CpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ调控作用的研究

doi:10.11843/j.issn.0366-6964.2019.06.019

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (6): 1284-1291.doi: 10.11843/j.issn.0366-6964.2019.06.019

CpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ调控作用的研究

翟亚军¹, 梁军², 魏单单¹, 孙华润¹, 潘玉善¹, 吴华¹, 刘建华¹, 胡功政^1*

1. 河南农业大学牧医工程学院, 郑州 450002;
2. 郑州市畜牧局, 郑州 450052

收稿日期:2018-12-10 出版日期:2019-06-23 发布日期:2019-06-23
通讯作者: 胡功政,主要从事细菌耐药机制与新兽药研制研究,E-mail:yaolilab@163.com
作者简介:翟亚军(1990-),女,河南郑州人,博士生,主要从事抗感染药物药理及细菌耐药性研究,E-mail:zyj90518@126.com
基金资助:
国家自然科学基金（31772799；31372481）

Regulative Mechanisms of CpxR on the Colistin Susceptibility of Salmonella enterica Serovar Typhimurium through the Colistin Resistance-related Genes pmrB and phoQ

ZHAI Yajun¹, LIANG Jun², WEI Dandan¹, SUN Huarun¹, PAN Yushan¹, WU Hua¹, LIU Jianhua¹, HU Gongzheng^1*

1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
2. Zhengzhou Animal Husbandry Bureau, Zhengzhou 450052, China

Received:2018-12-10 Online:2019-06-23 Published:2019-06-23

摘要/Abstract

摘要： 为了探索cpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ的调控作用，用overlapping PCR扩增得到cpxR、pmrB和cpxR、phoQ共表达基因cpxR-pmrB、cpxR-phoQ，用2倍微量肉汤稀释法对构建的pmrB、phoQ单基因过表达菌株JSΔacrBΔcpxR：：kan/ppmrB（JSΔΔ/pB）、JSΔacrBΔcpxR：：kan/pphoQ（JSΔΔ/pQ）和cpxR-pmrB、cpxR-phoQ共表达菌株JSΔacrBΔcpxR：：kan/pcpxR-pmrB（JSΔΔ/pRB）、JSΔacrBΔcpxR：：kan/pcpxR-phoQ（JSΔΔ/pRQ）进行黏菌素最小抑菌浓度（MICs）的测定，同时以各菌株在LB中的OD₆₀₀_nm值绘制生长曲线，以各菌株在不同浓度的黏菌素中的存活率绘制杀菌曲线。结果：成功构建的鼠伤寒沙门菌pmrB、phoQ的单基因过表达菌株JSΔΔ/pB、JSΔΔ/pQ和cpxR-pmrB、cpxR-phoQ共表达菌株JSΔΔ/pRB、JSΔΔ/pRQ的MIC结果显示，JSΔacrBΔcpxR：：kan/pcpxR（JSΔΔ/pR）的MIC值较JS下降93.75%，与本实验室前期研究结果一致。JSΔΔ/pB、JSΔΔ/pQ的MIC值较JS均上升3倍，而JSΔΔ/pRB、JSΔΔ/pRQ的MIC值较JS分别降低50%和75%。生长曲线结果显示JSΔΔ/pR的生长活性最低，JSΔΔ/pB、JSΔΔ/pQ、JSΔΔ/pRB和JSΔΔ/pRQ的生长活性均低于JSΔacrBΔcpxR：：kan/pHisA（JSΔΔ/pHisA）。黏菌素的杀菌曲线结果显示JSΔΔ/pB和JSΔΔ/pQ在不同浓度的黏菌素中的存活率显著高于黏菌素较敏感的JSΔΔ/pRB和JSΔΔ/pRQ。上述结果表明：CpxR能够通过PmrB和PhoQ调控鼠伤寒沙门菌对黏菌素的敏感性。

Abstract: To investigate the mechanisms of CpxR regulate the colistin susceptibility of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). The cpxR-pmrB or cpxR-phoQ co-expressed genes were acquired by overlapping PCR. The pmrB and phoQ single overexpressed strains and cpxR-pmrB or cpxR-phoQ co-expressed strains were constructed in this paper. The minimal inhibitory concentrations (MICs) of colistin for the above strains were measured by the 2-fold serial broth microdilution method. Growth curves were generated by OD_{600 nm} of strains in LB medium and colistin time-kill assay were carried out with survival rate of strains after incubated with different concentrations of colistin. The results showed that the MIC of cpxR overexpressed strain JSΔΔ/pR was dramatically decreased by 93.75% compared with JS which was consistent with the previous study. The MICs for pmrB and phoQ single overexpressed strains JSΔΔ/pB and JSΔΔ/pQ were increased by 300% compared with JS. However, the MICs for cpxR-pmrB or cpxR-phoQ co-expressed strains JSΔΔ/pRB and JSΔΔ/pRQ were decreased by 50% and 75%, respectively. Furthermore, the growth curves showed that unlike the low growth rate of JSΔΔ/pR, the growth rate of JSΔΔ/pB, JSΔΔ/pQ, JSΔΔ/pRB and JSΔΔ/pRQ were slightly lower than JSΔΔ/pHisA. Colistin time-kill assay showed that JSΔΔ/pB and JSΔΔ/pQ was more susceptible than JSΔΔ/pRB and JSΔΔ/pRQ when incubated with increasing concentrations of colistin. In conclusion, the results indicated that CpxR was able to regulate the colistin susceptibility of Salmonella Typhimurium through colistin resistance-related genes pmrB and phoQ.

中图分类号:

翟亚军, 梁军, 魏单单, 孙华润, 潘玉善, 吴华, 刘建华, 胡功政. CpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ调控作用的研究[J]. 畜牧兽医学报, 2019, 50(6): 1284-1291.

ZHAI Yajun, LIANG Jun, WEI Dandan, SUN Huarun, PAN Yushan, WU Hua, LIU Jianhua, HU Gongzheng. Regulative Mechanisms of CpxR on the Colistin Susceptibility of Salmonella enterica Serovar Typhimurium through the Colistin Resistance-related Genes pmrB and phoQ[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1284-1291.

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参考文献

[1] ZSCHIEDRICH C P, KEIDEL V, SZURMANT H. Molecular mechanisms of two-component signal transduction[J].J Mol Biol, 2016, 428(19):3752-3775.
[2] KAWADA-MATSUO M, KOMATSUZAWA H. Role of Streptococcus mutans two-component systems in antimicrobial peptide resistance in the oral cavity[J]. Jpn Dent Sci Rev, 2017, 53(3):86-94.
[3] HU W S, CHEN H W, ZHANG R Y, et al. The expression levels of outer membrane proteins STM1530 and OmpD, which are influenced by the CpxAR and BaeSR two-component systems, play important roles in the ceftriaxone resistance of Salmonella enterica serovar Typhimurium[J]. Antimicrob Agents Chemother, 2011, 55(8):3829-3837.
[4] HUANG H, SUN Y W, YUAN L, et al. Regulation of the two-component regulator CpxR on aminoglycosides and β-lactams resistance in Salmonella enterica serovar Typhimurium[J]. Front Microbiol, 2016, 7:604.
[5] WANG-KAN X, BLAIR J M A, CHIRULLO B, et al. Lack of AcrB efflux function confers loss of virulence on Salmonella enterica serovar Typhimurium[J]. mBio, 2017, 8(4):e00968-17.
[6] SCHUSTER S, VAVRA M, SCHWEIGGER T M, et al. Contribution of AcrAB-TolC to multidrug resistance in an Escherichia coli sequence type 131 isolate[J]. Int J Antimicrob Agents, 2017, 50(3):477-481.
[7] BAUCHERON S, TYLER S, BOYD D, et al. AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium DT104[J]. Antimicrob Agents Chemother, 2004, 48(10):3729-3735.
[8] EAVES D J, RICCI V, PIDDOCK L J V. Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium:role in multiple antibiotic resistance[J]. Antimicrob Agents Chemother, 2004, 48(4):1145-1150.
[9] GIRAUD E, CLOECKAERT A, KERBOEUF D, et al. Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar Typhimurium[J]. Antimicrob Agents Chemother, 2000, 44(5):1223-1228.
[10] HORIYAMA T, NISHINO K. AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli[J]. PLoS One, 2014, 9(9):e108642.
[11] NISHINO K, YAMADA J, HIRAKAWA H, et al. Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to β-lactams[J]. Antimicrob Agents Chemother, 2003, 47(9):3030-3033.
[12] LIU Y Y, WANG Y, WALSH T R, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China:a microbiological and molecular biological study[J]. Lancet Infect Dis, 2016, 16(2):161-168.
[13] ANJUM M F, DUGGETT N A, ABUOUN M, et al. Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in great Britain[J]. J Antimicrob Chemother, 2016, 71(8):2306-2313.
[14] QUESADA A, UGARTE-RUIZ M, IGLESIAS M R, et al. Detection of plasmid mediated colistin resistance (MCR-1) in Escherichia coli and Salmonella enterica isolated from poultry and swine in Spain[J]. Res Vet Sci, 2016, 105:134-135.
[15] LI X P, FANG L X, SONG J Q, et al. Clonal spread of mcr-1 in PMQR-carrying ST34 Salmonella isolates from animals in China[J]. Sci Rep, 2016, 6:38511.
[16] CATRY B, CAVALERI M, BAPTISTE K, et al. Use of colistin-containing products within the European Union and European Economic Area (EU/EEA):development of resistance in animals and possible impact on human and animal health[J]. Int J Antimicrob Agents, 2015, 46(3):297-306.
[17] LEE J Y, KO K S. Mutations and expression of PmrAB and PhoPQ related with colistin resistance in Pseudomonas aeruginosa clinical isolates[J]. Diagn Microbiol Infect Dis, 2014, 78(3):271-276.
[18] ZHAI Y J, HUANG H, LIU J H, et al. CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin[J]. J Antimicrob Chemother, 2018, 73(11):3016-3024.
[19] 黄慧,陈丽鹏,孙亚伟,等. 亚MIC庆大霉素下CpxR对鼠伤寒沙门菌生长特性和acrD基因转录的影响[J]. 中国兽医学报, 2016, 36(6):995-1000.
HUANG H, CHEN L P, SUN Y W, et al. Influence of CpxR on growth characteristics and AcrD gene transcription of Salmonella enterica serovar typhimurium at the subinhibitory concentrations of gentamicin[J]. Chinese Journal of Veterinary Science, 2016, 36(6):995-1000. (in Chinese)
[20] 黄慧,刘保光,孙亚伟,等. 鼠伤寒沙门菌cpxR和acrB双基因缺失菌株的构建及其对抗菌药物敏感性分析[J]. 畜牧兽医学报, 2016, 47(3):595-602.
HUANG H, LIU B G, SUN Y W, et al. Construction of cpxR and acrB double gene deletion strain of Salmonella enterica serovar Typhimurium and analysis of its susceptibility to antibacterial agents[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(3):595-602. (in Chinese)
[21] Clinical and Laboratory Standards Institute. M100-S26 Performance standards for antimicrobial susceptibility testing;Twenty-sixth informational supplement[S]. Wayne, PA:CLSI, 2016.
[22] HIRAKAWA H, NISHINO K, HIRATA T, et al. Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli[J]. J Bacteriol, 2003, 185(6):1851-1856.
[23] HIRAKAWA H, NISHINO K, YAMADA J, et al. β-Lactam resistance modulated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli[J]. J Antimicrob Chemother, 2003, 52(4):576-582.
[24] MAHONEY T F, SILHAVY T J. The Cpx stress response confers resistance to some, but not all, bactericidal antibiotics[J]. J Bacteriol, 2013, 195(9):1869-1874.
[25] BIALEK-DAVENET S, LAVIGNE J P, GUYOT K, et al. Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae[J]. J Antimicrob Chemother, 2015, 70(1):81-88.
[26] RAJAMOHAN G, SRINIVASAN V B, GEBREYES W A. Molecular and functional characterization of a novel efflux pump, AmvA, mediating antimicrobial and disinfectant resistance in Acinetobacter baumannii[J]. J Antimicrob Chemother, 2010, 65(9):1919-1925.
[27] HJORT K, NICOLOFF H, ANDERSSON D I. Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica[J]. Mol Microbiol, 2016, 102(2):274-289.
[28] BARON S, HADJADJ L, ROLAIN J M, et al. Molecular mechanisms of polymyxin resistance:knowns and unknowns[J]. Int J Antimicrob Agents, 2016, 48(6):583-591.

CpxR对鼠伤寒沙门菌的黏菌素耐药相关基因pmrB和phoQ调控作用的研究

Regulative Mechanisms of CpxR on the Colistin Susceptibility of Salmonella enterica Serovar Typhimurium through the Colistin Resistance-related Genes pmrB and phoQ

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