Isolation , molecular characterization , and antibiotic resistance patterns of Vibrio parahaemolyticus isolated from coastal water in the Eastern Province of Saudi Arabia

Vibrio parahaemolyticus is a Gram-negative halophilic marine microbe that causes gastroenteritis, wound infections, and septicemia in humans. Since the emergence of the pandemic clone O3:K6, V. parahaemolyticus has become a globally well-known pathogen. In this study, 375 seawater samples collected from the Eastern coast of Saudi Arabia were tested for the presence of V. parahaemolyticus. Three hundred and forty samples were determined positive for V. parahaemolyticus using traditional microbiological techniques. The genes toxR and tlhwere detected via polymerase chain reaction (PCR) in 41 isolates from 23 samples (6%). Thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (tdh) are the most common virulence genes associated with V. parahaemolyticus. As such, four isolates were tdhþ (1%) and another four were trhþ (1%). No evidenceofpandemiccloneswasdetectedusinggroup-specificPCR (GS-PCR). Sampleswere tested for antibiotic susceptibility against 28 agents. The vast majority of samples exhibited high resistance to carbenicillin (98%), ampicillin (88%), and cephalothin (76%). The multiple antibiotics resistance index was>0.2 for 35% of the isolates. The results of this study confirm the presence of V. parahaemolyticus in the Eastern coast of Saudi Arabia. This is the first report of tdhþ and trhþ isolates from this area. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2017.361 s://iwaponline.com/jwh/article-pdf/16/1/57/239993/jwh0160057.pdf Lubna Ghenem Nasreldin Elhadi (corresponding author) Department of Clinical Laboratory Science, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, P.O. Box 2435, Dammam 31441, Kingdom of Saudi Arabia E-mail: nmohammed@iau.edu.sa


INTRODUCTION
Vibriosis is a human illness caused by pathogenic species of the family Vibrionaceae (CDC ). The aquatic bacterial species Vibrio parahaemolyticus is the most frequently reported cause of vibriosis in the USA (Fabbro et al. ; CDC ). Several studies have found that V. parahaemolyticus isolation is positively correlated with mean water temperature (Blackwell & Oliver ; Rosec et al. ; Urquhart et al. ). The most common clinical manifestation of V. parahaemolyticus is gastroenteritis following ingestion of raw seafood contaminated by virulent strains (Quilici et al. ). In patients suffering from pre-existing medical conditions, vibriosis can progress to life-threatening septicemia (Ceccarelli et al. ). In addition, V. parahaemolyticus may also cause skin infections (Fabbro et al. ). Fortunately, V. parahaemolyticus-associated gastroenteritis is self-limiting in most patients (Bennett et al. ). However, when needed, CDC recommends that patients receive tetracycline or ciprofloxacin antibiotic treatment (Elmahdi et al. ). As a result of the excessive use of antibiotics in human and aquaculture systems, vibrios have begun to acquire antibiotic resistance genes similar to many other bacterial genera (Elmahdi et al. ). Several studies have reported a high rate of V. parahaemolyticus resistance to ampicillin (Ottaviani Furthermore, one concerning report characterized a multidrug resistance (MDR) conjugative plasmid, acquired by a strain of V. parahaemolyticus, that mediates resistance to multiple antibiotics, including ampicillin, ceftriaxone, cefotaxime, nalidixic acid, kanamycin, chloramphenicol, and streptomycin (Liu et al. ). The transmission of MDR genes via conjugative plasmids jeopardizes the effectiveness of disease control and treatment and poses a significant threat to public health (Liu et al. ; Li et al. ). Since the status and occurrence of V. parahaemolyticus in the Eastern coastal environment of Saudi Arabia is not well characterized, the objectives of this study were: (i) to determine the incidence of V. parahaemolyticus in seawater samples collected from the Arabian Gulf coast; (ii) to confirm their species identity using PCR targeting toxR and tlh; (iii) to determine their potential pathogenicity using PCR assays targeting tdh and trh genes; (iv) to screen for pandemic clones of V. parahaemolyticus using GS-PCR; and (v) to study the antibiotic susceptibility patterns of the V. parahaemolyticus strains isolated from the Arabian Gulf.

Site of study
The Arabian Gulf is a semi-enclosed marine environment that covers an area of about 240,000 km 2 . It has high levels of salinity and experiences intense fluctuations in water temperatures (Sheppard et al. ). Since the Arabian Gulf serves as a major global hub for oil transportation, its ecosystem is continuously stressed by the discharge of hydrocarbon pollutants and crude oil spills (Mahmoud et al. ). The industrial and sewage discharges combined with the Gulf's low water exchange rates has made this sea one of the most anthropogenically impacted regions in the world (Naser ). In this study, seawater was collected from 17 different locations along the coastline of the Arabian Gulf, as illustrated in Figure 1. The sample locations include public beaches, fishing areas, and recreational water sources. During sample collection, each location was divided into two to three equidistant sampling sites using GPS.

Water sample collection and transportation
Throughout a one-year time period (February 2015 to February 2016), a total of 375 surface water samples were collected using sterile 500 mL screw-cap bottles (Fischer, UK). The number of samples collected from the surface water of each location is listed in Table 1. The water pH and temperature of each sampling site were measured using a multi-parameter water quality meter (YSI-50 series, Horiba, USA). The samples were transported in portable coolers at ambient temperature and immediately analyzed on arrival at the laboratory.

Sample treatment
Sample treatment was based on the US FDA Bacteriological Analytical Manual (BAM) method for V. parahaemolyticus isolation with a few modifications (Kaysner & DePaola ). Briefly, samples were enriched in both 1% and 3% NaCl enriched alkaline peptone water (APW). To prepare the APW (pH ± 8.5), Peptone and NaCl were dissolved in water and dispensed into screw-cap bottles. Then, 25 mL of each seawater sample was added to 225 mL of the prepared APW and incubated for 18 hours at 35 C.

Cultivation and identification
Enriched samples were streaked on both thiosulfatecitrate-bile salts-sucrose agar (TCBS) (Oxoid, UK) and CHROM Vibrio agar (CHROM, France) and incubated at 37 C for 18-24 hours. A minimum of three to five typical colonies of V. parahaemolyticus were purified on tryptic soy agar supplemented with 2% NaCl and incubated at 37 C overnight. Colonies were biochemically confirmed as V. parahaemolyticus using Gram stains, oxidase tests, string tests, urease tests, and Kligler iron agar. Phenotypic characterization was achieved by using API 20 E, API NE, and API 10 S strip tests (Bio-Mérieux, France). Glycerol stocks of pure colonies were prepared and stored at À80 C for subsequent molecular testing.

Genomic DNA extraction
To subculture V. parahaemolyticus colonies, 1 mL of glycerol stock was transferred to Luria-Bertani (LB) broth (2% NaCl) and incubated overnight in a thermal shaker (Stuart shaking incubator S1500, UK). A modified version of the boiling extract method was used to extract genomic DNA (Silvester et al. ). Briefly, 1.5 mL of LB culture was transferred to Eppendorf tubes and centrifuged (10,000 rpm, 4 C, 1 minute). The remaining pellets were diluted 1:10-fold in sterile distilled water and vortexed for 1 to 2 minutes. The resulting suspension was boiled at 100 C for 15 minutes to lyse the cells and free crude DNA. The tubes were immediately stored at À20 C for further use.

Virulence detection
The virulence of all toxR þ and tlh þ isolates was further assayed via PCR amplification of the tdh and trh genes. Lastly, 10 μL of the final mixture were mixed with 1 μL of dye solution ethidium bromide (Promega, USA) and resolved on a 1% agarose gel via electrophoresis. Group-specific PCR All V. parahaemolyticus isolates that are tdh þ or trh þ were tested for the presence of toxRS gene using GS-PCR

Multiple antibiotic resistance index and MDR definition
The multiple antibiotics resistance (MAR) index was calculated as described by Krumperman () using the formula a/b where 'a' represents the number of antibiotics to which the particular isolate is resistant and 'b' represents the total number of multiple antibiotics to which the particular isolate has been exposed. MDR was defined as the nonsusceptibility of the organism to at least one agent in three or more categories of antimicrobials (Magiorakos et al. ).

Water physical parameters
Water temperatures ranged from 14.6 C to 32.4 C throughout the sampling period (February 2015 to February 2016).
The lowest seawater temperature from which V. parahaemolyticus was isolated was 16.9 C and the highest was 32.4 C.
In regards to the pH, the lowest and highest values at which V. parahaemolyticus was detected was pH 8.2 and 8.8, respectively.
Presumptive V. parahaemolyticus identification

Confirmation of V. parahaemolyticus identity by toxR
and tlh PCR Forty-one isolates from 23 samples (6%) were found to harbor both toxR and tlh genes. They were isolated from 23 samples (6%). The results are summarized in Table 2.

Virulence characterization
Four (1%) V. parahaemolyticus isolates were positive for the tdh gene and four (1%) other isolates harbored the trh gene.
None of the isolates were positive for both tdh and trh genes.
The urease test also serves as a method for determining virulence, for which two (0.5%) isolates produced weak positive results.

Group-specific PCR
All isolates positive for tdh and trh were further assayed for the toxRS gene using GS-PCR, but none was positive.
Detailed information regarding the characterization of V. parahaemolyticus isolates (n ¼ 41) from the coast of the Eastern Province of Saudi Arabia is listed in Table 3.

Antibiotic susceptibility testing
Based on the antibiotic susceptibility tests, the resistance rate of the 41 V. parahaemolyticus isolates in our study was 98% to carbenicillin, 88% to ampicillin, and 76% to cephalothin.
In contrast, all V. parahaemolyticus isolates were sensitive to piperacillin/tazobactam, ceftazidime, chloramphenicol, gentamicin, imipenem, meropenem, nalidixic acid, levofloxacin, and sulfamethoxazole/trimethoprim. The results of the antibiotic susceptibility tests are listed in Table 4.

Multiple antibiotic resistance index and MDR
The values of the MAR index ranged from 0.03 to 2.8.

Physical parameters
The strong association between the occurrence of V.      (7) 13 (  ; there was no significant difference in the distribution of the resistance patterns between virulent (tdh þ / trh þ ) and non-virulent isolates (tdh À /trh À ).
The frequency of dangerous antibiotic-resistant bacteria has been increasing over the past several decades (Fair & Tor ). In the case of V. parahaemolyticus this may be explained by the excessive use of antimicrobial agents in order to protect the industrial aquatic produce from infectious diseases and massive stock damages (Xu et al. ).