Genome analysis of clinical genotype Vibrio vulnificus isolated from seafood in Mangaluru Coast, India provides insights into its pathogenicity

Figures & data

Table 1. Summary of Vibrio vulnificus genomes used in the study.

Sl. No.	Strain name	Country of origin	Year of isolation	Source of isolation	GenBank Accession No.
1	93U204	Taiwan	2004	Tilapia, diseased	GCA_000746665
2	95-8-6	Denmark	1995	Eel	PDNE00000000
3	95-8-7	Denmark	1995	Eel	PDGM00000000
4	95-8-161	Denmark	1995	Eel	PDGN00000000
5	960426-1_4C	Denmark	1996	Eel	PDGL00000000
6	ATCC 27562	USA	1979	Estuarine	GCA_002224265
7	BAA87	Israel	1996	Wound infection	JDSE00000000
8	CG64	Taiwan	2011	Seawater	JZEU00000000
9	CICESE 316	Mexico	2011	Oyster	QKYO00000000
10	CICESE 325	Mexico	2011	Oyster	QKYP00000000
11	CMCP6	South Korea	2016	Clinical	GCA_004355205
12	E64MW	UK	–	Environmental	AFSX00000000
13	FORC_017	South Korea	2018	Clinical—blood	GCA_001675245
14	JY1305	USA	–	Oyster	AFSW00000000
15	JY1701	UK	–	Environmental	AFSY00000000
16	LSU1015	USA	–	Clinical—wound	PDNQ00000000
17	LSU1657	USA	–	Clinical—wound	PDFZ00000000
18	LSU2098	USA	–	Clinical—wound	PDFY00000000
19	MO6-24/O	South Korea	–	Human	GCA_000186585
20	SC9629	South Korea	2011	Seafood	JYGG00000000
21	SC9740	South Korea	2011	Seawater	JZEP00000000
22	SC9761	South Korea	2011	Seafood—oyster	JZEO00000000
23	YJ016	Taiwan	–	Clinical	GCA_000009745
24	E4010 (This study)	India	2018	Oyster	WRPB00000000

Figure 1. (A) Heat-map of Euclidean distance matric calculated based on percentage values of average nucleotide identity (ANI) values of different strains of Vibrio vulnificus reveals two major groups. The strain sequenced in this study is highlighted in the red box. Details of genomes retrieved from NCBI GenBank are provided in Table 1. The values indicate the extent of dissimilarity from dark pink to green color; (B) MLST based phylogenetic analysis of V. vulnificus genomes and details of their country, source, and ST shows differential branching pattern based on vcg alleles. (vcg-C—red; vcg-E—green). the newly sequenced isolate is marked with ‘a star’; (C) Core genome-based phylogenetic analysis of V. vulnificus genomes showing two distinct lineages based on vcg alleles (vcg-C marked in red and vcg-C marked in green color).

Table 2. Summary of the (A) assembly and annotation; (B) virulence genes identified by VFDB; (C) antimicrobial resistance genes identified by PATRIC in the genome of Vibrio vulnificus E4010 genome.

(A)
Sl.No	Attributes             Values
1.	Genome size	4,875,404 bp
2.	Total number of reads	2,701,430
3.	Contigs	87
4.	Maximum contig size	429,948 bp
5.	N50	250,841 bp
6.	G + C content (%)	46
7.	Coding sequences	4400
8.	tRNA	102
9.	rRNA	29
10.	ncRNA	4
11.	NCBI accession number	WRPB01000000
12.	MLST sequence type	ST-570
13.	ANIb % (against CMCP6)	98.17
14.	ANIb % (against YJ016)	98.00
15.	dDDH % (against CMCP6)	90.5
16.	dDDH % (against YJ016)	89.4
(B)
VF class	Virulence factors	Related genes identified in E4010
Adherence	Mannose-sensitive hemagglutinin (MSHA type IV pilus), Type IV pilus	mshA–mshN, pilB–pilD
Antiphagocytosis	Capsular polysaccharide	cpsA–cpsD, cpsF, cpsH–cpsJ, hp1, wbfV/wcvB, wecA, wza–wzc
Chemotaxis and motility	Flagella	cheA, cheB, cheR, cheV, cheW, cheY, cheZ, lag, flaA–flaC, flaE, lag, flaI, flgA–flgN, flhA, flhB, flhF, flhG, fliA, fliD–fliL, fliN–fliS, flrA–flrC, motA, motB, motX, motY
Enzymes	Metalloproteases	hap/vvp
Iron uptake	Heme receptors, periplasmic binding protein-dependent ABC transport systems, vibriobactin biosynthesis, vibriobactin utilization	hutA, hutR, vctC, vctD, vctG, vctP, viuC, viuD, viuG, viuP, vibA–vibF, vibH, viuA, viuB
Quorum sensing	Autoinducer-2	luxS
Secretion system	EPS type II secretion system	epsC, epsE–epsN, gspD
Toxin	Hemolysin/cytolysin, RTX toxin, Thermolabile hemolysin	vvhA, rtxA–rtxD, tlh
(C)
AMR mechanism	Related genes identified in E4010
Antibiotic activation enzyme	katG
Antibiotic inactivation enzyme	catB family
Antibiotic target in susceptible species	alr, ddl, dxr, EF-G, EF-Tu, folA, dfr, folP, gyrA, gyrB, Iso-tRNA, kasA, murA, rho, rpoB, rpoC, s10p, s12p
Antibiotic target protection protein	qnrB family
Antibiotic target replacement protein	fabV
Efflux pump conferring antibiotic resistance	mdtL, Tet (35), tolC/opmH
Gene conferring resistance via absence	gidB
Protein altering cell wall charge conferring antibiotic resistance	gdpD, pgsA
Regulator modulating expression of antibiotic resistance genes	H-NS, oxyR

Figure 2. Schematic representation of virulence and fitness genes identified in Vibrio vulnificus E4010.

Figure 3. (A) BLAST atlas for chromosome 1 and 2 of Vibrio vulnificus E4010 generated using BRIG with CMCP6 as reference; (B) Schematic representation of Multifunctional autoprocessing repeats-in-toxin (MARTX) gene clusters in V. vulnificus.

Figure 4. Basic PPI network of Vibrio vulnificus E4010 constructed using STRING consisting of 375 nodes and 1606 edges with clusters highlighted.

Figure 5. (A) the degree distribution shows a scale-free topology and measures up to a power law; (B) the average path length distribution (APL = 3.905) of the Vibrio vulnificus E4010 secretory network.

Table 3. The nodes with BC and/or degree values more than the mean plus standard deviation, and the majority of the proteins/nodes in the secretome network of Vibrio vulnificus E4010.

Name	BetweennessCentrality	ClosenessCentrality	ClusteringCoefficient	Degree
VV93_v1c37740	0.052259	0.370297	0.418357	46
VV93_v1c08230	0.031516	0.364522	0.440244	41
VV93_v1c15230	0.016923	0.350187	0.479082	39
VV93_v1c38150	0.012309	0.350187	0.489879	39
VV93_v1c00420	0.011222	0.354167	0.508772	39
VV93_v1c34010	0.033399	0.3692	0.422475	38
VV93_v1c14430	0.030783	0.358238	0.41394	38
VV93_v1c29010	0.025644	0.350187	0.442943	37
VV93_v1c13620	0.008062	0.347261	0.539039	37
VV93_v1c28640	0.007351	0.345656	0.522523	37
VV93_v1c45320	0.127174	0.373253	0.106349	36
VV93_v1c41410	0.027762	0.359962	0.418487	35
VV93_v1c11280	0.003939	0.338156	0.604278	34
VV93_v1c09620	0.054645	0.369931	0.4375	33
bamC	0.048895	0.332149	0.387097	31
VV93_v1c21290	0.045545	0.362403	0.367742	31
VV93_v1c23110	0.024793	0.341865	0.6	31
VV93_v1c20740	0.015004	0.332444	0.524631	29
VV93_v1c13550	0.011469	0.327496	0.583744	29
VV93_v1c30830	0.018287	0.329225	0.560847	28
VV93_v1c09230	0.006439	0.330973	0.571429	28
AIL70004.1	0.002905	0.329515	0.701058	28
VV93_v1c27660	0.070245	0.346296	0.169231	26
VV93_v1c15720	0.008373	0.320205	0.658462	26
VV93_v1c20800	0.006278	0.315346	0.649231	26
VV93_v1c28020	0.073653	0.338768	0.16	25
surA	0.038047	0.321306	0.237154	23
VV93_v1c05840	0.010784	0.330973	0.604743	23
VV93_v1c00830	0.007517	0.335727	0.675889	23
VV93_v1c22970	0.007349	0.328936	0.687747	23
VV93_v1c10670	0.007108	0.337545	0.494071	23
VV93_v1c22490	0.006563	0.335125	0.506494	22
luxP	0.016389	0.327209	0.657143	21
VV93_v1c32790	0.005421	0.33156	0.6	21
VV93_v1c33530	0.047597	0.348555	0.126316	20
VV93_v1c33510	0.030682	0.353832	0.242105	20
VV93_v1c04260	0.027533	0.293103	0.163158	20
VV93_v1c31910	0.018138	0.321306	0.231579	20
lolB	0.014632	0.290147	0.257895	20
AIL70622.1	0.00737	0.332149	0.567251	19
flgT	0.007103	0.325784	0.48538	19
lolA	0.006986	0.286371	0.374269	19
VV93_v1c20810	0.004725	0.2992	0.699346	18
VV93_v1c22540	0.034587	0.318298	0.345588	17
VV93_v1c10080	0.02802	0.330681	0.213235	17
VV93_v1c30240	0.011091	0.337545	0.5	17
VV93_v1c06430	0.008978	0.291051	0.397059	17
VV93_v1c11600	0.002581	0.303079	0.838235	17
VV93_v1c06340	3.81E-04	0.309859	0.830882	17

Figure 6. The secretome network of Vibrio vulnificus E4010 contains 10 clusters, ranked in size (number of proteins) and density (interconnectivity). Sixty-three (about 18%) of the network’s 575 total proteins are found in clusters.

Data availability statement

The draft genome of Vibrio vulnificus E4010 was earlier deposited in DDBJ/ENA/GenBank and given the accession WRPB00000000. The version described in this paper is version WRPB01000000. The new MLST profiles of V. vulnificus E4010 (pubMLST id: 775) have also been submitted to the pubMLST database (https://pubmlst.org).