Method for Specific Identification of the Emerging Zoonotic Pathogen Vibrio vulnificus Lineage 3 (Formerly Biotype 3)

Vibrio vulnificus is a zoonotic pathogen that is spreading worldwide due to global warming. Lineage 3 (L3; formerly biotype 3) includes the strains of the species with the unique ability to cause fish farm-linked outbreaks of septicemia. The L3 strains emerged recently and are particularly virulent and difficult to identify. Here, we describe a newly developed PCR method based on a comparative genomic study useful for both rapid identification and epidemiological studies of this interesting emerging group.

ABSTRACT

Vibrio vulnificus is a zoonotic pathogen that is spreading worldwide due to global warming. Lineage 3 (L3; formerly biotype 3) includes the strains of the species with the unique ability to cause fish farm-linked outbreaks of septicemia. The L3 strains emerged recently and are particularly virulent and difficult to identify. Here, we describe a newly developed PCR method based on a comparative genomic study useful for both rapid identification and epidemiological studies of this interesting emerging group. The comparative genomic analysis also revealed the presence of a genetic duplication in the L3 strains that could be related to the unique ability of this lineage to produce septicemia outbreaks.

INTRODUCTION

Vibrio vulnificus is a zoonotic pathogen that inhabits marine and estuarine water ecosystems located in temperate and warm zones (1). Global warming is causing V. vulnificus to spread to new geographic areas around the world (2–4). For this reason, the species has been proposed as a biological barometer of climate change (5). V. vulnificus can cause septicemia with a high probability of death in immunocompromised patients, especially those with a high iron content in their blood (1, 6–8). Septicemia is acquired after contact of a wound with seawater or aquatic animals (mainly diseased farmed fish) or after ingestion of raw shellfish (6, 9–11). Epidemiological data from the United States confirm that both hospitalization and mortality rates associated with V. vulnificus infections are among the highest of all water/foodborne infectious diseases (6, 11). The most complete epidemiological data set on the human diseases caused by V. vulnificus comes from the COVIS system from the United States (https://www.cdc.gov/vibrio/surveillance.html?CDC_AA_refVal=https%3A%2F%2Fwww.cdc.gov%2Fnationalsurveillance%2Fcholera-vibrio-surveillance.htm).

The species has been classically subdivided into three biotypes (Bts) (12, 13). Bt3 was the last group described and clusters together the only strains of the species associated with fish farm-linked outbreaks of septicemia (13). This biotype emerged in Israel in 1996 and, since then, has remained a major threat to humans handling farmed fish in that country (14, 15), especially during the summer months when water temperature rises (1). Interestingly, there are no reports of human infections caused by Bt3 strains outside Israel. One of the reasons could be that biotyping fails as a consequence of the numerous exceptions to the original biotype descriptions that have been found (16). In fact, the only methods that can clearly distinguish these strains from the rest of the species and from other pathogenic vibrios are genomic-based methods, such as genome sequencing, pulsed-field gel electrophoresis, or multiple locus sequence typing (MLST), which are costly and time consuming (17).

A new biologically meaningful classification system for V. vulnificus has recently been proposed (18). This new system is based on a phylogenetic analysis of the single nucleotide polymorphisms (SNPs) present in the core genome of the species and divides V. vulnificus into five well-supported phylogenetic lineages (L). According to this new system, Bt3 isolates belong to L3 and constitute a genetically homogeneous group, clearly distinguishable from the rest (15, 18).

The aim of this work was to design a rapid and sensitive method for the specific identification of V. vulnificus L3. For this purpose, specific L3 markers were sought out by comparing the genomes of representative strains of the main lineages of the species. Once one of them was selected, a new PCR method was designed with two target genes, one for the identification of the species and the other for the identification of the lineage. The new PCR method showed 100% reliability and sensitivity after being tested with more than 200 strains from different origins and lineages. The comparative genomic analysis also revealed the presence of a genetic duplication in the L3 strains that could be related to the unique ability of this lineage to produce septicemia outbreaks.

MATERIALS AND METHODS

Core genome L3.

We first identified the unique genes of L3. To this end, we downloaded 12 genomes from NCBI (https://www.ncbi.nlm.nih.gov/) from the three major lineages (18) (Table 1). All genomes were annotated using the software Prokka (19) with default options independently from their assembly level (complete/closed genome or contigs). With the annotation files, the core genome for each lineage was established using Proteinortho (20), using fasta amino file (.faa) from Prokka.

TABLE 1.

List of V. vulnificus strains downloaded from NCBI and used for Proteinortho analysis

Strain	Level	Lineage	Source of isolation	ANI (%)a
YJ016	Complete	1	Human blood	96.41
CMCP6	Complete	1	Human blood	96.26
ENV1	Complete	1	Oyster	97.12
FORC016	Complete	1	Human blood	96.39
MO6-24/O	Complete	1	Human blood	96.54
CECT4999	Complete	2	Eel	97.17
95-8-161	Contig	2	Eel	97.10
ATCC 27562	Complete	2	Human blood	97.06
NV22	Contig	2	Seawater	97.13
12	Contig	3	Human blood	99.87
BAA87	Contig	3	Human blood	99.86
VVyb1	Contig	3	Human blood

^aCompared with VVyb1.

Briefly, Proteinortho takes several annotated genomes (nucleotides or proteins) and performs a blast-based search for homologies between genomes. It allows the user to obtain a list of homologous genes between strains or species. In our case, we used Proteinortho to search for homologous genes inside L3 and exclusive to this group.

Gene selection and primer design.

Among the unique genes of L3, we chose a gene encoding a protein with a putative function, pgiA. Next, we confirmed by in silico BLASTN (v2.9.0) analysis among the 12 strains that the selected gene was specific to L3 strain genomes and designed primers compatible (similar melting temperature [T_m]) with those specific for the species target gene, vvhA (21). Basically, we searched the area shown in Fig. 1 that corresponds to a part of a hypothetical protein plus pgiA plus a noncoding region. With this information, the primers we designed were 5′-AGCCACTCTCCTGCGAGCAG-3′ and 5′-GAGGTGTGCGAGCGCCGAT-3′.

FIG 1.

Schematic representation of pgiA_L3 and surrounding genes in the Vvyb1 L3 strain. Each arrow corresponds to a gene detected and annotated with Prokka software. The purple rectangle represents the 834-bp amplified sequence by this PCR method. This sequence is formed by a part of the sequence for a hypothetical protein, pgiA_L3, and a part of a noncoding region. HP, hypothetical protein.

After that, we studied the PgiA protein. First, we performed a BLASTP analysis in the NCBI database to look for similar sequences in the database and make sure that the function of the gene corresponds to the one Prokka provided. Besides, we compared the sequence with other Vibrio species.

DNA extraction and PCR conditions.

To make sure the selected gene was only present in L3 strains, we tested all the V. vulnificus strains available in our laboratories (n = 217) coming from different sources (environmental and clinical) and countries and belonging to the five lineages (Fig. 2). The DNA was extracted by resuspending overnight cultures of bacteria in phosphate-buffered saline (PBS), boiling them at 100°C for 10 min, and storing them with ice a minimum of 5 min before using them.

FIG 2.

Doughnut and pie charts with V. vulnificus sources (n = 217). Most strains were isolated from diseased organisms (clinical). The rest were isolated from environmental or healthy organisms. Percentages in pie charts correspond to the total of strains, whereas percentages in the doughnut correspond to each category.

We established the PCR conditions as follows: 95°C for 5 min, followed by 30 cycles each at 94°C for 10 s, 55°C for 5 s, and 72°C for 5 s, with a final step of 72°C for 2 min. For PCR revelation, we loaded and ran the PCR-amplified product on a 2% agarose gel with DNA Safe stain for 25 min at 100 V.

RESULTS

To identify the unique genes of L3, we first downloaded representative genomes from the three major lineages. Table 1 shows the genomes used for this purpose, the lineage to which they belong, and the source of isolation and average nucleotide identity (ANI) values compared with those of the VVyb1 strain. Orthologous gene detection was performed by running Proteinortho, and we found that there was a total of 292 genes specific to L3, most of them encoding genes annotated as “hypothetical proteins” by Prokka (see Table S1 in the supplemental material). For the new PCR method, we selected a gene encoding a protein with a putative function, pgiA. Interestingly, we found two similar but different open reading frames (ORFs) annotated as pgiA in the genomes analyzed, one present in all lineages and the other exclusive to L3 (pgiA_L3) (Table S1). Furthermore, pgiA_L3 is located in the middle of two copies of a DNA segment next to several genes encoding tRNA (Table 2). This duplicated zone putatively encodes lactate uptake and utilization as well as resistance to phospholipase A2 (22, 23).

TABLE 2.

List of identified genes in the vicinity of pgiA_L3 and putative function^a

Gene	Annotationb	Putative function (reference)
lonA	ATP-dependent protease La type II	Involved in virulence regulation (31)
yejF	Putative ABC transporter ATP-binding protein YejF	Counteracts antimicrobial peptides (32)
dmlR	HTH-type transcriptional regulator DmlR	Possible regulator for the operon lutABC (33)
lutP-1	l-Lactate permease	Putatively involved in improved growth from lactate (sepsis biomarker) in the blood from septicemic patients (22, 30)
lutA-1	Lactate utilization protein A
lutB-1	Lactate utilization protein B
lutC-1	Lactate utilization protein C
lplT-1	Lysophospholipid transporter LplT	Resistance against host phospholipase A2, a sepsis biomarker (23, 29)
pgiAL3	Glucose-6-phosphate isomerase	Protease activity (34), molecular mimicry (35)
csy3	CRISPR-associated protein Csy3
lutP-2	l-Lactate permease	Putatively involved in improved growth from lactate (sepsis biomarker) in the blood from septicemic patients (22, 30)
lutA-2	Lactate utilization protein A
lutB-2	Lactate utilization protein B
lutC-2	Lactate utilization protein C
lplT-2	Lysophospholipid transporter LplT	Resistance against host phospholipase A2, a sepsis biomarker (23, 29)
ompA	Outer membrane protein A	Serum resistance and pathogenicity (36)
	tRNA-Ser(tga)
	tRNA-Ser(tga)
	tRNA-Phe(gaa)
	tRNA-Asn(gtt)

^aInformation pertaining specifically to pgiA_L3 is in boldface type. Information pertaining to the duplicated genes putatively related to sepsis is underlined.

^bAnnotation with Prokka or BLAST (best hit when Prokka annotation was “hypothetical protein”).

After in silico analysis, we isolated the DNA from all V. vulnificus strains available in our laboratories (217 in total). The strains came from different sources and locations (Fig. 2); 66% of them were clinical samples (from humans or animals), whereas 29% came from environmental isolations. We then applied the newly developed PCR method. Figure 3 shows a representative electrophoresis gel for some of the strains used in this study, including L3 and non-L3 strains. For all tested strains, we observed the 519- bp band corresponding to the species-specific gene (vvha) while only the samples from the L3 strains showed the second band, the 834-bp band corresponding to the pgiA_L3 gene plus the selected adjacent areas (Fig. 1). No false-positive or -negative result was obtained for any of the 217 strains tested. This result indicated a 100% sensitivity and reliability for the new PCR.

FIG 3.

Detection of V. vulnificus L3 by multiplex PCR. The PCR targets the species-specific gene vvha (amplified fragment; 519 bp) and the pgiA_L3 plus surrounding zones (see Fig. 1) (amplified fragment, 834 bp). Lanes A to K, L3 strains 12, 32, 97, 162, 1003, 11028, Vvyb1, Yb88, Yb95, V247, and V233. Lanes L and M, non-L3 strains YJ016 (L1) and CECT4999 (L2). N, negative control. Colors were changed to black and white for a better visualization.

DISCUSSION

This paper describes a newly developed PCR method useful for the identification of V. vulnificus L3, the only group of the species that causes septicemia outbreaks linked to fish farms (13). The marker gene for the identification of L3 was selected after comparing the genomes of representative strains of the main phylogenetic lineages of the species. The PCR was then designed and validated with more than 200 V. vulnificus strains belonging to the five lineages described, as well as some others that are yet to be analyzed. It should be noted that the new PCR method showed 100% reliability and sensitivity, which makes it a powerful tool not only in diagnosis but also in taxonomic and epidemiological studies. Interestingly, L3 has only been reported in Israel. Applying this methodology to clinical and environmental samples (fish, water, and shellfish), it would be possible to know whether this lineage is present only in Israel or has spread to other geographical areas, which would help to understand the epidemiology of the species (6). In recent years, increases in both the number of V. vulnificus strains present in coastal waters and clinical cases of human vibriosis have been reported worldwide (4, 7, 24, 25). These increases have been linked to global warming. In this scenario, rapid and sensitive detection methods, such as this new PCR, will be the key to identifying and preventing present and future outbreaks due to the species and, in particular, to this lineage.

The selected marker gene has turned out to be a variant of the pgiA gene, two variants being present in the L3 strains. Probably, pgiA_L3 has been acquired by horizontal gene transfer from other Vibrio species, since the protein has a great similarity with other Vibrio PgiA (for example, V. aestuarianus; similarity, 90.37%). Remarkably, pgiA_L3 is located in the middle of two copies of a putative genomic island, as suggested by the size of the fragment (11 kb) and the vicinity of genes encoding tRNA (26, 27). The putative island encodes lactate uptake (lutP) and utilization (lutABC) as well as for resistance to phospholipase A2 (lplT) (22, 23). Interestingly, both lactate and phospholipase A2 are considered biomarkers of sepsis, because their levels in the plasma of patients with sepsis have been suggested to predict mortality (28–30). Therefore, the presence of this duplicated putative genomic island could be related to the ability of L3 to cause septicemia outbreaks (13). Further studies are under way to find out the relevance of this hypothetic island in the virulence and abilities of L3. Finally, the application of the designed PCR in any clinical and research laboratory would allow researchers to not only diagnose cases of disease due to L3 but also understand the epidemiology of the lineage and the species, especially in a changing environment due to global warming.