Resistance Evolution And Mechanism Of Pseudomonas Aeruginosa Under Sublethal Ampicillin Exposure

Bacterial antibiotic resistance has become a global problem.Although bacterial resistance to high concentrations of antibiotics has been widely studied,the evolution and driving mechanism of bacterial resistance to low concentrations of antibiotics are still relatively rare.It is worth noting that low concentrations of antibiotics exist in most environments.Therefore,Pseudomonas aeruginosa was used here as a model strain to study the antibiotic resistance evolution and driving mechanism of Pseudomonas aeruginosa under sublethal ampicillin treatment by continuous passage experiment,combined with genomic sequencing,quantitative PCR,gene knockout and transcriptome analysis.This study not only enriches the understanding of bacterial resistance evolution,but also provides a reference for solving the public health problem of antibiotic resistance.The main results are as follows:(1)Pseudomonas aeruginosa could rapidly develop antibiotic resistance evolution under sublethal concentration of ampicillin,and the rate of resistance evolution was significantly different under different concentrations of antibiotics.After 6 days of low concentration treatment(15-30?g/m L),the evolution of bacterial resistance tended to be stable,and the continuous passage did not lead to the significant increase of the minimum inhibitory concentration(MIC).On the 21st day,the MIC of the low concentration group was3-5 times of that of the wild strain,and the MIC of the high concentration group(150-300?g/m L)was 15-18 times of that of the wild strain.(2)There were differences in antibiotic resistance evolution characteristics of Pseudomonas aeruginosa under low and high concentrations of antibiotics.The results of genome resequencing showed that mpl mutation occurred frequently in low concentration group(15-30?g/m L),amp D mutation mainly occurred in high concentration treatment group(150-300?g/m L);further gene knockout results showed that MIC and?-lactamase activities of?mpl gene knockout strains were 8000±1000?g/m L and 2.3±0.5 m U/mg respectively.The MIC and?-lactamase activities of?amp D knockout strains were20000±5000?g/mL and 4.6±0.6 mU/mg respectively,which were similar to those of high concentration treatment group.The results showed that the evolution of antibiotic resistance in low concentration group and high concentration group might be related to mpl and amp D mutation and enrichment of related mutants,respectively.(3)The resistance evolution of Pseudomonas aeruginosa treated with sublethal ampicillin may be related to the quorum sensing changes caused by mpl and amp D mutations.Compared with wild strian,the transcriptional level analysis of?mpl and?amp D showed that las A,las R,rhl A,rhl B,rhl I and rhl R were significantly up-regulated.The growth OD₆₀₀of?mpl in 2000?g/m L ampicillin decreased from 0.3 to 0.15 and?amp D decreased from 0.4 to0.2 compared with the control group.The addition of quencher significantly reduced the resistance to antibiotics,suggesting that quorum sensing was related to the resistance of Pseudomonas aeruginosa to sublethal ampicillin treatment.(4)The up-regulation of Rhl IR is one of the important reasons for the resistance of Pseudomonas aeruginosa to?mpl and?amp D.However,the MIC of?mpl?rhl I and?mpl?rhl R decreased from 8000?g/m L to 2000?g/m L and 1000?g/m L to 1000?g/m L,while the MIC of?amp D?rhl I and?amp D?rhl R decreased from 20000?g/m L to 5000?g/m L and3000?g/m L respectively.The MIC of?mpl?rhl I and?amp D?rhl I increased to 5000?g/m L and 10000?g/m L respectively after adding C4-HSL signal molecule into Rhl IR mutants.Compared with Las IR system,Rhl IR system contributes more to antibiotic resistance of Pseudomonas aeruginosa.