Characterization of potentially pathogenic Vibrio spp. by rpoB-DHPLC

Question Rising sea water temperature due to global warming enhances the conditions for Vibrio spp. to grow and disperse even in temperate waters of the North and Baltic Sea. Because of the increased incidence of Vibrio infections in the last years, a rapid and accurate method is required to analyze and identify complex Vibrio spp. populations, specifically potential pathogenic Vibrio species, in environmental samples. A PCR-DHPLC (Denaturing High Performance Liquid Chromatography) has been developed based on the rpoB gene of the genus Vibrio, which is a promising method to not only identify but also separate Vibrio spp. in mixed samples due to the different running behavior of amplified PCR products. Methods To facilitate the identification of potential human-pathogenic species we designed Vibrio specific primers based on rpoB sequences of Vibrio spp. strains isolated at Helgoland Roads (North Sea). These primers were combined to amplify fragments of ~500 bp of this rpoB gene. Using the PCR products of four different Vibrio species, we systematically improved the DHPLC conditions, including column temperature and acetonitrile gradient. Finally, we compared the PCR-fragment separation with and without a 40-bp clamp attached to the amplification primers. Results and Conclusions We developed five primer-sets for different regions of the targeted rpoB gene and verified the primer-sets by successfully amplification of 20-23 different Vibrio species (in total 31). We could show that for optimal separation of the amplified fragments by DHPLC the column temperature plays a crucial role and needs to be adapted for each PCR-fragment individually, either with or without GC-clamp. The attached GC-clamp was essential for partial denaturing of all fragments at the same temperature, but generated ambiguous retention peaks. We will illustrate the development of this method in detail

Vibrio in the German Bight: Spatial and temporal variations

Mesophilic bacteria of the genus Vibrio naturally exist in marine environments. Being the main water-borne human pathogens, the focus of this work was put on the species V. cholerae, V. parahaemolyticus and V. vulnificus. These are known to cause serious illnesses associated with food poisoning or wound infections. Although Vibrio infections occur mainly in tropical areas, potentially pathogenic Vibrio spp. have been detected in temperate northern European waters in recent years. Clinical cases in these regions seem to follow extreme weather events, especially heat waves. This study covers monthly samplings from spring 2015 until spring 2017 in a sampling transect between Helgoland and Cuxhaven in the German Bight. Species-specific detection and quantification was conducted using the MPN-PCR method targeting the functional genes toxR or vvhA. Additional evaluation of virulence-associated genes gave insight about the presence of pathogenic strains of these species. Besides recurring patterns of Vibrio abundances during summer seasons, the results showed an increase in pathogenic strain occurrences throughout the sampling period. This study reveals the urgency of a regulated Vibrio monitoring and an understanding of environmental dependencies of Vibrio occurrences

Potentially human pathogenic Vibrio spp. in a coastal transect: Occurrence and multiple virulence factors

An increase in human Vibrio spp. infections has been linked to climate change related events, in particular to seawater warming and heatwaves. However, there is a distinct lack of research of pathogenic Vibrio spp. occurrences in the temperate North Sea, one of the fastest warming seas globally. Particularly in the German Bight, Vibrio investigations are still scarce. This study focuses on the spatio-temporal quantification and pathogenic characterization of V. parahaemolyticus, V. vulnificus and V. cholerae over the course of 14 months. Species-specific MPN-PCR (Most probable number – polymerase chain reaction) conducted on selectively enriched surface water samples revealed seasonal patterns of all three species with increased abundances during summer months. The extended period of warm seawater coincided with prolonged Vibrio spp. occurrences in the German Bight. Temperature and nitrite were the factors explaining variations in Vibrio spp. abundances after generalized additive mixed models. The specific detection of pathogenic markers via PCR revealed trh-positive V. parahaemolyticus, pathogenic V. vulnificus (nanA, manIIA, PRXII) and V. cholerae serotype O139 presence. Additionally, spatio-temporally varying virulence profiles of V. cholerae with multiple accessory virulence-associated genes, such as the El Tor variant hemolysin (hlyAET), acyltransferase of the repeats-in-toxin cluster (rtxC), Vibrio 7th pandemic island II (VSP-II), Type III Secretion System (TTSS) and the Cholix Toxin (chxA) were detected. Overall, this study highlights that environmental human pathogenic Vibrio spp. comprise a reservoir of virulence-associated genes in the German Bight, especially in estuarine regions. Due to their known vast genetic plasticity, we point to the possible emergence of highly pathogenic V. cholerae strains. Particularly, the presence of V. cholerae serotype O139 is unusual and needs urgent continuous surveillance. Given the predictions of further warming and more frequent heatwave events, human pathogenic Vibrio spp. should be seriously considered as a developing risk to human health in the German Bight