Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (14): 2862-2874.doi: 10.3864/j.issn.0578-1752.2022.14.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

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

WANG XueYang1(),JIANG JunYao1,YANG Lu1,SHAO DongYan1,WU CongMing1,SHEN JianZhong1,SHEN YingBo1,2(),WANG Yang1()   

  1. 1. College of Veterinary Medicine, China Agricultural University, Beijing 100193
    2. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101
  • Received:2021-06-22 Accepted:2021-12-20 Online:2022-07-16 Published:2022-07-26
  • Contact: YingBo SHEN,Yang WANG E-mail:543114316@qq.com;wangyang@cau.edu.cn;shenyb@cau.edu.cn

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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 effects 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 the 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 by using PI dye and reactive oxygen species (ROS) detection kit, respectively. Colistin negative group and the three treatment groups (0.02, 1, and 4 μg·mL-1) were subjected to RNA sequencing as control, low, medium and high concentration groups, respectively, and the gene differential expression was analyzed by Deseq2 software. All statistical analysis were conducted by Prism v8.2.0 software. 【Result】 A formula was established to calculate the conjugative frequency in this study, and it was 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 with 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 and 4μg·mL-1 colistin could elevate the cell membrane permeability in donor and recipient bacteria, and the 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 promoted 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 might 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 could lead to the failure on colistin treatment for mcr-1-positive pathogens.

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

Table 1

Profiles on primers of real time quantitative PCR used in this study"

基因
Gene		 引物序列（5′-3′）
Primer sequence		 标准曲线
Standard curve		 片段大小
Size		 退火温度
Temperature
807 F: GAGCATCCACCTCAAAGCAG Y=-3.328X+38.293 181 bp 60℃
R: CGACCCAGATTCTTTCAACC
mcr-1 F: AAAGACGCGGTACAAGCAAC Y=-3.3521X+39.177 213 bp 60℃
R: GCTGAACATACACGGCACAG
16S F: CGGTGAATACGTTCYCGG Y= -3.545X+ 41.182 143 bp 60℃
R: GGWTACCTTGTTACGACTT

Fig. 1

Amplification curve, melt curve and standard curve of chromosome "807" gene in donor strain ZJ807"

Fig. 2

Principal component analysis amongst groups"

Fig. 3

Effects on conjugative frequency of IncI2 plasmid with colistin A: Dynamics on conjugative frequency of IncI2 plasmid under the same colistin concentration. B: Conjugative frequency of IncI2 plasmid under different colistin concentrations at the same time. C: Conjugative frequency of IncI2 plasmid under 4 μg·mL-1 colistin. D: Growth curve of donor bacteria under different colistin concentrations. Data are showed as mean and error bar represents standard deviation. P* value was detected using one-way ANOVA and corrected using "Bonferroni" method. *, **, *** and **** represent P*<0.05, P*<0.01, P*<0.001 and P*<0.0001, respectively"

Fig. 4

Effects of colistin on pilus generation and type IV secretion system (IncI2 plasmid) in donor bacteria A: Fold changes on expression of core genes related to type IV secretion system in IncI2 plasmid. B: Fold changes on expression of core genes related to adhesive-pilus generation in donor bacteria. Low, Medium and High refer to 0.02, 1 and 4 μg·mL-1 of colistin, respectively"

Fig. 5

Effect of colistin on cell membrane in donor and recipient bacteria A: Fold changes on cell membrane permeability in donor and recipient bacteria. B: Fold changes on core genes related to cell membrane in donor and recipient bacteria. Data are showed as mean and error bar represents standard deviation. P* value was detected using one-way ANOVA and corrected using "Bonferroni" method. *, ** and *** represent P*<0.05, P*<0.01 and P*<0.001, respectively. Low, Medium and High refer to 0.02, 1 and 4 μg·mL-1 of colistin, respectively"

Fig. 6

Effects of colistin on ROS generation and SOS response in the donor and recipient bacteria A: Fold changes on ROS production. B: Fold changes on expression of core genes related to ROS production and SOS response in donor and recipient bacteria. Data are showed as mean and error bar represents standard deviation. P value was detected using one-way ANOVA and corrected using "Bonferroni" method. *, ** and *** represent P<0.05, P<0.01 and P<0.001, respectively. Low, Medium and High refer to 0.02, 1 and 4 μg·mL-1 of colistin, respectively"

Fig. 7

Model depicting the mechanisms underlying the IncI2 plasmid-mediated conjugative transfer promoted by colistin"

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