In vitro synthesis of AI-2 and its effect on tetracycline resistance of Vibrio parahaemolyticus

LIN Cai-Yun1,2 JIANG Yan-Hua2 YAO Lin2 LI Feng-Ling2 QU Meng2 WANG Lian-Zhu2 XU Jia-Chao1

(1.College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong, China 266003)
(2.Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong, China 266071)

【Abstract】[Background] Antimicrobial resistance of bacteria has become more and more serious due to the abuse of antimicrobials. As a major foodborne pathogen, Vibrio parahaemolytics also exhibits a certain level of antimicrobial resitance. Quorum sensing system can regulate the antimicrobial resistance of bacteria, which provides a new pathway to study the mechanism and control technique of antimicrobial resistance of V. parahaemolytics. [Objective] To study the effect of signaling molecule autoinducer-2 (AI-2) on tetracycline resistance of V. parahaemolyticus strains from seafood. [Methods] AI-2 was synthesized in vitro by a reaction using the critical enzymes, S-ribosylhomocysteinase (LuxS) and S-adenosylhomocysteine nucleosidase (Pfs), which were prepared by prokaryotic expression. The effect of AI-2 on tetracycline resistance of V. parahaemolyticus was determined by colony counting method. The effect of AI-2 at different concentrations on the transcriptional levels of tetracycline resistance genes in V. parahaemolyticus was assayed by reverse transcription and real-time quantitative PCR. [Results] LuxS and Pfs enzymes were obtained by prokaryotic expression. The bioactive AI-2 could be synthesized in vitro by adding LuxS and Pfs to the substrate S-adenosylhomocysteine (SAH), with the fluorescence intensity of about 6 times as much as that of positive control. When treated with tetracycline at subinhibitory concentration, AI-2 could significantly promote the growth of V. parahaemolyticus strains, and AI-2 at the concentrations of 6, 15 and 30 μmol/L could increase the transcriptional levels of tetracycline resistance genes in V. parahaemolyticus strains to a certain extent. [Conclusion] AI-2 could enhance the tetracycline resistance of V. parahaemolyticus, which provided a reference for further study on the antimicrobial resistance mechanism of V. parahaemolyticus and developing control techniques targeting AI-2 on the antimicrobial resistance of V. parahaemolyticus.

【Keywords】 Vibrio parahaemolyticus; Signaling molecule AI-2; S-ribosylhomocysteinase; S-adenosylhomocysteine nucleosidase; Tetracycline resistance; Tetracycline resistance gene tet;


【Funds】 National Natural Science Foundation of China (31601566) Central Public-interest Scientific Institution Basal Research Fund of Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (20603022020004) National Key Research and Development Program of China (2017YFC1600703)

Download this article

(Translated by QI RS)


    [1] Fujino T, Okuno Y, Nakada D, et al. On the bacteriological examination of Shirasu-food poisoning [J]. Medical Journal of Osaka University, 1953, 4: 299–304

    [2] FAO, WHO. Risk assessment of Vibrio parahaemolyticus in seafood: interpretative summary and technical report. Microbiological risk assessment series No. 16 [R]. Rome: FAO/WHO, 2011

    [3] Li WW, Wang ST, Liang JJ, et al. Analysis of foodborne disease outbreaks in China mainland in 2013 [J]. Chinese Journal of Food Hygiene, 2018, 30 (3): 293–298 (in Chinese)

    [4] Fu P, Liu ZT, Liang JH, et al. Analysis of foodborne disease outbreaks in mainland China in 2014 [J]. Chinese Journal of Food Hygiene, 2018, 30 (6): 628–634 (in Chinese)

    [5] Fu P, Wang LS, Chen J, et al. Analysis of foodborne disease outbreaks in China mainland in 2015 [J]. Chinese Journal of Food Hygiene, 2019, 31 (1): 64–70 (in Chinese)

    [6] Elmahdi S, Da Silva LV, Parveen S. Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: a review [J]. Food Microbiology, 2016, 57: 128–134

    [7] Jiang YH, Yao L, Li FL, et al. Research progress on antibiotic resistance of Vibrio parahaemolyticus and its mechanism [J]. Chinese Fishery Quality and Standards, 2013, 3 (4): 96–102 (in Chinese)

    [8] Zhang DF, An H, Zhang J, et al. Research progress of the drug-resistance and its mechanism of Vibrio parahaemolyticus [J]. Science and Technology of Food Industry, 2018, 39 (9): 311–317, 324 (in Chinese)

    [9] Waters CM, Bassler BL. Quorum sensing: cell-to-cell communication in bacteria [J]. Annual Review of Cell and Developmental Biology, 2005, 21: 319–346

    [10] Bassler BL, Wright M, Silverman MR. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway [J]. Molecular Microbiology, 1994, 13 (2): 273–286

    [11] Ahmed NAAM, Petersen FC, Scheie AA. AI-2 quorum sensing affects antibiotic susceptibility in Streptococcus anginosus [J]. Journal of Antimicrobial Chemotherapy, 2007, 60 (1): 49–53

    [12] Yu YC, Wang LL, Pan Q, et al. Construction and biological characteristics of Salmonella Enterica Serovar Enteritidis luxS mutant [J]. Chinese Journal of Animal and Veterinary Sciences, 2018, 49 (6): 1291–1298 (in Chinese)

    [13] Rajan R, Zhu JG, Hu XB, et al. Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with a catalytic 2-ketone intermediate [J]. Biochemistry, 2005, 44 (10): 3745–3753

    [14] Beeston AL, Surette MG. Pfs-Dependent regulation of autoinducer 2 production in Salmonella enterica Serovar Typhimurium [J]. Journal of Bacteriology, 2002, 184 (13): 3450–3456

    [15] Schauder S, Shokat K, Surette MG, et al. The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule [J]. Molecular Microbiology, 2001, 41 (2): 463–476

    [16] Taga ME, Xavier KB. Methods for analysis of bacterial autoinducer-2 production [J]. Current Protocols in Microbiology, 2011, 23 (1): 1C.1.1–1C.1.15

    [17] Han XG, Lu CP. In vitro biosynthesis of autoinducer 2 of Steptococcus suis Serotype 2 using recombinant LuxS and Pfs [J]. Enzyme and Microbial Technology, 2009, 44 (1): 40–45

    [18] Federle MJ, Bassler BL. Interspecies communication in bacteria [J]. Journal of Clinical Investigation, 2003, 112 (9): 1291–1299

    [19] Guo MH, Fang ZJ, Sun LJ, et al. Regulation of thermostable direct hemolysin and biofilm formation of Vibrio parahaemolyticus by quorum-sensing genes luxM and luxS [J]. Current Microbiology, 2018, 75 (9): 1190–1197

    [20] Han XG, Bai H, Liu L, et al. Cloning and expression of luxS and pfs and in vitro biosynthesis autoinducer 2 of avian pathogenic Escherichia coli from Anhui Province [J]. Acta Microbiologica Sinica, 2012, 52 (9): 1167–1172 (in Chinese)

    [21] Zhang Y, Li MY, Huo L, et al. Biosynthesis of autoinducer-2 and determination of its bioactivity in vitro [J]. International Journal of Stomatology, 2016, 43 (5): 519–523 (in Chinese)

    [22] Zhao J, Zhang FR, Cui Y, et al. In vitro biosynthesis and activity detection of quorum sensing signal molecule AI-2 of Aeromonas hydrophila [J]. Microbiology China, 2015, 42 (10): 1858–1865 (in Chinese)

    [23] Yang J. Synthesis of AI-2 in vitro and effect of AI-2 on bacteriocin synthesis of Lactobacillus plantarum [D]. Harbin: Master’s Thesis of Northeast Agricultural University, 2015 (in Chinese)

    [24] Wang Y, Wang YX, Sun LY, et al. The LuxS/AI-2 system of Streptococcus suis [J]. Applied Microbiology and Biotechnology, 2018, 102 (17): 7231–7238

    [25] Liu ZC. The regulatory mechanism of AI-2 quorum sensing of avian pathogenic Escherichia coli chlortetracycline drug resistance [D]. Hefei: Master’s Thesis of Anhui Agricultural University, 2018 (in Chinese)

    [26] Xue T, Yu LM, Shang F, et al. Short communication: the role of autoinducer 2 (AI-2) on antibiotic resistance regulation in an Escherichia coli strain isolated from a dairy cow with mastitis [J]. Journal of Dairy Science, 2016, 99 (6): 4693–4698

    [27] Li WC. Regulatory mechanism of AI-2 quorum sensing on antibiotic resistance in avian pathogenic Escherichia coli [D]. Hefei: Master’s Thesis of Anhui Agricultural University, 2017 (in Chinese)

    [28] Pereira CS, Thompson JA, Xavier KB. AI-2-mediated signalling in bacteria [J]. FEMS Microbiology Reviews, 2013, 37 (2): 156–181

    [29] Lü XL, Hu JG, Chen ZG, et al. Inactive luxS influence on biological characteristics of O1, O2 and O78 serotypes isolates in avian pathogenicity Escherichia coli [J]. Acta Microbiologica Sinica, 2018, 58 (2): 238–246 (in Chinese)

This Article


CN: 11-1996/Q

Vol 47, No. 05, Pages 1321-1331

May 2020


Article Outline


  • 1 Materials and methods
  • 2 Results
  • 3 Discussion
  • 4 Conclusions
  • References