Abstract
A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the blaCARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Homologous analysis showed that blaCARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this blaCARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of blaCARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by blaCARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification.
Highlights
Vibrio sp. are gram-negative and halophilic bacteria that inhabit the estuarine and marine environment and some species can cause gastrointestinal diseases in human (Austin, 2010; Scallan et al, 2011; Letchumanan et al, 2014)
Many of the targeting genes used in these approaches are Accurate Confirmation of V. parahaemolyticus phylogenetic markers or those involved in virulence, yet some of which are not highly species-specific as different Vibrio species may share similar sequences, reducing the accuracy and specificity of such detection methods
This phenomena is in agreement with previous findings that tlh was distributed among V. alginolyticus (Xie et al, 2005), and that similar virulence-related genes in V. parahaemolyticus existed in other Vibrionaceae species(Klein et al, 2014)
Summary
Vibrio sp. are gram-negative and halophilic bacteria that inhabit the estuarine and marine environment and some species can cause gastrointestinal diseases in human (Austin, 2010; Scallan et al, 2011; Letchumanan et al, 2014). In our routine identification of V. parahaemolyticus, we noticed that PCR targeting tlh often could not differentiate organisms in the V. harveyi group, especially V. parahaemolyticus and V. alginolyticus. This phenomena is in agreement with previous findings that tlh was distributed among V. alginolyticus (Xie et al, 2005), and that similar virulence-related genes in V. parahaemolyticus existed in other Vibrionaceae species(Klein et al, 2014). Our laboratory recently identified a β-lactamase that contributed to intrinsic ampicillin resistance in V. parahaemolyticus (Chiou et al, 2015) The gene encoding this enzyme is an intrinsic gene in V. parahaemolyticus and is more conserved in this species compared to other gene markers. Species-specific β-lactamase genes are being explored as targets for development of combined genetic and phenotypic bacteria identification approaches. We attempted to develop a reliable and simple PCR assay targeting blaCARB−17 for detection and identification of V. parahaemolyticus
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