The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms

Figures & data

Figure 1. V. vulnificus phylogeny. The phylogenetic tree was reconstructed using the maximum-likelihood method and the generalized time-reversible model (GTR + F+R5) of evolution. Bootstrap support values from 1000 replicates are indicated in the corresponding nodes as percentages. L, lineage.

Figure 2. Plasmids in pv. piscis lineages and clades. The genes for conjugative transference, the survival in fish blood kit (ftpb and fpcrp), and the MARTX toxin are represented in green, red, and orange respectively, while the additional genes common to all pv. piscis plasmids are represented in brown. (A) Linear comparison among pCladeT and the original pv. piscis plasmids, pFv and pConj, performed with Easyfig [38]. (B) Ring representation of the pv. piscis plasmids from the clades and lineages emerged in the Eastern Mediterranean. From inside to outside, pL3 (used as reference; black ring), pClade A, pCladeT y pL5. The gene annotation of the pL3 is represented in the multicoloured external ring.

Table 1. Isolation data, lineage and identification of the new isolates in comparison with those of the control strains.

	Isolation data			Identification^d
Strain^a	Source^b	Geographic location/year of isolation	Phylogenetic Lineage^c	Species PCR/Api20E	Pathovar/clade E	Public Health Hazard
New isolates
VV3, VV4, VV5	Diseased tilapia	Eastern Mediterranean /2016	?	+/+ (99.3%)	-/-	+
TI417	Diseased tilapia	Eastern Mediterranean /2019	?	+/+ (99.3%)	-/-	+
Control strains
YJ016	Human blood	Asia/1993	L1	+/+ (99.3%)	-/-	+
CECT 529^T	Human blood	USA/1980	L2	+/+ (99.3%)	-/-	+
CECT 4999	Diseased eel	Europe/1999	L2/clade E	+/¿? (54.4%)	+/+	+
CECT 5198	Diseased eel	Europe/2000	L2/clade A	+/+ (99.3%)	+/-	-
95-8-161	Diseased eel	Europe/1995	L2/clade I	+/+ (99.3%)	+/-	+

^a CECT, Spanish Type Culture Collection; T, type strain.

^b All the new isolates were recovered as pure cultures from internal organs of moribund tilapia. VV3 was isolated from farm A, VV4 and VV5 from farm B and TI417 from farm C.

^c Phylogenetic lineage determined by Roig et al [11]. L1 includes biotype 1 strains, L2 biotypes 1 and 2 strains and L3 biotype 3 strains. L2-Clade E includes all the zoonotic strains reported to date.

^d Identification of the isolates was performed at species, pathovar (piscis) and zoonotic clade (clade E) level by PCR targeting vvhA [20], fpcrp (formerly vep07) [21] and seq 61 [21], respectively. Positive (+), negative (-), and doubtful (¿?) identification. The value in parentheses indicates the probability of a good identification according to the API20E profile (5146105, probability 99.3%; 5006005, probability 54.4%).

^e The public health hazard of the strain was determined by PCR targeting a polymorphism of pilF [22]. Discrimination is based on the amplification of a variable region located within the gene pilF resulting in a 338 bp fragment.

Table 2. Results obtained in the in vivo and ex vivo virulence assays performed with the new isolates in comparison with those of the control strains.

	Resistance to ^a		Virulence for^b
			Tilapia		Mouse
Strain	TP	HS + Fe	i.p. injection	Immersion	i.p. injection
Tilapia isolates
VV3	+ (9.1 × 10^3%)	+ (7.0 × 10^2%)	+ (3.3 × 10^6)	+ (3.3 × 10^5)	+ (1.5 × 10^6)
VV4	+ (8.3 × 10^3%)	+ (1.1 × 10^3%)	+ (8.4 × 10^5)	+ (8.4 × 10^5)	+ (9.2 x 10^5)
VV5	+ (8.5 × 10^2%)	+ (3.1 × 10^3%)	+ (2.5 × 10^6)	+ (4.2 × 10^5)	+ (2.0 × 10^6)
TI417	+ (9.5 × 10^2%)	+ (1.7 × 10^3%)	NT	+ (1.0 × 10^6)	NT
Control strains
CECT 4999	+ (8.5 × 10^2%)	+ (1.9 × 10^3%)	- (> 10^7)	- (> 10^8)	+ (1.0 × 10^6)
YJ016	-(0%)	+ (4.9 × 10^3%)	- (> 10^7)	- (> 10^8)	+ (1.0 × 10^6)
CECT 529^T	-(0%)	+ (5 × 10^2%)	- (> 10^7)	- (> 10^8)	- (> 10^7)

^a Resistance to tilapia plasma (TP) and iron-overloaded human serum (HS+ 10 μM of FeCl₃ [13]) after 4 h at 28°C (tilapia) or 6 h at 37°C (human) is coded as follow: +, survival ≥100%; -, survival <100%. Data in parentheses correspond to the medium percent survival from three independent experiments.

^b The medium value of 50% lethal dose (LD₅₀) from two different experiments is presented in parentheses as cfu/g (i.p. injection) or /ml (inmersion). Results are coded as follows: fish and mouse injection; - (≥10⁷), + (< 10⁷); fish immersion; - (≥10⁸) + (< 10⁸) [10, 24].

NT, not tested.

Figure 3. rtxA1 gene structure. (A) Schematic representation of rtxA1 gene from pv. piscis L2-clade E and L1-clade T (rtxA1 _{L2-clade E} and rtxA1_{L1-clade T}) (B) Comparison among the rtxA1 genes of the clades and lineages emerged in the Eastern Mediterranean. The images were constructed with Easyfig and the blue scale indicates nucleotide Blast similarity. Carboxi and amino terminal modules are coloured in grey, cysteine protease domain (CPD) in turquoise, Domain X (DmX) in pink, ExoY-like adenylate cyclase domain (ExoY) in black, makes caterpillars floppy-like domain (MCF) in orange, alpha/beta hydrolase domain (ABH) in purple, Rho GTPase-inactivation domain (RID) in green and domain of unknown function at the first position (DUF1) in yellow.

Table 3. Genotypic and phenotypic characterization of selected strains from previously described lineages and/or clades possessing a pFv-related plasmid.

Lineage/clade^a	Selected strain	Isolation data^b		API20E profile^c	PCR for^d:					Serology^e
		Source	Year		species	pv. piscis	clade E	ftbp	PHH	SerE	SerA	SerI	SerO	SerT
L1-clade A	yb158	Healthy tilapia	2005	5146105	+	+	-	+	+	-	-	-	-	+
	V246	Human blood	2005	5146105	+	+	-	+	+	-	-	-	-	+
L3	12	Human blood	1996	4146005	+	+	-	+	+	-	-	-	+	-
L5	V252	Human blood	2004	5346005	+	+	-	+	-	-	-	-	-	-

^a Phylogenetic lineage determined by Roig et al [11]. L3 and L5 are clonal complexes.

^b Data of isolation for the selected strain

^c Probability of identification of V. vulnificus: 4146005, 75.5%; 5146105, 99.3%.

^d The target genes for PCR were: vvhA, species; fpcrp, pv. piscis (PCR designed in this work); seq61, zoonotic clade E [21]; ftbp, fish transferrin binding protein [12]; a pilF polymorphism, public health hazard (PHH) [22]. Discrimination is based on the amplification of a variable region located within the gene pilF resulting in a 338 bp fragment.

^e +, AgO agglutination in less than 1 min and ELISA titter (the reciprocal of the highest dilution of the antiserum giving OD two times higher than that of the negative control) higher than 60,000. -, no agglutination and ELISA titer lower than 15,000.

Table 4. In vivo and ex vivo virulence assays performed with selected strains from previously described lineages and/or clades possessing a pFv-related plasmid.

Lineage/ clade^a	Selected strain	Resistance to^b		Virulence for^c
		TP	HS + Fe	Tilapia
L1-clade A	yb158	+ (5.2 × 10^3%)	+ (4.0 × 10^3%)	+ (2.5 × 10^7)
	V246	+ (1.5 × 10^3%)	+ (1.6 × 10^3%)	NT
L3	12	+ (7.1 × 10^3%)	+ (7.3 × 10^2%)	+ (1.3 × 10^7)
L5	V252	+ (9.2 × 10^2%)	+ (7.7 × 10^2%)	+ (2.5 × 10^6)

^a Phylogenetic lineage determined by Roig et al [11]. L3 and L5 are clonal complexes.

^b Resistance to tilapia plasma (TP) and iron-overloaded human serum (HS+ 10 μM of FeCl₃ [13]) after 4 h at 28°C (tilapia) or 6 h at 37°C (human) is coded as follow: +, survival ≥100%; -, survival <100%. Data in parentheses correspond to the medium percent survival from three independent experiments.

^c Virulence was determined by immersion challenge. The medium value of 50% lethal dose (LD₅₀) from two different experiments is presented in parentheses as cfu/ml. Results are coded as follows: - (≥10⁸) +(< 10⁸) [10, 24].

NT, non tested.

Figure 4. Evolutionary history of the genes for the “survival in fish blood kit”. Molecular phylogenetic analysis of ftbp (A) and fpcrp (B) was performed using the maximum likelihood method based on the Tamura 3-parameter model [47]. The tree is drawn to scale, with branch lengths measured as the number of substitutions per site. The main host (tilapia or eel) is shown in each tree.

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