4. Discussion
Lumpfish across hatcheries and deployment sites frequently show signs of systemic bacterial infection, including skin lesions, gill hemorrhages, and bacterial aggregations in lymphoid organs (i.e., spleen, liver, head kidney) [
10]. In the United Kingdom, Iceland, and Norway, several bacterial outbreaks have been reported in lumpfish hatcheries and at cage sites, and the most frequent pathogen detected is
V. anguillarum [
46]. Thus, it is not surprising that this pathogen was found to be present in Atlantic Canada.
V. anguillarum serotypes O1, O2, and O3 are the most prevalent strains among the 23 serotypes currently described [
18,
19]. Agglutination assays indicated that
V. anguillarum J360 is O2, similar to other
V. anguillarum strains isolated from lumpfish infections in the North Atlantic [
46]. The biochemical profile obtained using API20NE showed that
V. anguillarum J360 was able to reduce sugars, urea, and produce indole, suggesting a 99% possibility for
V. fluvialis (
Table S3). Although the biochemical profile did not identify
V. anguillarum J360, its phenotypic characterization is consistent with other
V. anguillarum isolates [
25,
26], except that
V. anguillarum J360 was positive for urease (
Table S4). This result is coincident with the presence of the urease encoding gene in chromosome-II (DYL2_19555). In addition,
V. anguillarum J360 was not able to grow in TCBS-selective media, suggesting susceptibility to bile salts or a poor adaptation to the culture medium (
Table 2) [
47].
V. anguillarum J360 showed a thermo-inducible α-hemolysin activity at 28 °C, but no hemolytic activity was observed at temperatures below 15 °C (
Figure 1E). Hemolytic activity is an important virulence factor for
V. anguillarum [
48]. For instance, severe hemorrhages are a typical clinical sign of
V. anguillarum infection in fish, including lumpfish [
46]. Coincidently, in the current study,
V. anguillarum J360 was shown to be highly virulent in lumpfish, and infected lumpfish displayed severe hemorrhagic symptoms at 5 dpi (
Figure 2B), similar to other strains described in Marco-Lopez et al. 2013. Koch’s postulates for
V. anguillarum J360 showed that lumpfish infected with 1 × 10
6 and 1 × 10
7 CFU/dose reached 100% mortality within 10 dpi at 10 °C (
Figure 2C). In addition,
V. anguillarum was re-isolated from the spleen, liver, and head-kidney, confirming Koch’s postulates. The original
V. anguillarum outbreak in cultured lumpfish and the infection assays in the current study showed similar clinical signs (
Figure 2B).
V. anguillarum hemolytic activity was evident during infection. However, the lumpfish is a cold-water fish typically cultured between 6 and 12 °C [
49]. These results contradicted with the
V. anguillarum thermo-inducible hemolytic activity at 28 °C (
Figure 1E). Perhaps, internal fish conditions (e.g., innate and adaptive immunity) triggered
V. anguillarum hemolytic activity. These results suggest that its regulatory mechanisms need further analysis.
V. anguillarum J360 possesses two chromosomes, a large plasmid and a small plasmid (
Figure 3 and
Table 4).
Vibrio spp. and
V. anguillarum genomes selected for phylogenetic and comparative genomics analysis possess two chromosomes and one large plasmid (
Table 1). Typically, serotype O1 harbors a virulent plasmid, called pJM1 or pJM1-like, and serotypes O2 and O3 strains possess a nonvirulent large plasmid [
50,
51] or they do not harbor large plasmids [
20,
37].
V. anguillarum J360 is a O2 serotype that does not harbor a virulence plasmid (
Figure S5), suggesting that this strain could increase its virulence if a virulence plasmid is acquired.
The total genome size of
V. anguillarum J360 is 4,561,566 bp (
Table 4), which is larger than the currently available
V. anguillarum genomes (
Table 1). This may suggest that
V. anguillarum J360 may have acquired genetic material through horizontal gene transfer and/or adapted to its lumpfish host or to environmental conditions in Atlantic Canada.
Phylogenetic distance based on the whole genome alignment analysis of chromosome-I and chromosome-II showed that
V. anguillarum J360 is closely related to
V. anguillarum VIB43, and distantly related to
V. anguillarum VIB12 (
Figure 4A,B). Interestingly,
V. anguillarum VIB43 and VIB12 were isolated from the same host species, sea bass (
Dicentrarchus labrax), but from different locations.
V. anguillarum VIB43 was isolated in Scotland and
V. anguillarum VIB12 was isolated in the Mediterranean Sea [
20]. The ANI analysis between
V. anguillarum J360 and
V. anguillarum VIB43 showed a 99.93% identity for chromosome-I and 99.95% for chromosome-II (
Figure 4C,D and
Figure S2A,B), which suggest that these two strains share a common ancestor.
Additionally, the whole genome phylogenetic analysis showed that
V. anguillarum J360 and VIB43 are not closely related to
V. anguillarum 775, M3, and NB10, which is contradictory to previous MLSA studies that indicated that
V. anguillarum VIB43 and VIB12 are closely related to those
V. anguillarum strains [
20,
50]. The MLSA computed the phylogenetic stress based on concatenated conserved sequences [
51,
52]. In this study, we used nine conserved housekeeping genes, including 16S rRNA,
ftsZ,
gapA,
gyrB,
mreB,
pyrH,
recA,
rpoA, and
topA (
Table S1). In contrast to the whole genome phylogenetic analysis, the MLSA showed that
V. anguillarum J360 clusters alone, and the closest related strain is
V. anguillarum NB10 isolated from rainbow trout (
Oncorhynchus mykiss) in the Gulf of Bothnia, Sweden [
6], which is distantly related to
V. anguillarum 775 and M3 strains (
Figure S3,
Table 1).
V. anguillarum M3 was isolated from Japanese flounder (
Paralichthys olivaceus) in Shandong, China, and classified as closely related to
V. anguillarum 775 isolated from Coho salmon (
Oncorhynchus kisutch) in the Pacific coast of USA [
7,
8]. By contrast, MLSA phylogenetic analysis based only on the 16S rRNA gene showed that
V. anguillarum NB10 is closely related to
V. anguillarum M3 [
6].
In contrast to the MLSA analysis, the whole genome phylogenetic analysis of
V. anguillarum strains is in concordance with the geographic origin of the strain isolation. For instance, according to the whole genome phylogenetic analysis,
V. anguillarum J360 and
V. anguillarum VIB43, both isolated in the North Atlantic Ocean, are highly related, and closely related to
V. anguillarum strains 90-11-286, S3, and JLL237 isolated in Finland (
Table 1;
Figure 4). Actually, these geographic locations are a natural habitat for lumpfish populations [
53].
In contrast to the whole genome phylogenetic analysis, the MLSA uses protein-coding genes, which evolved at a slow but constant rate, and it could have better resolution, especially at the species level [
51,
52]. However, the selection and number of coding genes, and alignment method, are variable for MLSA. We found that the phylogenetic analysis using whole genomes is more reliable than MLSA, and the robustness of our analyses showed to be consistent with two different software. In addition, whole genome analyses allow homologous regions, deletion, translocation, and inversion events to be identified.
Genome alignment and synteny analysis between
V. anguillarum J360 and
V. anguillarum VIB43 showed a high similarity within the chromosome sequences, but inversions and unmatched regions were also observed (
Figure 5A,B). This suggests that homologous recombination events play an important role in
V. anguillarum evolution, perhaps influenced by insertion sequence (IS) elements such as chromosomal integrons or “super integrons” (SIs) describing
Vibrio spp. and several Gram-negative species [
20]. We determined that there are five LCBs in chromosome-I (
Figure 5C) and two LCBs in chromosome-II (
Figure 5D) shared between
V. anguillarum J360 and VIB43. Further analysis revealed that all the LCBs present in chromosome-I have small inversion events (
Figure 5A,C). In addition, we found that LCBs-1 and -4 have genome gaps (GGs) or unmatching regions in both strains (
Figure 5A and
Figure S4A). The GGs identified in LCB-1 of
V. anguillarum J360 chromosome-I are not present in
V. anguillarum VIB43 LCB-1 (
Figure S4A). These identified GGs possess several genes that encode for IS families transposases (IS66, ISL3, IS3, IS5) and site-specific integrases previously described in the
V. anguillarum VIB43 genome, and with high similarity to
V. anguillarum NB10, 775, and ATCC-6855 genomes [
6,
20]. We found that the unique GGs in LCB-1 of
V. anguillarum J360 possess genes related to iron uptake and iron homeostasis (
Figure S4A), suggesting that these genes could be acquired by horizontal gene transfer.
In
V. anguillarum J360 chromosome-II, two GGs were identified in LCB-1 and LCB-2 (
Figure S4B), and both GGs have an IS630-like element belonging to the ISVa15 transposase family. This IS630-like element is not present in
V. anguillarum VIB43 LCBs. According to the description of Holm et al. (2018), ISVa3–ISVa20 are new insertion sequence (IS) elements in the
V. anguillarum genomic repertory that are responsible for the divergency within strains. This suggests that
V. anguillarum J360 and
V. anguillarum VIB43 could be derived from a common ancestor and adapted to local environmental conditions and host species.
Pathogenesis-associated genes were found in both chromosomes, but chromosome-I harbors most of the virulence genes and their respective transcriptional regulators (
Table 6). No virulence-associated genes were found in the large plasmid pVaJ360-I or in the small plasmid pVaJ360-II. The
V. anguillarum virulence plasmid pJM1 possesses intrinsic virulence genes associated with iron uptake, like anguibactin biosynthesis (
angA-angE,
vabA-E) and anguibactin transport (
fatA-fatD) [
8,
54,
55]. By contrast, all the
V. anguillarum J360 iron homeostasis-related genes are in its chromosomes. For instance, genes related to ferric-anguiobactin and siderophore uptake (e.g.,
exbB and
exbD2, respectively) are present in chromosome-I. Comparative genomic analyses showed that the large virulent plasmids pJM1, P67-NB10, and p65-ATCC have high similarity (
Figure S5A,B). However, the large plasmid pVaJ360-I and the small plasmid pVaJ360-II of
V. anguillarum J360 do not present similarity (
Figure S5A) or identity (
Figure S5B) with other reported plasmid sequences, nor possess virulence-associated genes. This suggests that
V. anguillarum J360 does not harbor plasmids previously described in
V. anguillarum, including serotype O2 strains [
50].
Hemolysins are important virulence factors for
V. anguillarum species, and contribute to its attachment, tissue colonization, and iron homeostasis, thus increasing its pathogenicity [
56,
57].
V. anguillarum J360 has four hemolysin genes, and these are consistent with the hemorrhagic clinical signs observed in lumpfish during the infection assays (
Figure 2B). In addition, a thermolabile hemolysin gene is present in chromosome-II, which can be related to the thermo-inducible hemolytic phenotype of
V. anguillarum J360 (
Figure 1E).
The resistance of
V. anguillarum J360 to ampicillin (
Table 2) relates to the presence of a class C beta-lactamase (
ampC)-encoding gene in chromosome-II. Metalloproteases such as
pmbA,
tldD, and
ftsH genes, which are associated with carbon storage, hydrolysis of peptide bonds, and virulence, were also identified.
V. anguillarum J360 does not possess some of the virulence genes present in
V. anguillarum strains isolated from the Pacific coasts, including the metalloproteases
empA and
prtV [
8,
58]. Nonetheless,
V. anguillarum J360 harbors a M6 family metalloprotease (DYL72_17780) similar to the
prtV gene, associated to gelatinase activity (
Table S3).
In addition, genes that encoded for secreted enzymes such as phospholipase and lipases were found in chromosome-II, which correlates with the lipase (C
14)-positive phenotype observed in the enzymatic profile (
Table S3).
V. anguillarum J360 possesses several genes associated with flagella and motility, such as the operons
fliRQPONMLKJIHGFE (DYL72_03140-DYL72_03205),
flgLKJIHGFEDCB (DYL72_03685-DYL72_03735), and
motYBA (DYL72_12660, DYL72_00275, DYL72_12090) located in chromosome-I, which is consistent with the mot
+ phenotype of
V. anguillarum J360 (
Table 2).
Virulence factors present in other
V. anguillarum genomes (e.g.,
V. anguillarum 775 and
V. anguillarum M3) were not identified in the
V. anguillarum J360 genome. These include mannose-sensitive hemagglutinin type 4 pilus (MHSA) [
8,
54],
vstA-
vstH genes for a Type VI secretion system [
55], and
virA-
virB genes related to lipopolysaccharides synthesis [
58].
In concordance with the locations of the virulence factors, transcriptional regulators such as
luxR,
lysR,
cysB,
nhaR, and
hfq were mostly located in chromosome-I, and an additional copy of
luxR was found in chromosome-II. LuxR belongs to a transcriptional activators family, that together with an N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), mediates the signal transduction mechanisms of quorum-sensing genes such as
luxICDABE operon [
59]. The duplication of
luxR in
V. anguillarum J360 suggests that quorum-sensing plays an important role in the biology of this strain.
Genomic Islands (GIs) have been identified in
V. anguillarum species, for instance,
V. anguillarum NB10 possesses 29 GIs [
6],
V. anguillarum 775 possesses 10 GIs [
8], and
V. anguillarum J360 has 21 GIs (
Figure 6). We found that GI-19 in chromosome-I (2,994,299..3,011,196 nt) of
V. anguillarum NB10 [
6] has some homologous regions with
V. anguillarum J360 GI-14 in chromosome-I (2,988,473..3,000,617 nt). In addition, we determined that GI-25 (546,066..578,220 nt) and GI-26 (639,559..674,888 nt) in chromosome-II of
V. anguillarum NB10 [
6] share similar genetic context with GI-18 (552,330..612,508 nt) and GI-19 (622,682..673,456 nt) in chromosome-II of
V. anguillarum J360, respectively. However, the low similarity between GIs of
V. anguillarum J360 and
V. anguillarum NB10 suggests a relatively distant relationship, consistent with the whole genome phylogenetic analysis (
Figure 4).
In contrast, 19 GIs were identified in
V. anguillarum VIB43 (
Figure S6, Supplementary files 3 and 4), which showed high similarities with the GIs founded in
V. anguillarum J360. For instances, GI-1, GI-4, and GI-12 present in
V. anguillarum VIB43 chromosome-I (
Figure S6) possess several homologous regions with GI-8, GI-10, and GI-3 of
V. anguillarum J360 chromosome-I (
Figure 6), respectively (
Supplementary files 1 and 3). In addition, GI-6 shared homologous regions with GIs 11 and 12; however, all these regions are flanked by IS66 or IS66-like family transposases (ISVa9, ISVa15, ISVa11), suggesting that these regions are hot spots for recombination events (
Supplementary files 1 and 3). GI-12 of
V. anguillarum VIB43 and GI-3 of
V. anguillarum J360 are highly conserved (
Supplementary 1 and 3). Similar results were observed in chromosome-II. GI-15 and GI-16 of
V. anguillarum VIB43 (
Figure S6) showed several homologous regions with GI-16 and GI-17 of
V. anguillarum J360 (
Figure 6), respectively (
Supplementary files 2 and 4). GI-17 of
V. anguillarum VIB43 has several homologous regions with GIs-18 and GI-19 of
V. anguillarum J360 (
Supplementary files 2 and 4). The homologous regions between GIs-17 of
V. anguillarum VIB43, and GI-18 and GI-19 of
V. anguillarum J360 encode for several virulence factors like lipocalin, Hcp tube protein (T6SS), interferase toxin, damage-inducible protein J, secretion proteins, and toxins. These homologous regions are flanked by several IS elements (
Supplementary files 2 and 4), which indicates that these three GIs are genomic pathogenic islands, perhaps acquired through horizontal gene transference [
60]. These results support the hypothesis that these IS elements (IS66, ISL3, IS3, IS5)
6 are responsible for the genomic gaps (GGs) and genomic rearrangements previously mentioned, as well as support the 0.05–8% of genomic differences observed at the identity analyses.