The Differences Of Fitness And Metabolic Bases Of Sensitive And Resistant Escherichia Coli Under The Stress Of Doxycycline

The widespread use of veterinary antibiotics leads to residues of antibiotics in the environment and microbial antibiotics resistance.Thus,it is worthy to pay attention to the effects and mechanisms of veterinary antibiotic stress on antibiotic-sensitive and antibiotic-resistant bacteria coexisting in the environment.Based on this,this paper studied the difference of fitness of antibiotic-sensitive and antibiotic-resistant Escherichia coli K12co-cultured under the stress of doxycycline,and explored the mechanism by comparing the metabolic differences between the two strains.The results would provide a reference for scientific assessment of the environmental effects of veterinary antibiotics and their resistant bacteria.Escherichia coli K12 was used as the object in this research,the 0^th generation sensitive strain and the 38^th generation resistant strain which was sub-cultured with gradient concentration of doxycycline.The green fluorescent protein labeling plasmids p UC19-EGFP and the red fluorescent protein labeling plasmids p UC19-m Cherry were cross-introduced to construct the different fluorescent protein-labeled doxycycline sensitive and resistant Escherichia coli strains.The drug sensitivity,growth performance and biofilm formation ability determined.The co-culture experiments were carried out at the initial inoculation ratio of 1:0,2:1,1:1,2:1 and 0:1(sensitive strain:resistant strain,V:V)and under doxycycline of0 mg/L,1 mg/L and 2 mg/L,respectively,The metabolism of carbon,nitrogen and energy of the different fluorescent protein labeled sensitive and resistant Escherichia coli were determined.The main results of this paper were as follows:(1)Four fluorescent protein-labeled strains of sensitive bacteria K12-G0-EGFP(green)and K12-G0-m Cherry(red),resistant bacteria K12-G38-EGFP(green)and K12-G38-m Cherry(red)were successfully constructed.The MIC of doxycycline against the sensitive strains K12-G0-EGFP and K12-G0-m Cherry was 4 mg/L,and the MIC of doxycycline against the resistant strains K12-G38-EGFP and K12-G38-m Cherry was 128 mg/L.With the increase of the doxycycline concentration,the inhibition on the growth of sensitive strains became stronger,but there was no significant effect on the resistant strains.(2)With the increase of the doxycycline concentration,the adaptability of sensitive strains in the co-culture decreased,and the adaptability of resistant strains increased.Without the stress of doxycycline,the adaptability of sensitive strains were higher than that of drug-resistant strain and showed a competitive advantage.At the initial inoculation ratio of 2:1,1:1 and 1:2,the proportions of sensitive strains were 88.03%?88.13%,77.12%?96.80%and 82.54%?94.73%at the end of co-culture process,respectively.With the increase of the concentration of doxycycline,the adaptability of sensitive strains decreased,and the adaptability of resistant strains increased.When the concentration of doxycycline was 1 mg/L,the proportions of resistant strains in the end of co-culture process were 72.55%?85.74%,51.12%?74.39%and 62.31%?77.85%at the initial inoculation ratio of 2:1,1:1 and 1:2,respectively.When the concentration of doxycycline was 2 mg/L,the sensitive strains were completely replaced by resistant strains within 12 h.(3)Compared with sensitive strain,the overall utilization of carbon resources of drug-resistant Escherichia coli significantly decreased in the absence of doxycycline or low doxycycline stress(P<0.05).The amino acid carbon source was preferred to be used.Due to the reduction of central carbon generation and increasing of bypass metabolism function,which in turn reduces the level of energy metabolism and ultimately leads to a decrease in growth performance and adaptability of resistant strain.This in turn reduces the level of energy metabolism,which ultimately leads to a decrease in growth performance and adaptability.In summary,the stress of doxycycline directly affects the adaptability of sensitive and resistant Escherichia coli.At low concentrations of doxycycline,the central carbon metabolism and energy metabolism of resistant strain reduced,which leads to a decline in growth performance and adaptability.