Study On The Evolving Mechanism Of Escherichia Coli Resistance Under Doxycycline Stress And Gradient Stress

The veterinary antibiotics not only cause the problem of veterinary antibiotic residues in the environment,but also form a stress on the environmental microorganisms,resulting in a more serious problem of drug resistance pollution.In this paper,Doxycycline?DOX?and Escherichia coli K-12 were selected as the research subjects.The strains with different levels of drug resistance were obtained through stress tests,and the adaptability of the strains during the stress process was analyzed from the apparent structure and biological function.Then the mechanism of its resistance evolution were explored at the genetic level.This study can provide a theoretical basis for microbial evolution of veterinary antibiotic resistance prediction and resistance control.Doxycycline stress and acclimation tests were performed in CK,S1,S2,S3 and G2groups,each group has 3 replicates.The LB medium was selected as the basal medium.CK,S1,S2,and S3 groups were set up,and added 0?l/mL,1?l/mL,2?l/mL,and 3?l/mL constant concentrations of doxycycline respectively.Gradient group G2 was set,which was initially added 2?l/mL doxycycline and added 2?l/mL doxycycline after each generation.Domestication tests were conducted.Each generation acclimates 24h.10%inoculum was transferred to the next-generation medium until the G2 group reached the maximum doxycycline tolerant concentration.From the 0_th generation onwards,samples were taken every other generation for MIC/IC₅₀ index testing.The highest resistance to DOX after acclimation and the 0_th generation of the original sensitive strain W,the 20th generation of the medium resistance strain M and the 38th generation of the high resistance strain E in the G2 group were selected to detect and compare the apparent morphology,structural morphology,strain function,genome and transcriptome and other indicators.The results obtained in this paper are as follows:?1?The drug resistance of E.coli increased significantly under doxycycline stress and gradient stress?P<0.05?.Among them,the drug resistance of the gradient concentration group was the most significant.The MIC of Doxycycline in E.coli increased from 4?g/mL to 48,48,48,and 96?g/mL for S1,S2,S3,and G2 groups,respectively,and the IC₅₀increased from 2.180±0.080?g/mL to 30.600±.0.995,30.227±1.531,32.510±0.590,and64.797±0.749?g/mL.?2?Compared with the original susceptible strains,the colonies of medium resistance strain M and high resistance strain E did not change in the apparent morphology.All of them were milky white,smooth round or elliptical,and all the cells were short rods,but the colony sizes were 0.0244±0.0120 cm² and 0.0252±0.0013 cm²,respectively,which were significantly smaller than those of sensitive strains?0.1517±0.0063 cm²,P<0.05?.And there were more small neurites at the edge of the cell membrane.In the presence of doxycycline,some of the original susceptible strains W were lysed and died,and the intracellular material increased.The medium resistance strain M had normal morphology,but increased internal matter,and the high resistance strain E had no change.?3?Compared with the original sensitive strains,the stationary phase growth density(OD₆₀₀)of medium resistance strain M and highly resistance strain E was 0.863±0.022 and0.774±0.023,respectively,which was significantly lower than that of the original sensitive strain 0.923±0.018?P<0.05?;the amount of biofilm formation(OD₅₉₀)was 0.148±0.004 and0.152±0.008,respectively,which was significantly higher than that of the original sensitive strain 0.119±0.012?P<0.05?.The degradation rate of doxycycline in the 1?g/mL degradation system was 39.83±4.16%and 41.46±3.75%,respectively,which was not significantly different from that of the original sensitive strain W?39.82±4.85%?.However,the degradation rates of the three were all lower than those of the non-inoculation medium?47.17±3.12%??P>0.05?,which all inhibited the degradation of doxycycline.The utilization intensity of total carbon sources was significantly reduced?P<0.05?,but there was no significant difference in the utilization intensity of different carbon sources?P>0.05?.?4?The tetracycline resistance genes tet A,tetO,tetM,tetG,tetW,and tet X were not detected in the original sensitive strain W,medium resistance strain M,and high resistance strain E,but multidrug resistance genes acrD,acrR,marA,and marR were detected.SNP was detected in the multidrug resistance gene regulators,30S ribosomal protein S10 gene and IS5 transposase gene from medium resistance strain M and high resisatcne strain E under doxycycline stress-adaptation.And the metabolic pathways such as cell metabolism,organic nitrogen compound metabolism and peptide biosynthesis were significantly downregulated?P<0.05?.In summary,under doxycycline stress and gradient stress,the resistance of E.coli increased,the colony sizes,growth performance and intensity of using carbon decreased.The reason is related to the change of cell wall structure,the increase of biofilm formation and increase of mutation of 30s ribosomal protein S10...