Current Trends in Microbiology Vol. 12, 2018 Interactions between the human pathogen Vibrio parahaemolyticus and common marine microalgae Savannah L. Klein, Katherine E. Haney, Thomas M. Hornaday, India B. Gartmon and Charles R. Lovell* Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, 29208, USA. ABSTRACT KEYWORDS: Vibrio parahaemolyticus, tdh, trh, T3SS2, T6SS, microalgae. Vibrio parahaemolyticus is a gastrointestinal pathogen that is abundant in coastal marine environments. Elevated numbers of V. parahaemolyticus cells INTRODUCTION have been correlated with marine microalgae Vibrio parahaemolyticus, a common organism in blooms, particularly blooms of diatoms and coastal environments, is a significant and sometimes dinoflagellates, but the nature of the relationship pandemic human pathogen responsible for an between V. parahaemolyticus and microalgae is estimated 34,000 cases of seafood-associated unknown. We performed in vitro assays using 27 gastroenteritis per year in the United States [1]. Most environmental V. parahaemolyticus strains and cases of V. parahaemolyticus-induced gastroenteritis various phototrophs; a diatom, a dinoflagellate, are self-limiting and relatively mild, but infections unarmored and armored forms of a coccolithophore, can be deadly in immunocompromised individuals. and two species of cyanobacteria. The V. The common mode of transmission of this parahaemolyticus strains we employed contained bacterium to the human host is ingestion of raw or different combinations of virulence-correlated genes, undercooked shellfish, primarily oysters. In addition, the hemolysin genes tdh and trh, the Type III some strains of V. parahaemolyticus can infect Secretion System 2 (T3SS2) marker gene vscC2, wounds and some produce systemic infections, and the Type VI Secretion System (T6SS) marker while others are apparently non-pathogenic. gene vipA1. We determined that all V. Elevated densities of V. parahaemolyticus most parahaemolyticus strains, even strains in which no often occur during the warm months and at warm virulence factor genes were detected, were able to locations [2, 3] but recently large vibriosis outbreaks cause decreases in diatom, dinoflagellate, and have occurred at locations not considered typical unarmored coccolithophore biomass in vitro. for this organism [4, 5]. No correlation between content of any virulence V. parahaemolyticus not only persists but can gene and damage to microalgae was apparent. increase in population size very rapidly in coastal We hypothesize that marine microalgae represent marine environments [3, 6, 7]. It is not understood a reservoir of nutrients that the copiotroph how this copiotrophic organism acquires carbon V. parahaemolyticus can utilize in salt marsh and other nutrients in coastal marine ecosystems environments, which are often poor in labile carbon where levels of utilizable soluble (labile) carbon and energy sources. This helps to explain the and energy sources are typically quite low [8-10]. recent correlations between V. parahaemolyticus Even considering the known catabolic versatility and microalgae blooms in such environments. of this species [3], rapid growth opportunities in many coastal ecosystems would seem infrequent *Corresponding author: [email protected] at best. The abundance of V. parahaemolyticus as Savannah L. Klein et al. free-living cells in water is typically low (< 2,000 2011 as described previously [27]. The North cells per liter) [6], but this organism can be very Inlet-Winyah Bay National Estuarine Research abundant in surficial sediment and in infaunal Reserve protects the third largest watershed on the burrows [3, 6]. V. parahaemolyticus also occurs at east coast of the United States; and North Inlet is higher levels in shellfish [2, 7, 11], and in a bar built oligotrophic salt marsh where human association with algal blooms [12-16]. Thus, impact is negligible [28, 29]. Samples were diluted significant reservoirs exist even when no outbreak and plated directly onto Thiosulfate Citrate Bile is underway [17]. The population expansion of Salts Sucrose agar (TCBS) (BD, NJ). The V. parahaemolyticus that predicates an outbreak presumptive identification of all V. parahaemolyticus may be supported by means other than the typically strains used in this study was confirmed by recA low abundance, and largely refractory, dissolved sequence analysis [27] using the PCR primers and organic carbon pool found in relatively low protocols of Thompson et al. [30]. human impact coastal marine systems. Virulence gene PCR screening Potential for a V. parahaemolyticus outbreak has often been predicted on the basis of local temperature, Two virulence-related hemolysin genes, tdh and trh, have been correlated with pathogenesis in salinity, turbidity, and chlorophyll a concentrations V. parahaemolyticus, and these hemolysin genes are [18-20]. In addition, some correlations between V. parahaemolyticus densities and certain algal taxa, frequently used as molecular markers for strain specifically diatoms and dinoflagellates [12, 21, 22], virulence [27, 31]. Additional virulence factors, have been reported and elevated levels of specifically secretion systems, have been discovered with recent sequencing of V. parahaemolyticus V. parahaemolyticus can occur during dinoflagellate genomes [32-34]. The Type III Secretion System and diatom blooms [13, 14]. The interaction between microalgae and V. parahaemolyticus could be (T3SS2) has also been implicated in V. commensalistic, based on soluble exudates released parahaemolyticus virulence [32] and the outer from algal cells lysed by viruses [23, 24], inefficient membrane protein gene, vscC2 is a useful marker for this structure [35, 36]. grazing by zooplankton [25, 26], or from undamaged The Type VI Secretion System (T6SS) has also algal cells. Or, perhaps, the microalgae themselves serve as supplemental carbon sources that been detected in some V. parahaemolyticus isolates V. parahaemolyticus can utilize. [37]. This secretion system has not been implicated We examined the ability of V. parahaemolyticus in the pathogenicity of V. parahaemolyticus to to cause damage to healthy phototrophs. Chlorophyll humans, but T6SS producing V. parahaemolyticus strains have been shown to cause damage to other a served as an indicator of phototroph biomass. prokaryotes in vitro when incubated on a surface Six phototrophs, including three species of microalgae and two species of cyanobacteria were incubated [37]. Its impacts on eukaryotic microalgae are with several strains of V. parahaemolyticus. The presently unknown. Strains were screened for the phototrophs employed are abundant in marine T6SS marker gene vipA1 using the PCR primers environments and present a variety of cell wall and protocols of Salomon et al. [37]. surface structures and properties, providing insight V. parahaemolyticus strains were grown overnight into associations between susceptibility of the at 37 ºC in Saline Luria-Bertani Broth (SLB; per L microalgae to V. parahaemolyticus predation and 27 g NaCl, 10 g Tryptone, 5 g Yeast Extract) and cell wall features. boiled extracts (15 min at 95-100 ºC) were prepared. All PCR reactions were completed within three MATERIALS AND METHODS days of DNA extraction and 1 μl of boiled DNA extract was used per reaction. PCR products were V. parahaemolyticus strain isolation and resolved on a 1.5% agarose gel and sequenced using characterization an ABI Prism 3730 DNA analyzer to confirm V. parahaemolyticus strains were isolated from the gene identity. Sequences were analyzed using the pristine North Inlet estuary near Georgetown, SC, Kimura 2 parameter model with Mega version 7 USA (33°20’N, 79°12’W) in August and September [38]. Sequence data obtained from this work were Vibrio parahaemolyticus interactions with phototrophs submitted to the NCBI GenBank and assigned the final and initial time points was determined by the accession numbers KX171447- KX171449. formula: ((Tfinal – Tinitial) / (Tinitial)) x 100. Before and after incubation, aliquots were observed under a Cultivation of microalgae and cyanobacteria Nikon Eclipse TS100 microscope to determine the Phototroph cultures were obtained from the Bigelow effect of V. parahaemolyticus clinical strains ATCC National Center for Marine Algae and Microbiota 17802T, ATCC 33846, and environmental strain (Bigelow Center, East Boothbay, ME). Three species 5-10-J5-4 on unarmored E. huxleyi. Microalgal cell of eukaryotic microalgae were used in this project, counts were performed using a hemocytometer after the diatom Thallasiosira pseudonana (CCMP 1335), the 24-h co-incubation. the dinoflagellate Prorocentrum minimum (CCMP Each V. parahaemolyticus strain was tested in 12 695), and two strains of the coccolithophore, wells per 96 well plate. For true replication, each Emiliania huxleyi (CCMP 371 and CCMP 373). 96 well plate was repeated three times. Controls T. pseudonana and P. minimum are common in included replicates of phototrophs in appropriate North Inlet and E. huxleyi CCMP 371 is a coccolith- media and replicates of phototrophs in artificial producing (armored) form that causes extensive seawater (with no V. parahaemolyticus added), blooms. E. huxleyi CCMP 373 is an unarmored against which experimental replicates were compared. mutant phenotype. Two species of cyanobacteria Vibrio pacinii, an avirulent Vibrio [43], was used were also used, Prochlorococcus marinus (CCMP as a non-V. parahaemolyticus, heterotrophic bacterial 1986) and