Molecular epidemiology of photobacterium damselae subsp. damselae outbreaks in marine rainbow trout farms reveals extensive horizontal gene transfer and high genetic diversity

fmicb-09-02155 September 17, 2018 Time: 16:40 # 1 ORIGINAL RESEARCH published: 19 September 2018 doi: 10.3389/fmicb.2018.02155 Edited by: Zhe Zhao, Hohai University, China Reviewed by: Carmen Amaro, Universitat de València, Spain Peng Luo, South China Sea Institute of Oceanology (CAS), China Qingpi Yan, Jimei University, China *Correspondence: Carlos R. Osorio cr[email protected] Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Received: 23 March 2018 Accepted: 22 August 2018 Published: 19 September 2018 Citation: Terceti MS, Vences A, Matanza XM, Dalsgaard I, Pedersen K and Osorio CR (2018) Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity. Front. Microbiol. 9:2155. doi: 10.3389/fmicb.2018.02155 Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity Mateus S. Terceti1, Ana Vences1, Xosé M. Matanza1, Inger Dalsgaard2, Karl Pedersen3 and Carlos R. Osorio1* 1Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 2National Institute of Aquatic Resources, Technical University of Denmark, Kongens Lyngby, Denmark, 3National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the toxR gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPRCas systems and putative new mobile genetic elements. This study provides sound evidence that P. damselae subsp. damselae outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen. Keywords: Photobacterium damselae, vibriosis, damselysin, phobalysin, hemolysin, rainbow trout INTRODUCTION The marine bacterium Photobacterium damselae subsp. damselae has been associated with disease in a number of marine animals, and also in humans. It has been reported as a primary pathogen causing diseases in turbot (Scophthalmus maximus) (Fouz et al., 1992), sea bream (Sparus aurata) (Vera et al., 1991) and other sparid fish species (Company et al., 1999;Labella et al., 2006), sea bass (Dicentrarchus labrax) (Abdel-Aziz et al., 2013;Uzun and Ogut, 2015), rainbow trout (Oncorhynchus mykiss) (Pedersen et al., 1997, 2009), shrimp (Exopalaemon carinicauda) (Liu et al., 2016), and etc. The geographical distribution of this bacterium is increasing and nowadays it Frontiers in Microbiology | www.frontiersin.org 1September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 2 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae constitutes an emerging pathogen in aquaculture (Abdel-Aziz et al., 2013;Khouadja et al., 2014;Terceti et al., 2016;Sharma et al., 2017;Eissa et al., 2018;Tao et al., 2018). Its pathogenicity is attributed to the production of up to four different toxins (Osorio et al., 2018), and two main categories of strains can be distinguished. On the one side, strains harboring the virulence plasmid pPHDD1 produce the plasmidencoded toxins damselysin (Dly) and phobalysin P (PhlyP) (Rivas et al., 2011), in addition to the chromosome I-encoded toxins phobalysin C (PhlyC) and the phospholipase PlpV (Vences et al., 2017). On the other side, strains lacking pPHDD1 only produce PhlyC and PlpV. Dly is a phospholipase-D active against sphingomyelin (Kreger et al., 1987) and PlpV is believed to be a phospholipase-A2 (Osorio et al., 2018), whereas PhlyP and PhlyC are pore-forming toxins (Rivas et al., 2015b). These four toxins are secreted via the type II secretion system (Rivas et al., 2015a;Vences et al., 2017). The highest virulence for fish is believed to be due to the additive functions of PhlyP plus PhlyC, and to the synergistic effect that both Dly and PlpV exert with the pore-forming toxins PhlyP and PhlyC (Rivas et al., 2013, 2015b;Vences et al., 2017). Strains with pPHDD1 exhibit wide hemolytic haloes on sheep blood agar plates whereas plasmidless strains cause narrow hemolytic haloes, and the two types of strains can be distinguished by this phenotypical test. Experimental inoculations have clearly demonstrated that strains harboring pPHDD1 are more virulent than plasmidless strains (Terceti et al., 2016;Vences et al., 2017). Studies conducted before the discovery of pPHDD1 had already suggested that fish farm outbreaks could be caused by the two types of strains (with wide and narrow hemolytic haloes, respectively) coexisting in the fish samples (Pedersen et al., 2009;Labella et al., 2010). Later studies proved that pPHDD1 occurs only within a fraction of P. damselae subsp. damselae strains (Rivas et al., 2014). A recent study has revealed that the P. damselae subsp. damselae populations which caused outbreaks in sea bass fish farms in the Turkish Black Sea lacked this plasmid, and it was also demonstrated that they constituted a multiclonal population with high genetic diversity (Terceti et al., 2016). During the summer seasons of 1994 and 1995, P. damselae subsp. damselae was isolated for the first time as causative agent of outbreaks in rainbow trout fish farms, in Denmark (Pedersen et al., 1997). The outbreaks were coincident with periods of water temperatures up to 5◦C higher than normal, from the beginning of July until mid-August. A few years later, in 2006, the Danish rainbow trout fish farms were again the scenario of P. damselae subsp. damselae outbreaks during an unusually warm summer season (Pedersen et al., 2008, 2009). The epidemiological analyses of strains from these 3 years uncovered a high genetic heterogeneity. Among six strains from 1994, three distinct ribotype patterns were identified, and the nine strains from 1995 yielded four distinct ribotypes, which were in turn different from those of the 1994 outbreaks (Pedersen et al., 1997). Notably, the analysis of 16 strains from the 2006 outbreaks revealed that each strain had a distinct PFGE pattern (Pedersen et al., 2009), providing sound evidence of a high genetic heterogeneity in the populations causing the outbreaks. These previous studies had not analyzed the distribution of virulence-related genes, since genetic diversity was evaluated by DNA-fingerprinting techniques. Nevertheless, the hemolytic phenotypes clearly differentiated a group of strains with strong hemolytic activity from a group of strains with weak hemolytic activity, and it was found that strongly hemolytic strains were 10,000 times more virulent (differences in four logarithmic units in the LD50) than the weakly hemolytic strains (Pedersen et al., 2009) (Table 1). Isolates from the rainbow trout outbreaks constitute a fantastic biological sample for analysis of genetic diversity in this pathogen, since they all come from the same fish host and the same area. In the present study, we have undertaken an in-depth genetic study of these 31 strains, and found evidence that different P. damselae subsp. damselae genotypes coexisted at the same time causing the outbreaks. Analysis of the whole genome sequences of four selected strains revealed a massive genetic heterogeneity. A number of mobile elements including pPHDD1 plasmid, putative prophages, as well as other virulence-related gene clusters and CRISPR-Cas systems showed a differential presence among isolates. From these results it is concluded that P. damselae subsp. damselae outbreaks can be caused by multiclonal populations rather than by specialized clonal lineages, and horizontal gene transfer has played a major role in shaping the genetic diversity within this subspecies. MATERIALS AND METHODS Bacterial Strains and Culture Conditions In two previous studies, a total of 31 P. damselae subsp. damselae strains were collected from head kidneys of diseased rainbow trout (Oncorhynchus mykiss) at several fish farms in Denmark (Pedersen et al., 1997, 2008). In 1994, six isolates were collected from six fish from two different farms; in 1995, nine isolates from nine fish from three different farms; and in 2006, 16 isolates from a total of seven different fish farms (Table 1). Strains were grown on tryptic soy agar or broth, supplemented with 1% NaCl (TSA-1 and TSB-1, respectively) and cultured at 25◦C. Hemolysis and Motility Assays Hemolysis assays on agar plates were conducted by picking a colony of each isolate previously grown on TSA-1, and inoculating it on sheep blood agar plates (Oxoid). For swimming motility assays, single isolated colonies of a 18 h culture agar plate for each strain were picked with a sterile plastic tip and stabbed into motility agar, which was prepared with TSB-1 broth supplemented with 0.25% bacteriological agar. For hemolysis and motility assays, pictures were taken at 24 h post-inoculation of the plates. Experiments were repeated three times to ensure that the hemolytic haloes and motility radius of the strains were reproducible. Assays for Phospholipase and Gelatinase Activities The phospholipase/lecithinase activity was assayed using agar plates supplemented with egg yolk emulsion as a lecithin Frontiers in Microbiology | www.frontiersin.org 2September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 3 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae TABLE 1 | Photobacterium damselae subsp. damselae strains used in this study, isolated from head kidney of rainbow trout (Oncorhynchus mykiss) in Denmark. Strain (short code) Strain (original code) Farm Year of isolation LD50 ∗pPHDD1 plasmid § Phobalysin C (hlyAch gene) Sucrose phenotype on TCBS U Collagenase (colP gene) Reference DK2 940804-1/1 A 1994 − + G−Pedersen et al., 1997 DK3 940804-1/2 A 1994 + + G−Pedersen et al., 1997 DK4 940804-2/1a B 1994 + + G−Pedersen et al., 1997 DK5 940804-2/3 B 1994 + + G−Pedersen et al., 1997 DK6 940804-2/4 B 1994 + + G−Pedersen et al., 1997 DK7 940804-2/5a B 1994 + + G−Pedersen et al., 1997 DK8 950810-3/2 C 1995 − + G−Pedersen et al., 1997 DK9 950810-3/4 C 1995 − + G−Pedersen et al., 1997 DK10 950810-3/5 C 1995 + + G−Pedersen et al., 1997 DK11 950823-1/3b D 1995 − + G+Pedersen et al., 1997 DK12 950823-1/5 D 1995 − + G+Pedersen et al., 1997 DK13 950825-2/4a E 1995 − + G−Pedersen et al., 1997 DK14 950828-1/3 D 1995 − + G−Pedersen et al., 1997 DK15 950901-2/2b E 1995 − + G+Pedersen et al., 1997 DK16 950901-2/5b E 1995 − + G+Pedersen et al., 1997 DK18 206308-4 N/A 2006 3.6 ×104+ + G−Pedersen et al., 2009 DK19 206306-2 N/A 2006 + + G−Pedersen et al., 2009 DK20 206328-2 N/A 2006 3.9 ×103+ + G−Pedersen et al., 2009 DK21 206306-6 N/A 2006 + + G−Pedersen et al., 2009 DK22 206328-5 N/A 2006 + + G−Pedersen et al., 2009 DK23 206303-14 N/A 2006 − + G−Pedersen et al., 2009 DK24 206302-7 N/A 2006 + + G−Pedersen et al., 2009 DK25 206320-5 N/A 2006 − + G+Pedersen et al., 2009 DK26 206276-1 N/A 2006 1.5 ×108− − G+Pedersen et al., 2009 DK27 206302-2 N/A 2006 − − G+Pedersen et al., 2009 DK28 206266-1 N/A 2006 − − G+Pedersen et al., 2009 DK29 206317-1 N/A 2006 + + G+Pedersen et al., 2009 DK30 206303-1 N/A 2006 1.5 ×107− + G+Pedersen et al., 2009 DK31 206308-1 N/A 2006 − + G+Pedersen et al., 2009 DK32 206352-6 N/A 2006 − + Y−Pedersen et al., 2009 DK33 206351-4 N/A 2006 − + G−Pedersen et al., 2009 N/A, not available. ∗LD50 for rainbow trout expressed in cfu, data from Pedersen et al. (2009).§pPHDD1 detection included PCR for dly and hlyApl genes and for pPHDD1 oriV. UG, green colony; Y, yellow colony. Frontiers in Microbiology | www.frontiersin.org 3September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 4 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae source. Ten microliters of TSB-1 overnight cultures for each P. damselae subsp. damselae strain were spotted onto TSA-1 plates supplemented with 3% egg yolk extract (Oxoid), and results were evaluated after 24 h of culture at 25◦C. Hydrolysis of lecithin by the phospholipase yields water-insoluble diglycerides that cause the appearance of an opaque precipitate. The gelatinase activity assay was carried out by spotting 10 µl of a TSB1 overnight culture onto TSA-1 plates supplemented with 1% gelatin (Oxoid), and results were developed after 48 h of incubation at 25◦C by covering the agar plate surface with a 12.5% (wt/vol) HgCl2solution. Hydrolysis of gelatin by the gelatinase enzyme causes the appearance of a translucent halo around the bacterial colony upon addition of HgCl2. Penicillin MIC Assay To determine the susceptibility to penicillin, exponentially grown cultures of isolates DK2, DK3, DK20, and DK29 were adjusted to an OD600 of 0.5 and seeded onto TSA-1 plates in the presence of E-test gradient benzylpenicillin strips (bioMérieux). PCR Relevant PCR primers used in this study are listed in Supplementary Table S1. PCR reactions were routinely performed with Kapa Taq DNA polymerase (Kapa) using a T-gradient thermocycler (Biometra). Routinely, the following thermal cycling conditions were used: 95◦C for 5 min, followed by 30 cycles of 95◦C for 30 s, 52.5◦C for 30 s and an elongation step of 1 min at 72◦C per kb. Molecular Phylogenetic Analysis Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). The evolutionary history of the strains was inferred using the Neighbor-Joining method (Saitou and Nei, 1987), and the analysis involved 31 toxR gene nucleotide sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. Genome Sequencing Genomic DNA of strains DK2, DK3, DK20, and DK29 was purified using the GNOME DNA kit (Q-biogene), and sequenced using an Illumina MiSeq sequencer with 100×coverage. Reads were trimmed for quality, adapters and ambiguous nucleotides, and were assembled using SPAdes 3.6 (Nurk et al., 2013). Draft genome sequences were annotated and compared with the Rapid Annotations using Subsystems Technology (RAST Server) (Aziz et al., 2008). For the comparative analysis and the identification of common vs. specific genes among strains, putative orthologous genes were defined as reciprocal best hit proteins with a minimum 90% identity. Search of acquired antibiotic resistance genes (ARGs) was carried out through the four assembled genomes using the pipeline ResFinder (version 2.1) (Zankari et al., 2012) available at the Center for Genomic Epidemiology1. The threshold value for presence of an ARG was set to 50% similarity expressed as percent sequence identity (ID) and 60% of alignment length (coverage) of resistance gene. A total of 15 categories of ARGs were assayed, which included the following antimicrobials: Aminoglycosides; Betalactams; Colistin; Fluoroquinolones; Fosfomycin; Fusidic acid; Glycopeptides; MLS-Macrolide, Lincosamide and Streptogramin B; Nitroimidazole; Oxazolidinone; Phenicol, Rifampicin; Sulfonamide; Tetracycline; and Trimethoprim. Accession Numbers DNA sequences have been deposited in GenBank database under accession numbers: PVXF00000000 (genome of strain DK2), PVXG00000000 (genome of strain DK3), PVXH00000000 (genome of strain DK20), and PVXI00000000 (genome of strain DK29). RESULTS A toxR Gene-Based Phylogenetic Analysis Provides Evidence for a Multiclonal Origin of the P. damselae subsp. damselae Strains Associated With Rainbow Trout Outbreaks In previous studies (Pedersen et al., 1997, 2008, 2009), P. damselae subsp. damselae was isolated as the causative agent of disease in marine rainbow trout farms in Denmark (Table 1). These studies revealed a lack of clonality among the strains, which exhibited a high diversity in their ribotype and PFGE patterns, suggesting that rainbow trout outbreaks were caused by genetically heterogeneous populations of P. damselae subsp. damselae. Albeit all the strains had been clearly assigned to P. damselae subsp. damselae by phenotypical tests in the aforementioned three previous studies, we wanted here to corroborate their taxonomic affiliation by testing for the presence of conserved gene markers. We proved that all the strains yielded positive amplification of the subspecies-specific ureC gene encoding a subunit of urease enzyme (Osorio et al., 2000), and all tested positive for the rstAB genes encoding a two-component regulatory system recently characterized in P. damselae subsp. damselae (Terceti et al., 2017) (data not shown). To demonstrate the hypothesis of the multiclonal origin of the Danish rainbow trout strains, here we PCR-amplified and sequenced the complete toxR gene in the 31 strains, and carried out a phylogenetic analysis. The toxR gene, which encodes a transmembrane transcriptional regulator of virulence genes, is considered a highly valuable molecular clock for fine-tuned discrimination of taxa within the Vibrionaceae due to its high variability (Osorio and Klose, 2000). As a result of the toxR-based analysis, we found that the 1994 outbreaks were caused by at least two different clones of P. damselae subsp. damselae (Figure 1), represented by strain DK2 on the one side, and DK3 to DK7 1http://cge.cbs.dtu.dk/services/ Frontiers in Microbiology | www.frontiersin.org 4September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 5 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae FIGURE 1 | Phylogeny of 31 P. damselae subsp. damselae strains isolated from outbreaks in Danish rainbow trout farms. Neighbor-joining tree based on the alignment of complete toxR gene sequences of 31 strains. Numbers at the nodes indicate bootstrap values (% of 1,000 replicates; only bootstrap values of >70 are shown). The year of isolation (1994, 1995, or 2006) is also indicated. A heatmap illustration is shown to the right of the tree, and includes information regarding the presence of virulence plasmid pPHDD1, virulence genes hlyAch,colP, and plpV, fimbrial gene cluster, twin-arginine (Tat) pathway protein, and three distinct CRISPR-Cas systems, 1, 2, and 3, respectively. Frontiers in Microbiology | www.frontiersin.org 5September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 6 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae on the other side, respectively. Since all fish examined within an outbreak were received and sampled at the same time, the isolation of five strains (DK3-7) with identical toxR sequences likely indicates that at the time of the outbreak one specific genotype proliferated and caused an acute mortality event in farm A and B. The nine strains from the 1995 outbreaks (DK8-16) from three farms depict a completely different landscape, as they are distributed in as many as five clusters in the phylogenetic tree. Interestingly enough, the study by Pedersen et al. (1997) already differentiated these nine strains into four biotypes, and there is almost a perfect correlation with those biotypes and the clusters determined in the present study: strains DK8, 9, and 14 (cluster G in Figure 1) correspond exactly to biotype 5 by Pedersen et al. (1997); DK11 and DK12 (cluster F) are biotype 7; DK15 and DK16 (cluster D) correspond to biotype 8; and, finally, strains DK10 and DK13 (biotype 6 in Pedersen et al., 1997) constitute an exception to the rule as they are distantly located in the toxR tree. Interestingly, the 1995 outbreaks also reveal that they were caused by multiclonal populations of P. damselae subsp. damselae. As an example, strains DK8–DK9 and DK10, isolated from the same farm, have very different toxR sequences and also different gene content (Table 1 and Figure 1). The 2006 outbreaks are represented by 16 strains collected from seven different fish farms, and these strains are distributed along almost all the clusters in the toxR-based phylogenetic FIGURE 2 | Representation of the four different categories of hemolytic phenotypes on sheep blood agar plates exhibited by the P. damselae subsp. damselae strains analyzed in this study: Large halo (LH), medium halo (MH), small halo (SH), and no hemolytic halo (NH). The individual strains and the complete clusters of strains belonging to each hemolytic category are listed at the left side of the pictures. The genotype of each hemolytic category is also detailed. The symbol 9 denotes pseudogene. Frontiers in Microbiology | www.frontiersin.org 6September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 7 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae tree. The first conclusion that can be drawn from the analysis of the 2006 outbreaks is their highly multiclonal nature. Some groups of strains seem to belong to the same genotype, as is the case of DK26-28 and DK20-22. The tree also reveals that some toxR genotypes from 2006 are identical to genotypes previously isolated in 1994 and 1995. However, none of the clones causing outbreaks in 1994 and 1995 became predominant enough as to displace other genotypes, and the 2006 outbreaks were indeed the most genetically diverse. It is also noteworthy that the majority of the clusters in the phylogenetic tree include strains from different outbreaks. P. damselae subsp. damselae Strains Contain Different Virulence Gene Repertoires Currently we know that P. damselae subsp. damselae can produce a number of virulence factors to cause pathogenicity in hosts. The four main virulence factors recognized so far have cytotoxic activity for different cell types (Osorio et al., 2018). Here, we found that the 31 rainbow trout strains could be divided into four distinct categories according to their haloes of β-hemolysis on sheep blood agar: a large β-hemolytic halo (LH) (7 strains), a medium halo (MH) (6 strains), a small halo (SH) (15 strains) or virtually absence of β-hemolytic halo (NH) (3 strains) (Figure 2). In order to ascertain the hemolysin gene content for each type of strain, we PCR tested for presence of each of the three major hemolysins, Dly, PhlyP and PhlyC, the ones that contribute to detectable phenotypes on sheep blood agar. Results demonstrated that 13 out of 31 strains (Table 1) tested positive for the three genes encoding Dly (dly gene), PhlyP (hlyApl gene), and PhlyC (hlyAch gene) hemolysins, and yielded also positive amplification of the pPHDD1 replication origin. These 13 strains, which correspond to the LH and MH strains, thus harbor a pPHDD1like plasmid (Figures 1,2). The totality of the 15 strains with small hemolytic halo (SH) tested positive for hlyAch gene exclusively, and were negative for pPHDD1 replication origin. These strains will be here referred to as “plasmidless” strains. The three nonhemolytic (NH) strains, DK26, DK27, and DK28, tested negative for the complete hlyAch gene but yielded partial amplification products of this gene instead, suggesting the presence of hlyAch pseudogenes. To further examine this possibility, we conducted a PCR amplification and sequencing of the region flanking hlyAch in the 31 P. damselae subsp. damselae strains. As a result, we found that the NH strains contained an IS630-family element inserted within the hlyAch promoter sequence (Figure 3). This IS630 element was inserted at the same base pair (position 153 upstream the ATG start codon of hlyAch) in the three strains, suggesting that they represent clonal colonies. These three strains also have identical toxR gene sequences (Figure 1). Since the fourth P. damselae subsp. damselae toxin, the phospholipase-A2 PlpV, does not produce detectable haloes on sheep blood agar by itself (Vences et al., 2017), we carried out a lecithinase agar test to gain evidence of the production of PlpV. As a result, we found that 13 strains yielded wide haloes, and these were coincident with the strains that tested positive for Dly and for the additional pPHDD1 gene markers, indicating that, as reported in a recent study (Vences et al., 2017), Dly phospholipase is a major contributor to lecithin degradation in this subspecies. The remaining 18 strains produced small lecithinase haloes (Supplementary Figure S1). The 31 strains tested positive for presence of plpV gene (Figure 1). These results are in agreement with the current knowledge that small haloes are caused by PlpV alone, whereas large haloes are the result of the combined lecithinase activities of Dly plus PlpV (Vences et al., 2017). Recently, a collagenase gene colP was reported to provide P. damselae subsp. damselae strains with the ability of degrading gelatin and collagen, and was shown to play a minor role in virulence (Vences et al., 2017). Using a PCR test specific for this gene, we here found that colP tested positive in 20 rainbow trout strains (Table 1 and Figure 1), which also proved to be positive in a gelatinase agar plate assay. The remaining 11 strains tested negative for colP and were also negative for gelatin degradation on plate assays (Figure 4). A PCR analysis of the genetic context upstream and downstream colP gene revealed a conserved gene content in all the strains, with the exception of DK32 that contained an insertion sequence instead of colP gene, without disrupting any of the flanking genes (Figure 4). The intergenic region where colP is inserted overlaps with the transcriptional terminators of the two flanking genes. This observation, together with the finding of a clean insertion of an IS element in DK32, suggests that this genomic spot is prone to DNA acquisition. Additional Phenotypic Tests Also Reveal Heterogeneity of the P. damselae subsp. damselae Strains As shown above, pPHDD1 plasmid and colP genes exhibit differential presence even within strains isolated from the same fish farm within an outbreak. We conducted additional phenotypical tests, which included the ability to degrade sucrose on TCBS agar. Only one strain (DK32) produced yellow colonies on TCBS, with the remaining 30 strains growing as green colonies (Table 1). Of interest was also the heterogeneity observed in the swimming motility phenotypes. Two strains were nonmotile (DK23 and DK31), and the rest exhibited different levels of swimming motility. For the majority of the strains, it was observed that those with identical toxR sequences also exhibited a similar motility phenotype (Supplementary Figure S2). Complete Genome Sequencing of Four Rainbow Trout Strains Uncovers a High Number of Strain-Specific Genes, Potential Virulence Factors, and Mobile Elements To gain an insight into the genomic divergences among strains, we obtained the draft genome sequences of DK2 and DK3 (from a 1994 outbreak in the same farm), DK20 and DK29 (from two different outbreaks in 2006). The general features of the four genomes are shown in Table 2. The genome size values and the %GC were similar to those reported for other strains of this subspecies (Vences et al., 2017). The core genome of the Frontiers in Microbiology | www.frontiersin.org 7September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 8 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae FIGURE 3 | Scheme of the variable genomic regions upstream the hlyAch gene encoding phobalysin C toxin in P. damselae subsp. damselae strains isolated from rainbow trout. The conserved kefA gene is represented as a black arrow, and two conserved tRNA genes are depicted as blue arrows. (A) Two strains DK24 and DK32 contain three extra genes (white arrows) located between a functional hlyAch gene and the two tRNA genes. (B) The three non-hemolytic strains DK26, DK27, and DK28 contain an IS630-family transposase gene inserted within the hlyAch promoter region, abolishing gene transcription and causing the loss of the hemolytic activity. (C) The remaining 28 isolates all are hemolytic and harbor a functional hlyAch gene, and there is no additional DNA between hlyAch and the two tRNA genes. four genomes was established in 3,493 genes. Most notably, each strain proved to contain a large number of specific genes, which ranged from the 330 genes unique to DK29 to the 104 genes unique to DK20 (Table 2). DK2 lacked pPHDD1 plasmid, and the four genomes lacked the large plasmid pPHDD203 encoding a type III secretion system (T3SS), which has been previously reported only in the type strain so far (CIP 102761, GenBank Acc. No. ADBS00000000). Among the strain-specific genes, special attention was paid to DNA sequences encoding functions related to potential mobile DNA, including prophages, plasmids, and others (Table 3). The genome sequence of DK2 revealed a total of 154 unique genes, all absent in the other three sequenced genomes. Most of these genes encoded putative hypothetical proteins, but some of them were annotated as putative phage proteins and restrictionmodification systems. Notably, a contig of 29,856 bp unique to this strain was found to include a group of genes predicted to participate in the synthesis and uptake of the siderophore vibrioferrin (Table 3), a siderophore originally identified in Vibrio parahaemolyticus (Yamamoto et al., 1994). Indeed, recent studies have shown that some strains of P. damselae subsp. damselae produced this siderophore and used it as iron scavenger (Balado et al., 2017;Puentes et al., 2017). Vibrioferrin production showed to be a variable trait in the subspecies, and a recent study has uncovered that many virulent strains do not produce this siderophore and test negative for pvs genes (Balado et al., 2017). We therefore designed two different primer pairs, targeted to the biosynthetic genes pvsB and psvD, respectively, and assayed the presence of these genes among the 31 rainbow trout strains. We found that these genes were exclusively found in DK2 (Figure 1). The observation that the contig containing the vibrioferrin gene cluster also contains a number of insertion sequences and other mobile element-related genes (Table 3), suggests that it might have been acquired by horizontal gene transfer by some P. damselae subsp. damselae lineages. Strain DK3 harbors 140 unique genes, including many phagerelated proteins (Table 3). An interesting feature of this strain is the existence of a putative fimbrial operon of five genes that proved to be absent in the other three genomes (see below). The genome sequence of DK20 showed 104 specific genes, the lowest number among the four genomes analyzed. Most of them accounted for hypothetical proteins, mobile-element related functions and phage proteins (Table 3). Strain DK29 contained the largest number of specific genes, which accounted for a total of 330. Not surprisingly, this strain has the largest genome of the four analyzed (Table 2). The majority of the unique genes were found to be clustered in several large contigs (Table 3). One contig contained a CRISPR-Cas system of the type I-F (see below). Five contigs contained phagerelated genes. Two contigs which accounted for ca. 80 kb of DNA unique to DK29 contained features of plasmid DNA including an IncF-type conjugative system, suggesting that they correspond to one or more putative novel plasmids as these DNA sequences showed little similarity to known sequences (data not shown). CRISPR-Cas Systems Identification of CRISPR-Cas systems in P. damselae subsp. damselae has been neglected in previous studies. The sequencing of four genomes in the present study uncovered several clusters encoding predicted Cas proteins (Figure 5). Strain DK29 contained two different CRISPR-Cas systems of types I-F and I-E, respectively, with the typical signature protein Cas3. The DK20 genome also encoded a I-E type system, virtually identical to that encoded by DK29 genome. Strain DK2 encoded two Cas proteins Frontiers in Microbiology | www.frontiersin.org 8September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 9 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae FIGURE 4 | Gelatine-degrading activity of P. damselae subsp. damselae correlates with presence of colP gene encoding a collagenase within a variable DNA region. Colony pictures at the right side of the panel depict gelatinase activity detection on agar plates. A black arrow points at the border of the translucent gelatine degradation halo. (A) Strains with capacity to degrade gelatine all test positive for internal primers for colP gene (data not shown), which is located in the same conserved genome position in all the isolates. A PCR using the primer pair ups-colp-F and downs-colp-R, designed within the flanking genes VDA_003255 and VDA_003256, respectively, produces an amplicon of 4,456 bp. (B) Gelatinase-negative strains test negative for internal primers of colP gene (data not shown), and yield a smaller amplification fragment of 1,805 bp when tested with ups-colp-F and downs-colp-R primer pair. (C) The colP-negative strain DK32 yields a different amplicon band due to the insertion of a transposase gene between VDA_003255 and VDA_003256. (D) Agarose gel electrophoresis of the three different amplicon sizes produced with primer pair ups-colp-F and downs-colp-R, revealing three different genotypes in the variable region encoding ColP collagenase. of a putative type I-F system. No Cas proteins could be deduced from the genome annotation of DK3. In order to gain an insight into the distribution of each of these three CRISPR-Cas systems among the P. damselae subsp. damselae collection, we designed tailored primer pairs to screen for a number of signature genes. The results uncovered a high genetic heterogeneity, with strains harboring one Cas protein clusters, other isolates harboring the three of them, and two strains testing negative for the three assayed Cas systems (Figure 1). Antibiotic Resistance Genes The four sequenced genomes possessed two conserved genes encoding putative beta-lactamases, which is in agreement with the resistance to penicillin observed in the four isolates (Supplementary Figure S3). These genes appear to be encoded on the chromosomes and not on plasmids, suggesting that they have been part of the P. damselae subsp. damselae genome for long. In a previous study (Pedersen et al., 2009), it was reported that strains DK20 and DK29 exhibited resistance to sulphonamides. However, the search of acquired ARGs using the pipeline ResFinder (version 2.1) (Zankari et al., 2012) and setting the threshold value for presence of an ARG to 50% similarity, yielded negative results for the four genomes sequenced in the present study. Visual inspection of annotation files produced by RAST server also failed to uncover additional ARGs. Moreover, none of the four genomes contained sequences with significant Frontiers in Microbiology | www.frontiersin.org 9September 2018 | Volume 9 | Article 2155 fmicb-09-02155 September 17, 2018 Time: 16:40 # 16 Terceti et al. Molecular Epidemiology of P. damselae subsp. damselae rainbow trout outbreaks harbor at least one of these systems. As a pathogen that also thrives as a free-living bacterium in seawater, P. damselae subsp. damselae lives in close contact with bacteriophages (Novianty et al., 2014;Yamaki et al., 2015), hence the abundance of CRISPR-Cas systems. Not surprisingly, much of the variable DNA content that showed to be specific of each of the four genomes analyzed here contained typical features of prophage DNA. Recently, it has been reported that CRISPR-Cas systems may also play a regulatory role on endogenous genes. Thus, in addition to protecting from phages, these systems are increasingly being recognized as regulators of bacterial virulence (Louwen et al., 2014;Ma et al., 2018). CONCLUSION The results presented here have provided an in-depth picture of the epidemiology of the outbreaks caused by P. damselae subsp. damselae in Danish rainbow trout farms in 1994, 1995, and 2006. An overview of the virulence gene repertoires plus the presence of additional markers has revealed a high degree of genetic variability within this subspecies. In addition, as illustrated in Figure 1, it can be concluded that P. damselae subsp. damselae outbreaks are caused by multiclonal populations. High-virulence (presence of pPHDD1 plasmid) and low-virulence (absence of pPHDD1) strains coexist within an outbreak. Therefore, further research is needed in order to clarify whether pPHDD1negative strains are in fact causative organisms of the disease, or whether they play a secondary role in infection. The variability of polysaccharide biosynthesis genes and other gene markers among strains is overwhelming, and horizontal gene transfer is believed to have played a major role in the diversification of this subspecies, since much of the strain-specific DNA had features related to plasmids, prophages and pathogenicity islands. P. damselae subsp. damselae is a fascinating microorganism, with a high genetic diversity, and constitutes a very good model for studying the role of horizontal gene transfer as a driving force in the evolution of bacterial pathogens. AUTHOR CONTRIBUTIONS MT and CO performed the experiments and wrote the manuscript. MT, AV, XM, and CO performed analysis and interpreted the results. KP and ID provided the strain collection and significantly contributed to data interpretation. CO designed the study and directed the research. All the authors read and approved the final manuscript. FUNDING This work has been supported by grant AGL2016-79738-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant GRC-2014/007 is also acknowledged. MT thanks the Brazilian Ministry of Education and CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior) for a predoctoral fellowship. XM thanks Xunta de Galicia for a predoctoral fellowship. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02155/full#supplementary-material REFERENCES Abdel-Aziz, M., Eissa, A. E., Hanna, M., and Okada, M. A. (2013). Identifying some pathogenic Vibrio/Photobacterium species during mass mortalities of cultured gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) from some Egyptian coastal provinces. Int. J. Vet. Sci. 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