Scientia Agricultura Sinica

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Colistin Promotes mcr-1-positive IncI2 Plasmid Conjugation Between Escherichia coli #br#

WANG XueYang1, JIANG JunYao1, YANG Lu1, SHAO DongYan1, WU CongMing1, SHEN JianZhong1, SHEN YingBo2WANG Yang1 #br#   

  1. 1College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; 2 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101
  • Published:2022-01-20

Abstract: Background】Colistin is a last line antibiotic for the treatment of clinical infections caused by multi-drug resistant Gram-negative bacteria, and it has also been extensively used in animal industry as a feed additive and therapeutic drug. In 2015, Chinese researchers discovered the plasmid-mediated colistin resistance gene mcr-1, indicating that this last defense line is at risk of being breached. However, the effect of bactericidal concentration and sub-inhibitory concentration of colistin on the transmission of mcr-1-positive plasmid is still unknown. 【Objective】This study used the most prevalent mcr-1-positive plasmid IncI2 as an object to explore the influence on the conjugative transfer frequency under different colistin concentrations. 【Method】The conjugation experiment under different colistin concentrations (0.02–4 μg·mL-1) was carried out by broth method. Real time quantitative PCR and the constructed formula were used to calculate the conjugative transfer frequency at different timepoints (1–24 h), and also different colistin concentrations. The cell membrane permeability and ROS production of donor and recipient bacteria under different colistin concentrations were detected using PI dye and reactive oxygen species (ROS) detection kit, respectively. Colistin negative group and the three treatment groups (0.02, 1, 4 μg·mL-1) were subjected to RNA sequencing as control, low, medium and high concentration groups respectively, and gene differential expression was analyzed by Deseq2 software. All statistical analysis were conducted by Prism v8.2.0 software. 【Result】We established a formula to calculate the conjugative frequency in our study, and found that the bactericidal concentration (4 μg·mL-1) of colistin significantly increased the conjugative transfer frequency of mcr-1-positive IncI2 plasmid by 3-10 times at different timepoints, whilst no significant difference on other concentrations. Transcriptome results showed that when compared to control group, the expression of genes related to type IV secretion system (T4SS) in IncI2 plasmid, including virB1, virB2, virB5 and traC were significantly increased in all colistin concentrations groups. In addition, the expressional level of type I fimbrium biosynthesis genes were significantly increased in all colistin groups. PI staining results showed that 2 μg/mL and 4 μg/mL colistin could elevate the cell membrane permeability in donor and recipient bacteria, and coincidently transcriptome results showed that the expressional levels of membrane-related genes including ompAX, bamDE, lolB, yiaD, csgEF were significantly up-regulated. However, ROS production and expressional level of related genes were not significantly increased after colistin treatment. 【Conclusion】This study revealed that colistin promotes the conjugative transfer frequency of mcr-1-positive IncI2 plasmid between E. coli by increasing the activity of bacterial T4SS, cell membrane permeability and pilus formation, suggesting the bactericidal concentration of colistin could increase the plasmid transmission in all survived mcr-1-positive E. coli. Therefore, the therapeutic use of colistin in animals may maintain the existence and transmission of mcr-1-positive plasmids. In addition, since colistin has been approved for clinical use in human medicine, this phenomenon may lead to the failure on colistin treatment for mcr-1-positive pathogens.


Key words: colistin,  , mcr-1,  , Escherichia coli, IncI2 plasmid, conjugation

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