Study site
The Arabian Gulf is a subtropical marine system that is very shallow with a maximum depth of around 60 m. Many studies report that the gulf environment is under different stresses that lead to alteration of the surroundings. The most significant natural alterations are the warming of the climate and salinity¹³. Due to the impacts of development in Arabian Gulf countries, the water is polluted by industrial wastes such as heavy metals, oil, and gases, as well as sewage discharge. The Arabian Gulf consider has high pollution due to the semi-enclosed ecosystem with low water exchange¹.

In this study, seawater samples were collected from 17 different locations that were divided into two or three sites, which were coordinated using GPS. The locations are along the coastline of the Arabian Gulf, and recreational areas were targeted, as shown in Fig. 1. The locations are Alaziziyah Beach (AZB), Corniche Tiba Jubail (CTJ), Dammam Corniche (DMC), Dammam Marina Front (DMF), Fanateer Corniche (FNC), Half-Moon Beach (HMF), Alkobar Corniche (KBC), Alkobar Marina Front (KBF), Albuhairah Beach (LAK), Almorjan Island (MOI), Palm Beach Jubail (PBJ), Qatif Corniche (QTC), Ras Tanura Corniche (RTC), Saihat Corniche (SEC), Alshibaly (SHB), Tarout Corniche (TRC), and Imam Abdulrahman Bin Faisal University (IAU).

Water sample collection and transportation
A total of 362 seawater samples were collected from the surfaces of the different locations between February 2015 and February 2016. The samples were collected using sterile 500-ml screw-cap bottles (Fischer, UK) and kept in insulated coolers. All samples were immediately transported to the microbiology research laboratory at Imam Abdulrahman Bin Faisal University and analyzed for the isolation of V. vulnificus.

Sample treatment and cultivation
All seawater samples were enriched with alkaline peptone water (APW) containing 1% NaCl, as described in the Bacteriological Analytical Manual (BAM) of the FDA⁹.  Briefly, 25 ml of the samples were added to 225 ml of prepared APW and then incubated for 18-24 hours at 37°C. After the enrichment step, full loops of enriched seawater samples were streaked onto thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Oxoid, UK) and Vibrio chromogenic agar (CHROM Agar, France) and incubated at 37°C for 18-24 hours. Since V. vulnificus varies in fermenting sucrose, the colonies appeared on TCBS in yellow and green colors. On the Vibrio chromogenic agar, the colonies appeared as turquoise. Around 3 to 5 typical colonies were selected and sub-cultured on tryptic soy agar (TSA) enriched with 3% NaCl and incubated at 37°C.

Biochemical identification
After the culture and isolation of pure colonies, presumptive V. vulnificus was confirmed by a series of biochemical tests. The confirmation includes Gram staining, oxidase tests, and Kligler’s iron agar (KIA). For typical V. vulnificus, the results must be Gram negative rods and produce a purple color on strips that indicate a positive oxidase test. When the selected colonies are emulsified in bile salt on a clean slide, they appear viscous. Pulling the loop gently produces a mucoid string mass, which indicates a positive string test, and the colonies have a K/A pattern with no gas or H₂S production. All the isolates of typical V. vulnificus according to biochemical tests were reconfirmed by API 20E strip tests (Bio Mérieux, France). The API suspension was prepared using 2% saline, and V. vulnificus ATCC 27562 strain was used as a positive control. The pure identified colonies of V. vulnificus were preserved in tryptic soya broth supplemented with 3% NaCl and glycerol and stored at -80°C.

Genomic DNA Extraction
For genomic DNA extraction, one loop full of the preserved presumptive V. vulnificus was sub-cultured on Luria-Bertani (LB) broth supplemented with 3% NaCl and then incubated at 37°C over night. The DNA was extracted according to Silvester et al. ( 2015) with little modification. Briefly, 1.5 ml of the incubated LB broth was centrifuged at 10,621 x g for 2 minutes, and the supernatant was decanted. The pellets were then suspended in sterile distilled water, followed by vortexing for 2 minutes. Finally, all sample suspensions were boiled in a water bath at 100°C for 15 minutes to lyse the bacterial cells and free the DNA. After boiling, all samples were kept at -20^oC to be used for detection of the vvhA gene and ERIC-PCR genotyping.

Detection of vvhA gene in Vibrio vulnificus
PCR amplification was performed to detect the vvhA gene as described elsewhere¹⁴. The total volume of the reaction mixture was 25 µl, which was composed of 1 µl of diluted DNA (1:10) template, 5 µl of 10X buffer (Promega, USA), 4 µl of 25 mM MgCl₂ solution (Promega, USA), 0.5 µl of 2.5 mM deoxynucleotide triphosphate (dNTP) solution (Promega, USA), 0.25 µl of Taq DNA polymerase (Promega, USA), 1 µl of forward primer 52 TTCCAACTTCAAACC GAACTATGA-32, 1 µl of reverse primer 52 ATTCCAGTCGATG CG AATACGTTG-32 , and 12.25 µl of deionized water (Promega, USA). V. vulnificus ATCC 27562 was used as a positive control, while V. alginolyticus ATCC 17749 was used as a negative control.

The reaction program began at 94°C for initial denaturation for 3 minutes, followed by 30 cycles of amplification including 1 minute at 94°C for denaturation, 1 minute at 65°C for annealing, and 1 minute at 72°C for extension. The 30 amplification cycles were followed by a final extension step at 72°C for 5 minutes. After that, 10 µl of the PCR reaction mixture was loaded on 1.5% agarose electrophoresis gel with 1 µl of ethidium bromide dye (Promega, USA) added to make it visible under UV light. The buffer used was 1X Tris Borate EDTA. The positive vvhA gene was determined after comparison with the positive control, which has a 205-bp amplicon.

Enterobacterial repetitive intergenic consensus (ERIC) PCR
ERIC-PCR was performed according to a previously described method¹⁵. The total volume of the reaction mixture was 25 µl, which was composed of 1 µl of diluted DNA (1:10) template, 5 µl of 10X buffer (Promega, USA), 4 µl of 25 mM MgCl₂ solution (Promega, USA), 0.5 µl of 2.5 mM dNTPs solution (Promega, USA), 0.25 µl of Taq DNA polymerase (Promega, USA), 2 µl of ERIC primer, and 12.25 µl of distilled water. The amplification reaction was 4 minutes at 94°C for pre-denaturation, followed by 35 cycles of 45 seconds at 94°C for denaturation, 1 minute at 52°C for  annealing, an extension step at 65°C for 8 minutes, and a final extension at 65°C for 10 minutes. After that, 10 µl of the PCR reaction mixture was loaded on 1% agarose electrophoresis gel with 1 µl of ethidium bromide dye added (Promega, USA) to make it visible under UV light. The buffer used was 1X Tris Borate EDTA.

Fingerprinting analysis
All gels were scanned for DNA bands, and the images were captured using a gel documentation system (Syngene G: Box). The gel images were analyzed using GelJ software to analyze the DNA fingerprint¹⁶. The dendrogram tree was constructed based on the unweighted average pair group method (UPGMA) using a Dice coefficient. The numerical index of discrimination (D) was calculated empirically using a previously described formula¹⁷:

where D is the discriminatory index, N is the total number of strains in the sample population, s is the total number of types described, and nj is the number of strains belonging to the j^th type.

Antibiotic susceptibility testing
The isolates of V. vulnificus recovered from sea water samples that were positive for the vvhA gene were tested against 24 antibiotics (Oxoid, England). The disc diffusion method was performed according to the CLSI protocols¹⁸. The following antimicrobial agents were tested: ampicillin (AMP: 25 µg), amoxicillin-clavulanic acid (AMC: 30 µg), piperacillin (PRL: 100 µg), piperacillin-tazobactam (TZP: 110 µg), aztreonam (ATM: 30 µg), cephalothin (KF: 30 µg), cefoxitin (FOX: 30 µg), cefotaxime (CTX: 30 µg), ceftazidime (CAZ: 30 µg), ceftriaxone (CRO: 30 µg), cefepime (FEP: 30 µg), imipenem (IPM: 10 µg), meropenem (MEM: 10 µg), amikin (AK: 30 µg), gentamicin (GN: 10 µg), streptomycin (S: 10 µg), tetracycline (TE: 30 µg), doxycycline (DO: 30 µg), ciprofloxacin (CIP: 5 µg), levofloxacin (LEV: 5 µg), chloramphenicol (C: 30 µg), trimethoprim-sulfate (SXT: 25 mcg), nalidixic acid (NA: 30 µg), and rifampicin (RD: 5 µg). The reference strain Escherichia coli ATCC 1175 was used as a control while performing the antibiotic susceptibility testing.

Multiple antibiotic resistance index
The Multiple Antibiotic Resistance (MAR) index was calculated as a/b, where a is the number of multiple antibiotics to which the isolates are resistant, and b is the total number of multiple antibiotics to which particular isolates have been exposed¹⁹.

Multidrug resistance
The categories of antibiotics used in this study were penicillins, cephalosporin, carbapenem, aminoglycosides, tetracycline, fluoroquinolone, amphenicol, folate inhibitor, quinolone, and rifampin. The strains with resistance to at least one antibiotic from three or more different classes of antimicrobial were considered as having multidrug resistance (MDR)²⁰.