Elsevier

Journal of Marine Systems

Volume 221, September 2021, 103574
Journal of Marine Systems

Fish as a winter reservoir for Vibrio spp. in the southern Baltic Sea coast

https://doi.org/10.1016/j.jmarsys.2021.103574Get rights and content

Highlights

  • Fish acts as Vibrio winter reservoir in the Baltic Sea.

  • Vibrio spp. often highest abundant in connection to invasive species round goby.

  • Quantitative Vibrio 16S rRNA gene ddPCR without Photobacterium falsification.

Abstract

Vibrio spp. are ubiquitous in aquatic environments. In temperate regions, including the Baltic Sea, these bacteria, which include facultative pathogenic species, proliferate during warm water periods and may thus pose a risk to human health. However, while present at relatively high abundances in the summer months, Vibrio spp. are rarely detected during cold water periods. Several potential winter reservoirs, such as fish and sediment, have been discussed, but the seeding sources that give rise to Vibrio during the summer months are unknown.

In this study, we employed a combination of cultivation-independent genus-specific digital droplet PCR, Illumina 16S rRNA gene sequencing, culture-dependent determinations of colony-forming units (CFU) and genus-specific strain isolation to identify possible winter reservoirs of Vibrio spp. Using these techniques, we demonstrate that in the southern Baltic Sea especially fish can serve as a winter reservoir for distinct Vibrio assemblages, including facultative pathogenic V. vulnificus. Particularly the invasive species round goby (Neogobius melanostomus), with its potential to appear in high individual densities, has the capacity to carry a comparable Vibrio spp. cell number like water and sediment, while only accounting for 1/1000 of the spatial share in the overall coastal environment. Thus, by providing a winter reservoir for distinct Vibrio communities, coastal fish and, especially areas in which round goby densities are high, may be hot spots of pathogenic Vibrio species.

Introduction

The genus Vibrio consists of more than 130 gram-negative, motile, metabolically and genetically diverse species (Gomez-Gil et al., 2013). Vibrio spp. are found in water (Thompson et al., 2004), sediment (Givens et al., 2014), attached to particles (Oberbeckmann et al., 2011), and on the surface and intestines of higher organisms, such as crustaceans (Vandenberghe et al., 1999), bivalves (Givens et al., 2014), and fish (Noguchi et al., 1987). The lifestyles of these bacteria range from symbiotic to pathogenic and include species able to infect humans (Elyakov et al., 1991; Farmer Iii and Hickman-Brenner, 2006; Jayasree et al., 2006). Among the latter are V. parahaemolyticus, V. cholerae, V. alginolyticus and V. vulnificus. While ubiquitous distributed in aquatic environments including subpolar regions as Iceland (Haley et al., 2012), with their preference for warmer water temperatures, these species do not pose a major threat for humans in temperate regions (Baker-Austin et al., 2010; Baker-Austin et al., 2017; Hlady and Klontz, 1996; Huq et al., 2005), but this may change as global warming increases sea surface temperatures and therefore potentially the range of Vibrio spp. (Baker-Austin et al., 2013). This is especially true for the Baltic Sea, which is relatively shallow and is warming 5–6 times faster than the global average (EEA, 2019). In addition to temperature, the brackish water of the Baltic Sea, with a salinity of <25, supports the growth of Vibrio species such as V. vulnificus (Kaspar and Tamplin, 1993; Takemura et al., 2014), the main causative agent of vibriosis at the German Baltic Sea coast (Hauk and Duty, 2015). A preview of potential future scenario of increase in Vibrio-based abundance was obtained during the recent unusually warm summers in the Baltic Sea area, during which cases of Vibrio infections increased (Baker-Austin et al., 2016; Hauk and Duty, 2015).

Water temperatures in the Baltic Sea in summer rise above 15–20 °C, which is warm enough for the proliferation of Vibrio spp. to levels allowing its simple detection (Böer et al., 2012; Böer et al., 2013). However, in winter, Vibrio spp. are rarely detected in the cold waters of the Baltic (Böer et al., 2013; Oberbeckmann et al., 2011). This winter disappearance has been explained by the ability of Vibrio spp. cells to enter a dormant state, so-called VBNC (viable but not cultivatable), when the water temperature drops below 10 °C (Baffone et al., 2003; Weichart et al., 1992; Wolf and Oliver, 1992). VBNC cells are still alive and metabolically active but their transfer to cultivation medium results in their death (Bloomfield et al., 1998). This prevents the detection of Vibrio spp. cells by cultivation-based approaches, which remains the most common method of Vibrio spp. quantification (Kong et al., 2004; Wolf and Oliver, 1992).

The absence or extreme scarcity of Vibrio spp. cells during cold water periods in temperate regions raises the question whether refuges exist where these bacteria are more protected and survive in higher abundances until water temperatures again increase. While sediment is perhaps the most obvious possible refuge (Böer et al., 2013; Chase et al., 2015), others are possible, given the flexibility in lifestyle and habitat of Vibrio spp. For example, biofilms are robust towards environmental changes (Kubota et al., 2008; Wai et al., 1998) and Vibrio spp. are able to attach to particles and chitin (Kirschner et al., 2011; Matz et al., 2005; Shime-Hattori et al., 2006) such that seston might also offer a winter reservoir for members of this genus. A study at the subtropical U.S Gulf coast by DePaola et al. (1994) showed that fish could provide an advantageous winter habitat, albeit one that is influenced by the lifestyle of the fish species, as absolute Vibrio spp. abundances on bottom-feeding fish were up to five magnitudes higher than on filter-feeding and carnivorous species.

Cod (Gadus morhua) and herring (Clupea harengus membras) are carnivorous and facultative-filter-feeding species, respectively, indigenous to the Baltic Sea and both are migrating species of high economic value. As a winter reservoir for Vibrio spp., they could therefore transport and spread bacterial populations over a wider area or re-seed coastal Vibrio assemblages after longer cold water periods. In addition, following the introduction of the non-indigenous, bottom-feeding round goby (Neogobius melanostomus) in the Baltic Sea in the 1990s (Skóra and Stolarski, 1993), this species has spread over large coastal areas of the southern Baltic Sea, reaching densities of 20 to >100 individuals per m2 in the Baltic Sea and freshwater habitats, respectively (Chotkowski and Marsden, 1999; EMI, 2017). Consequently, the round goby may be an important winter shelter, including for coastal Vibrio communities. However, despite these possible alternative reservoirs, the seeding banks of Vibrio spp. in the Southern Baltic Sea are practically unknown, although their identification would allow a better understanding of the spreading dynamics of Vibrio, including its pathogenic members, in temperate regions during warm water periods. Furthermore, it would enable predictions of coastal areas at probable risk, i.e., those where Vibrio could quickly reach high concentrations as the water temperature rises.

In this study, to gain insights into the potential winter reservoirs of Vibrio spp. we examined the abundance and composition of this bacterial genus in water, sediment, and seston and in three fish species (cod, herring, round goby) during two late summer/autumn and winter periods, also taking into account the naturally occurring salinity gradient of the southern Baltic Sea coast. To exclude false-positive results introduced by members of the genus Photobacterium, we used optimized cultivation-dependent and -independent approaches, combining 16S rRNA Illumina sequencing and in silico in house-improved specific primers for Vibrio spp. with quantitative digital droplet PCR and classical strain isolation methods.

Section snippets

Sampling

Sampling map was created using the free software Ocean Data View version 5.1.5 (Schlitzer, 2021). Samples were collected at four registered bathing areas (Regional Office for Health and Social Affairs of Mecklenburg-Western Pomerania / LAGuS-MV) along the Baltic Sea coast in Mecklenburg-Western Pomerania: Warnemünde (W), LAGuS-MV number 236; Lubmin (L), LAGuS-MV number 750; Karlshagen (K), LAGuS-MV number 703; and Niendorf (N), LAGuS-MV number 278 (Fig. 1). Sampling at these sites was conducted

Environmental conditions

Salinity at the four stations decreased from west to east, with the salinity of the W and N samples ranging from 11.6‰ to 15.3‰ and that of the L and K samples from 6.0‰ to 8.3‰ (Table 1). The exception was the water of the W samples from the summer of 2018, which had a salinity of 7.4‰. The water temperature in winter was at least 10 °C lower than in summer/autumn, ranging from 1 °C to 5.9 °C and from 10.6 °C to 16.8 °C, respectively. Again, the summer 2018 samples were the exception, as the

Discussion

The identification of the winter habitats of Vibrio spp. and its facultative pathogenic members in temperate coastal regions represents an important step in understanding Vibrio dynamics during the onset of warm water periods. Moreover, it can also allow better predictions of potential risk areas from which Vibrio spp. may be able to reach high or even harmful concentrations once water temperatures again become favourable.

Conclusion

Our study showed that fish has the potential to serve as a winter reservoir for Vibrio spp. in the southern Baltic Sea. Namely the round goby was one of the few sampling materials in which the potential human pathogenic V. vulnificus was detected. As the invasive species round goby can reach high abundances, especially close to the coastal shore, it might serve as a new winter reservoir for distinct coastal Vibrio communities and potentially pathogenic species. Therefore, areas of high round

Deposit of sequence data

Sequence data are deposed at the European Nucleotide Archive, ENA, under the accession number: PRJEB38826.

Declaration of Competing Interest

None.

Acknowledgments

The project was funded by the Forschungsstiftung Ostsee c./o. Ozeaneum Stralsund GmbH, project number 09/2014. We thank Daniel Oesterwind et al. from the Thünen-Institut für Ostseefischerei for their support and providing of fish traps. The excellent technical assistance of Jana Bull (University of Rostock) during library preparation and Illumina sequencing is greatly acknowledged. The purchase of the Illumina MiSeq in the lab of BK was kindly supported by the EU-EFRE (European Funds for

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