Development of determination methods for food pathogen Vibrio papahaemolyticus by detecting quorum sensing signals

Abstract

This study aimed to develop the methods for V.parahaemolyticus determination based on its AHL production property. The first two experiments were the development of methods for AHL determination by colorimetry method, and evaluation of the method efficiency by validation following USFDA guideline. The methods including AHL extraction and determination of AHL coloring complex by spectrophotometry (λmax= 520), called Procedure A, and determination of AHL existing in broth media without performing extraction step called Procedure B, were developed for AHL determination. The efficiency of both methods were not significantly different. Based on statistical analysis of validation parameters, both methods were accurate, precise and selective for AHL determination. Method sensitivity evaluation showed that, minimal AHL concentration could be detected by either procedure was 30 µM. These methods could detect AHL produced from V.parahaemolyticus cultured in NB containing minimal population approximately 6 logCFU/ml. However, the Procedure B was selected for the further works because it was more rapid and less laborious. The influences of intrinsic and extrinsic factors on AHL production properties were investigated using colorimetry (Procedure B) and HPLC analysis. It was found that strains, cultivation times and cultivation conditions could not influence on both quality and quantity of AHL produced from V.parahaemolyticus. Interestingly, 8% NaCl in cultural media could induce V.parahaemolyticus to produce 28 folds larger amount of AHL than the other factors, whereas it had not influence on AHL quality. Therefore, AHL which was 3-hydroxy-C4-HSL could be one of the V.parahaemolyticus identity. Finally, Procedure B and 8% NaCl peptone water as an enrichment selective media for growing V.parahaemolyticus and inducing its AHL production were investigated. It was found that this methodology could determine V.parahaemolyticus starting at 2 logCFU/ml within 20 hours both in single and co-cultured with other bacteria. Therefore, this new strategy could be an alternative for a simple and rapid identification of V.parahaemolyticus.