A key aspect in microbial taxonomy is the identification of microorganisms at the species or genus level. This is important to distinguish pathogenic species in health and environmental sciences, to identify beneficial or symbiotic species and also to study microbial diversity in environmental niches in Nature. Due to the widespread use and importance of bacterial identification, methods and processes should be accurate, affordable, fast and easy to use (Urakawa et al., 1997; Amaral et al., 2014).

The Vibrionaceae is a large family of marine Gram-negative Gammaproteobacteria, which includes organisms of different environmental importances, e.g., symbiotic (e.g., Vibrio fischeri), bioactive (e.g., V. coralliilyticus and Photobacterium galatheae), and pathogenic organisms (e.g., V. cholerae and Aliivibrio salmonicida). Several of the human pathogenic Vibrio species, such as V. cholerae and V. parahaemolyticus, are mesophilic organisms and changes in sea water temperature and salinity influence their distribution (Reid et al., 2011; Huehn et al., 2014; Le Roux et al., 2015; Xu et al., 2015). The increase in sea water temperature has been linked to a number of human illness outbreaks caused by Vibrio species (e.g., V. cholerae, V. parahaemolyticus, V. vulnificus) (Le Roux et al., 2015) and to several epidemics in marine animals, such as oysters, salmon, sea bass, eel, trout, shrimps, and corals, caused by V. salmonicida, V. anguillarum, P. damselae, V. vulnificus, and V. coralliilyticus (Ottaviani et al., 2012; Huehn et al., 2014; Le Roux et al., 2015). The rising sea water temperatures have also been linked to the increase in the numbers of V. cholerae associated with plankton in the North Sea (Vezzulli et al., 2012). The increased spread of Vibrio pathogens and their importance as infectious and/or food poisoning agents has a direct impact on health of mankind and requires changes in microbiological food control processes and clinical settings (Nair et al., 2007). Any survey or intervention to reduce risk requires that the organisms can be rapidly and correctly identified.

Identification of species from the Vibrionaceae family has primarily been based on Multi-Locus Sequencing Analysis (MLSA), which relies on the amplification and sequencing of up to nine genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA, and the 16S rRNA gene) (Thompson et al., 2005; Sawabe et al., 2007, 2013; Gabriel et al., 2014). This has been necessary due to the limitations of 16S rRNA gene phylogeny in this family, an issue addressed with coupling of other techniques such as restriction fragment length polymorphism analyses (Urakawa et al., 1997, 1999), but ultimately unsolvable due to several (7–15) different copies of 16S rRNA gene encoded in a single genome (Reen et al., 2006; Jensen et al., 2009; Machado and Gram, 2015). We have recently identified the ferric-uptake regulator gene (fur) as a new phylogenetic marker in the Vibrionaceae family, and developed a PCR based method for the amplification of the genomic region encoding the fur gene (Machado and Gram, 2015). Its use as a single phylogenetic marker in the classification of Vibrionaceae at the species level could reduce the timing and cost of strain identification. To enable the use of this potential worldwide, we here describe the design of an online platform, FurIOS 1.0, which allows an easy and fast identification of Vibrionaceae species using only their fur DNA sequence. We also use a collection of 191 Vibrionaceae strains to demonstrate its potential.

FurIOS is an easy-to-use online service that allows the identification of bacteria from the Vibrionaceae family at the species level using the fur gene as a single identification marker. This online service available to any user worldwide is an extra effort to implement the use of the recently developed method for the amplification of the fur gene sequence, with greater discriminatory power when compared to MLSA or 16S rRNA analyses. Here, we have applied this tool in the classification of environmental Vibrionaceae strains belonging to the global culture collection Galathea 3.

We identified at the species level 90% of the 191 Vibrionaceae strains from the Galathea 3 culture collection. The most abundant species was Vibrio splendidus, followed by V. tasmaniensis, V. crassostreae, and V. campbellii (Figure 2). The collection was based on culturing from marine samples and subsequent testing of antibacterial activity against the fish pathogen V. anguillarum (Gram et al., 2010), and this may explain the over-representation (70%) of the species belonging to the Splendidus clade (Sawabe et al., 2013). Several studies have shown the presence of V. splendidus in water samples through all seasons, with higher predominance in summer, and in locations ranging from the arctic to the tropics (Thompson et al., 2004; Jensen et al., 2009). These features reflect the great adaptability of this species (Jensen et al., 2009), which can also explain its over-representation in a global culture collection such as the Galathea 3 collection. The analysis also provided evidence that two V. splendidus strains ATCC 33789 and ZS-139 are indeed not V. splendidus species, which we confirmed using whole genome phylogenetic analysis (Table 1).

Besides the over-representation of strains from the Splendidus clade, this dataset allowed the evaluation of the potential of this identification method and web-tool in the classification of Vibrionaceae environmental samples at the species level, by means of a single PCR reaction and sequencing of its product. The method presented here can be used for identification of Vibrionaceae species in less than 24 h, and it requires minimal equipment and service. The analyses of the 191 sequences using the FurIOS web service took only 3 min, although this time may depend on the server usage at the moment of use. Sequence based identification has in other settings also been developed into online tools, which have been successfully used for several years for MLSA of pathogenic bacteria³,⁴,⁵. A tool based on MLSA has been under development for identification of Vibrio species⁶, however, it appears not to be functional. FurIOS is therefore the first functional online service developed for the classification of Vibrionaceae species and it is easier to implement and use than the MLSA based analyses, because it uses a single gene with great discriminatory power (Machado and Gram, 2015; Giubergia et al., 2016).

This identification exercise provided 20 isolates with an inconclusive species attribution (“other species” – Figure 2). These might constitute new species or species not yet represented in the FurIOS database. Although the number of species represented in the database is lower than the number of species so far described within this family, the use of the fur gene as an identification marker and the publication of more fur sequences will allow the improvement of the database by increasing the number of species represented. Newly published fur sequences can be sent to the corresponding author who will act as a database curator. Also publications making use of FurIOS will be checked for relevant sequences to be added. Supporting the possibility of “other species” being a new species is the case of strain S2757. Its fur gene sequence had an 89% identity to V. tubiashii DSM 19142 and it was therefore here classified as “other species.” This strain has been further studied and has been recently described as the type strain of the new species V. galatheae (Giubergia et al., 2016). In that study, 16S rRNA, MLSA, and fur gene analyses were performed and the fur gene, as a single gene analysis, provided as good discrimination as did the MLSA analysis (Giubergia et al., 2016). This exemplifies the use of the fur gene in the classification of a new species belonging to the Vibrionaceae family.

Although there might not be a perfect single gene for species identification or phylogenetic evaluation, the fur gene seems so far the best “single” gene identified in the classification of Vibrio species and the development of this tool is an effort to bring that to use and help the scientific community with identification of strains from the Vibrionaceae family. Yet, evaluation of specific phylogenetic relationships should be done using multiple genes, such as MLSA analyses. It is not clear why fur mutational rate allows distinction of Vibrionaceae at the species level, but we have observed indications of the same pattern in Pseudoalteromonas (Machado et al., 2015).

The recent findings have provided the Vibrionaceae taxonomy field with new and more accurate approaches to evaluate the phylogeny and taxonomy relatedness between the different species of this family. This comes as a great opportunity to re-evaluate the evolutionary theories in this family as well to discover and correctly classify newly identified species. Regarding FurIOS, its simplistic design and straightforward pipeline makes it suitable for any research environment, from academia to industry, and especially for quick species identification in clinical and food-safety settings, where Vibrionaceae strains are of high risk (e.g., microbiological control of sea food products).

HM designed the study and the database used, and together with SG performed the testing of the web-based tool. JC carried out the programming and together with KR the implementation of the web-based tool. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.