Identification and characterization Identification and characterization of Vibrio species by René Erler A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Committee Prof. Dr. Matthias S. Ullrich Molecular Microbiology Jacobs University Bremen Prof. Dr. Frank Oliver Glöckner Microbial Genomics & Bioinformatics MPI for Marine Microbiology & Jacobs University Bremen Dr. Antje Wichels Microbial Ecology Alfred Wegener Institute Dr. Gunnar Gerdts Microbial Ecology Alfred Wegener Institute Date of Defense: 19th January 2015 TABLE OF CONTENTS 2 TABLE OF CONTENTS TABLE OF CONTENTS SUMMARY ........................................................................................................................... 1 INTRODUCTION ................................................................................................................ 3 RESEARCH AIMS ............................................................................................................... 13 OUTLINE ............................................................................................................................. 15 CHAPTER I ......................................................................................................................... 19 VibrioBase: a MALDI-TOF MS database for fast identification of Vibrio spp. that are potentially pathogenic in humans EXCURSUS 1: Variability of MALDI-TOF MS measurements .......................................... 49 EXCURSUS 2: Application of VibrioBase in surveillance programs .............................. 50 CHAPTER II ....................................................................................................................... 51 Effective species identification of Vibrio spp. using mass spectrometric peaks as potential biomarkers CHAPTER III ....................................................................................................................... 77 Biogeographical mapping of V. cholerae, V. parahaemolyticus and V. vulnificus populations in the North and Baltic Seas using ERIC-PCR genotyping GENERAL DISCUSSION .................................................................................................. - 111 OUTLOOK ......................................................................................................................... 121 REFERENCES .................................................................................................................... 123 ACKNOWLEDGMENTS ................................................................................................... 141 STATUTORY DECLARATION ........................................................................................ 142 4 SUMMARY SUMMARY Potentially pathogenic Vibrio spp. in the marine environment are regarded to as emerging health risk in Europe, particularly infections caused by V. parahaemolyticus, V. vulnificus and non-O1/non-O139 V. cholerae strains. Thus, the inclusion of Vibrio spp. in surveillance programs and studies about the distribution of Vibrio spp. are urgently needed. This thesis contributes both: the evaluation of Matrix-assisted laser / desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as tool for species identification in environmental samples and the analysis of spatial patterns of Vibrio spp. populations in the North and Baltic Seas. Regarding MALDI-TOF MS, the specific reference database VibrioBase was developed. VibrioBase reveals Vibrio spp. identification agreements of 96.9% compared to the approved rpoB-sequence-based identification method as well as higher identifications scores and a better discrimination for closely related Vibrio spp. compared to the general BiotyperTM database of the manufacturer. Thus, the cost-effective and rapid MALDI-TOF MS method achieves accurate Vibrio spp. identification when used in combination with VibrioBase. In addition, single mass peaks were used to allow species identifications based on sensitive peaks (SIBOPS). The SIBOPS approach achieves 98% identification agreements with the conventional MALDI-TOF MS / VibrioBase system and is independend of the respective mass spectrometric eqipment and software tools. Furthermore, mass spectrometric biomarkers were found, that might be used for the direct identification of Vibrio spp. in environmental samples. Using ERIC-PCR genotyping, general patterns were not observed for the distribution of V. cholerae, V. parahaemolyticus and V. vulnificus in the North and Baltic Seas. Particularly the latter two species were separated into distinct regions, which are however not linked to geographical aspects and, to a minor extent, are asscociated with environmental parameters such as salinity. In consequence, MALDI-TOF MS was confirmed as a promising tool for species identifications from environmental samples. VibrioBase and SIBOPS are important steps towards the implementation of Vibrio spp. in surveillance programs executed by health authorities. Future reseach has to be done to acquire knowledge about ecological aspects of Vibrio spp. in temperate waters, in particular the linkage to plankton species in the North and Baltic Sea and the influence of bacteriophages for shaping Vibrio spp. populations. - 1 - INTRODUCTION - 2 - INTRODUCTION INTRODUCTION The genus Vibrio The family Vibrionaceae within the class of γ-proteobacteria includes chemoorganotrophic bacteria of the genera Listonella, Photobacterium and Vibrio. The bacteria of this family are gram-negative and mostly appear as rod-shaped cells with a single flagellum. Vibrio is the largest genus and consists of more than 90 species (Thompson et al. 2004). A common characteristic feature of Vibrio spp. is the presence of two chromosomes (Heidelberg et al. 2000; Schoolnik et al. 2000). The larger chromosom I contains genes for essential cell functions whereas the smaller chromosom II carries genes that are typically found on plasmids. In general, vibrios reveal a high rate of recombination and are regarded as a phylogenetically diverse group of species (Harth et al. 2007; Vos et al. 2009). Pathogenicity of Vibrio spp. At least twelve species are known to cause illness in humans. All of them can be cultivated at 37°C and differ in their virulence potential. Three Vibrio species are of particular clinical importance: V. cholerae, V. parahaemolyticus and V. vulnificus (Blake et al. 1980). The remaining species are the causative agents for rare infections in humans and are considered as weak pathogens: V. alginolyticus, V. harveyi, V. mimicus, V. metschnikovii, V. fluvialis, V. furnissii, V. hollisae, V. damselae and V. cincinnatiensis (Janda et al. 1988; Thompson et al. 2004). Globally, V. cholerae causes the most infections due to the consumption of contaminated water or food. According to the World Health Organization (WHO 2013), Cholera represents an estimated burden of 1.4 to 4.3 million cases, and 28,000 to 142,000 deaths per year, particularly on the African continent (Griffith et al. 2006; Mintz et al. 2013). The classical Cholera disease is associated with the toxin CTX that is responsible for the electrolyte imbalance and the secretion of water into the intestinal lumen, consequently leading to severe watery diarrhoea and dehydration (Muanprasat et al. 2013). The majority of Cholera cases is caused by two serotypes: O1 and O139 (Thompson et al. 2004). Infections with these highly pathogenic strains are rare in the northern hemisphere and only reported for travellers returning to America or Europe from Cholera-affected regions (Wittlinger et al. 1995). Other - 3 - INTRODUCTION strains are commonly referred to as non-O1/non-O139 V. cholerae and cause mild-to- moderate diarrhoea, but also severe septicemia was reported (Namdari et al. 2000; Dutta et al. 2013). Infections with non-O1/non-O139 strains were reported from countries in the Baltic region such as Finland (Lukinmaa et al. 2006) and Poland (Stypulkowska-Misiurewicz et al. 2006). In contrast, the main transmission route for pathogenic V. parahaemolyticus is the consumption of undercooked and raw shellfish. Infections with this species are the leading cause of bacterial seafood-borne gastroenteritis worldwide (Su et al. 2007). Two hemolysins are mainly responsible for the associated damage of intestinal epithelial cells: the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) (Honda et al. 1985; Nishibuchi et al. 1995; Makino et al. 2003; Ritchie et al. 2012). The prevalence of the hemolysin genes tdh and trh is high in clinical (>85%) and low (<15%) in environmental isolates (Ottaviani et al. 2013; Li et al. 2014). The O3:K6 serotype of V. parahaemolyticus is of particular clinical importance (Okuda et al. 1997; Nair et al. 2007). During the last two decades, these tdh-positive strains dispersed over the world and even caused outbreaks in regions with temperate waters such as Alaska and Chile (Gonzalez-Escalona et al. 2005; McLaughlin et al. 2005). Recently, O3:K6 strains were isolated from oysters, harvested in Southern England (Powell et al. 2013). However, no V. parahaemolyticus associated infections were reported from the North and Baltic Seas, so far. To some extend, gastroenteritis is also caused by V. vulnificus (De et al. 2011). But far more significant is the development of life-threatening septicemia from V. vulnificus, either caused by intestinal pathogenic strains or triggered by the sea-water-borne transmissions of V. vulnificus strains through open wounds. Such infections develop