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

Abstract:

[Background] Antimicrobial resistance of bacteria has become more and more serious due to the abuse of antimicrobials. As an important foodborne pathogen, Vibrio parahaemolytics also exhibited 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 resitance of V. parahaemolytics. [Objective] To study the effect of signaling molecule autoinducer-2 (AI-2) on tetracycline resistance of V. parahaemolyticus strains from seafoods. [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 colonies 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 positive control. When treating 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 antimicrobial resistance of V. parahaemolyticus.