and D-tyrosine,20 mmol/m L of D-cysteine,D-arginine,D-threonine,D-alanine and D-valine,25 mmol/m L of D-phenylalanine,30 mmol/m L of D-tryptophan and D-lysine and D-leucine,the D-serine at 40 mmol/m L all completely inhibited the biofilm formation of E.coli EC344-1.However,15 mmol/m L of D-tyrosine,10 mmol/m L of D-arginine and D-tryptophan,20mmol/m L of D-histidine and D-leucine,30 mmol/m L of D-serine,and 80 mmol/m L of D-aspartic acid completely inhibited the biofilm formation of E.coli EC125-1.The results showed that D amino acid had good biofilm inhibition,but there were strain differences.3.The combination of antibiotics and D-amino acids reduces the drug tolerance of E.coliCombining D-methionine,D-leucine,D-tyrosine,and D-tryptophan,with florfenicol,tetracycline,gentamicin,and amoxicillin,respectively,was applied to E.coli EC344-1 using the checkerboard method.The results showed that the minimum inhibitory concentration was reduced from 8 μg/m L to 4 μg/m L for the combination of amoxicillin with D-methionine and D-leucine and from 8 μg/m L to 2 μg/m L for the variety with D-tryptophan.The minimum inhibitory concentration decreased from 8μg/m L to 2μg/m L in combination with D-tyrosine and had no effect in combination with D-tyrosine.The minimum inhibitory concentration of gentamicin in a mixture with four amino acids decreased from 16 μg/m L to 2 μg/m L.The minimum inhibitory concentration of tetracycline in combination with D-methionine,D-tyrosine,and D-tryptophan decreased from 8 μg/m L to 4 μg/m L,and in combination with D-leucine,the minimum inhibitory concentration decreased from 8 μg/m L to 2 μg/ml.The minimum inhibitory concentration of florfenicol in a mixture with four amino acids decreased from 8 μg/m L to 4 μg/ml.This study showed that the antibiotics had a synergistic effect when combined with D-amino acids,and the sensitivity of E.coli to the antibiotics was enhanced.This study showed that 73 E.coli strains were dominated by groups D and B1,and the R1 core dominated the LPS core type.The isolates carried multiple virulence genes with strong pathogenicity.The isolated strains had a high rate of multi-drug resistance and carried multiple resistance genes,and 72.6% of the strains could produce biofilms.D-amino acid can effectively inhibit biofilm growth and effectively increase the minimum inhibitory concentration of isolated strains with the combination of tetracycline,florfenicol,amoxicillin,and gentamicin,which can provide a reference for the prevention and control of sheep-derived E.coli in Ta’er Reclamation,Xinjiang. |
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