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 tlh were 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 tdh+ (1%) and another four were trh+ (1%). No evidence of pandemic clones was detected using group-specific PCR (GS-PCR). Samples were 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 tdh+ and trh+ isolates from this area.

INTRODUCTION

Vibriosis is a human illness caused by pathogenic species of the family Vibrionaceae (CDC 2016). The aquatic bacterial species Vibrio parahaemolyticus is the most frequently reported cause of vibriosis in the USA (Fabbro et al. 2010; CDC 2016). Several studies have found that V. parahaemolyticus isolation is positively correlated with mean water temperature (Blackwell & Oliver 2008; Rosec et al. 2009; Urquhart et al. 2016). The most common clinical manifestation of V. parahaemolyticus is gastroenteritis following ingestion of raw seafood contaminated by virulent strains (Quilici et al. 2005). In patients suffering from pre-existing medical conditions, vibriosis can progress to life-threatening septicemia (Ceccarelli et al. 2013). In addition, V. parahaemolyticus may also cause skin infections (Fabbro et al. 2010).

Molecular techniques have become important for the detection of V. parahaemolyticus in water samples. The species-specific markers toxR and tlh have been proposed to confirm V. parahaemolyticus strains identity to the species level (Brasher et al. 1998; Bej et al. 1999; Kim et al. 1999). The thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin related-hemolysin gene (trh) are the most common virulence factors associated with gastrointestinal symptoms (Roque et al. 2009). Consequently, polymerase chain reaction (PCR) protocols for the detection of these virulence genes have been established (Tada et al. 1992).

Historically, V. parahaemolyticus cases were sporadically reported (de Jesús Hernández-Díaz et al. 2015) until there was a sudden surge of cases in India in 1996 caused by the novel serotype O3:K6 (Okuda et al. 1997). Since then, reports of gastroenteritis caused by O3:K6 have spread to Africa (Ansaruzzaman et al. 2008), Asia (Li et al. 2016), Europe (Martinez-Urtaza et al. 2005), and the American continent (Velazquez-Roman et al. 2014). The rapid spread of O3:K6 marked the first pandemic of V. parahaemolyticus and placed this pathogen at the front of the global public health agenda (Ceccarelli et al. 2013). The clonality of the pandemic O3:K6 isolates can be confirmed using group-specific PCR (GS-PCR) targeting the toxRS gene (Okuda et al. 1997).

Fortunately, V. parahaemolyticus-associated gastroenteritis is self-limiting in most patients (Bennett et al. 2015). However, when needed, CDC recommends that patients receive tetracycline or ciprofloxacin antibiotic treatment (Elmahdi et al. 2016). 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. 2016). Several studies have reported a high rate of V. parahaemolyticus resistance to ampicillin (Ottaviani et al. 2013; Shaw et al. 2014; Silvester et al. 2015; He et al. 2016). 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. 2013). 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. 2013; Li et al. 2015). 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.

MATERIALS AND METHODS

Site of study

The Arabian Gulf is a semi-enclosed marine environment that covers an area of about 240,000 km2. It has high levels of salinity and experiences intense fluctuations in water temperatures (Sheppard et al. 2010). 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. 2009). 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 2013). 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.

Figure 1

Locations of the 17 sampling sites in the Eastern Province of Saudi Arabia.

Figure 1

Locations of the 17 sampling sites in the Eastern Province of Saudi Arabia.

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.

Table 1

Total number of seawater samples collected from each location

Location Number of samples 
Alaziziyah Beach (AZB) 25 
Corniche Tiba Jubail (CTJ) 
Dammam Corniche (DMC) 10 
Dammam Marina Front (DMF) 20 
Fanateer Corniche (FNC) 35 
Half-Moon Beach (HMF) 50 
Alkhubar Corniche (KBC) 35 
Alkubar Marina Front (KBF) 15 
Albuhairah Beach (LAK) 40 
Almorjan Island (MOI) 
Palm Beach Jubail (PBJ) 30 
Qatif Corniche (QTC) 20 
Ras Tanura Corniche (RTC) 
Sayhat Corniche (SEC) 15 
Alshibaly (SHB) 25 
Tarout Corniche (TRC) 30 
University of Dammam (OUD) 10 
Total 375 
Location Number of samples 
Alaziziyah Beach (AZB) 25 
Corniche Tiba Jubail (CTJ) 
Dammam Corniche (DMC) 10 
Dammam Marina Front (DMF) 20 
Fanateer Corniche (FNC) 35 
Half-Moon Beach (HMF) 50 
Alkhubar Corniche (KBC) 35 
Alkubar Marina Front (KBF) 15 
Albuhairah Beach (LAK) 40 
Almorjan Island (MOI) 
Palm Beach Jubail (PBJ) 30 
Qatif Corniche (QTC) 20 
Ras Tanura Corniche (RTC) 
Sayhat Corniche (SEC) 15 
Alshibaly (SHB) 25 
Tarout Corniche (TRC) 30 
University of Dammam (OUD) 10 
Total 375 

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 2004). 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 thiosulfate-citrate-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 (BioMérieux, France). Glycerol stocks of pure colonies were prepared and stored at −80 °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. 2015). 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 −20 °C for further use.

Species identity confirmation of V. parahaemolyticus isolates

PCR targeting toxR and tlh genes was used to confirm the identity of V. parahaemolyticus to the species level. Positive (ATCC 17802) and negative (V. alginolyticus, ATCC 17749) controls were included in each run. A 368 bp region of the toxR gene (F-5′-GTC TTC TGA CGC AAT CGT TG-′3 and R-5′-ATA CGA GTG GTT GCT GTC ATG-′3) was amplified with the following thermocycler conditions: 20 cycles of 1 minute denaturation at 94 °C, 1.5 minutes of annealing at 63 °C, and 1.5 minute extension at 72 °C, followed by a final extension at 72 °C for 7 minutes as described by Kim et al. (1999). The thermocycler conditions for the 450 bp region of the tlh gene (F-5′-AAA GCG GAT GTA TCA GAA GCA CTG-3′ and R-5′-GCT ACT TTC TAG CAT TTT CTC TGC-3′) included 30 cycles of 1 minute at 94 °C, 1 minute of annealing at 58 °C, and 1 minute of extension at 72 °C, followed by a final extension at 72 °C for 3 minutes (Brasher et al. 1998; Bej et al. 1999). Finally, 10 μL of the final PCR mixture were mixed with 1 μL of dye solution Ethidium bromide (Promega, USA) and resolved on a 1% agarose gel via electrophoresis.

Virulence detection

The virulence of all toxR+ and tlh+ isolates was further assayed via PCR amplification of the tdh and trh genes. V. alginolyticus (ATCC 17749) was used as a negative control for both tdh and trh genes. V. parahaemolyticus (ATCC 17802) was the positive control for the tdh gene and V. parahaemolyticus (AQ 4037) was used as a positive control for trh. A 251 bp region of the tdh gene (F-5′-CCA CTA CCA CTC TCA TAT GC-3′and R-5′-GGTACTAAATGGCTGACATC-3′) were performed as outlined by Tada et al. (1992) with minor changes: 35 cycles of denaturation at 94 °C for 1 minute, annealing at 59 °C for 1 minute, and extension at 72 °C for 1 minute, followed by a final extension at 72 °C for 7 minutes. A 251 bp region of the trh gene (F-5′ GGC TCA AAA TGG TTA AGC G-3′ and R-5′-CAT TTC CGC TCT CAT ATG C-3′) was amplified using previously published methods (Tada et al. 1992). 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+ or trh+ were tested for the presence of toxRS gene using GS-PCR (Matsumoto et al. 2000). The sequence of the forward primer was 5′-TAATGAGGTAGAAACA-3′ and the reverse primer sequence was 5′-ACGTAACGGGCCTACA-3′. A positive (V. parahaemolyticus ATCC 17802) and a negative control (V. alginolyticus, ATCC 17749) were included in each run. The amplification program began at 96 °C for 5 minutes, followed by 25 cycles of 1 minute of denaturation at 94 °C, 2 minute of annealing at 45 °C, and 3 minutes of extension at 72 °C. The 25 cycles were followed by a final extension at 72 °C for 7 minutes. Finally, 10 μL of the final mixture were mixed with 1 μL of dye solution ethidium bromide (Promega, USA) and resolved in 1% agarose gel electrophoresis.

Antibiotic susceptibility testing

The isolates of V. parahaemolyticus were tested against 28 antimicrobial agents (Oxoid, UK). Susceptibility testing was performed using the disk diffusion method according to CLSI protocols. The following antibiotic agents were tested: ampicillin (AMP: 25 μg), ticarcillin (TE: 75 μg), carbenicillin (CAR: 100 μg), piperacillin (PRL: 100 μg), amoxycillin/clavulanic acid ‘Augmentin’ (AMC: 30 μg), piperacillin/tazobactam (TZP: 110 μg), cephalothin (KF: 30 μg), cefoxitin (FOX: 30 μg), cefaclor (CEC: 30 μg), ceftizoxime (ZOX: 30 μg), cefotaxime (CTX: 30 μg), ceftriaxone (CRO: 30 μg), ceftazidime (CAZ: 30 μg), cefepime (FEP: 30 μg), chloramphenicol (C: 30 μg), kanamycin (K: 30 μg), gentamicin (CN: 10 μg), streptomycin (S: 10 μg), amikacin (AK: 30 μg), tetracycline (TE: 30 μg), imipenem (IPM: 10 μ), meropenem (MEM: 10 μg), nalidixic acid (NA: 30 μg), ciprofloxacin (CIP: 5 μg), sulfamethoxazole/trimethoprim (SXT: 25 μg), aztreonam (ATM: 30 μg), and nitrofurantoin (F: 300 μg). The reference strain Escherichia coli (ATCC11775) was used as a control during antibiogram testing.

Multiple antibiotic resistance index and MDR definition

The multiple antibiotics resistance (MAR) index was calculated as described by Krumperman (1983) 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 non-susceptibility of the organism to at least one agent in three or more categories of antimicrobials (Magiorakos et al. 2012).

RESULTS

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

Using traditional microbiological techniques (i.e., culturing and biochemical tests), 84 of the 375 samples (22%) were determined to be positive for the presence of V. parahaemolyticus.

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.

Table 2

Number of V. parahaemolyticus isolates confirmed by PCR targeting toxR and tlh genes

Location No. of water samples Presumptive isolates (culture and biochemical) toxR tlh No. of positive water samples confirmed by PCR 
AZB 25 21 
CTJ 
DMC 10 
DMF 20 
FNC 35 35 
HMF 50 24 
KBC 35 40 
KBF 15 16 
LAK 40 50 
MOI 15 15 15 
PBJ 30 53 
QTC 20 12 10 10 
RTC 
SEC 15 
SHB 25 36 
TRC 30 10 
UOD 10 
Total 375 340 41 (12%) 41 (12%) 23 (6.13%) 
Location No. of water samples Presumptive isolates (culture and biochemical) toxR tlh No. of positive water samples confirmed by PCR 
AZB 25 21 
CTJ 
DMC 10 
DMF 20 
FNC 35 35 
HMF 50 24 
KBC 35 40 
KBF 15 16 
LAK 40 50 
MOI 15 15 15 
PBJ 30 53 
QTC 20 12 10 10 
RTC 
SEC 15 
SHB 25 36 
TRC 30 10 
UOD 10 
Total 375 340 41 (12%) 41 (12%) 23 (6.13%) 

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 = 41) from the coast of the Eastern Province of Saudi Arabia is listed in Table 3.

Table 3

Characterization of V. parahaemolyticus isolates (n = 41) collected from the coastal water environment of the Eastern Province of Saudi Arabia

Isolate no. Sample location Isolate code Date of isolation Water parameters
 
Urease test PCR
 
Temp (°C) pH toxtlh tdh trh toxRS 
KBC VP116 24/02/2015 19.9 8.8 − − − 
FNC VP10 07/04/2015 24.8 8.35 − − − − 
FNC VP55 07/04/2015 24.8 8.35 − − − − 
FNC VP56 07/04/2015 24.8 8.35 − − − − 
FNC VP57 07/04/2015 24.8 8.35 − − − − 
FNC VP58 07/04/2015 24.8 8.35 − − − − 
FNC VP59 07/04/2015 24.8 8.35 − − − − 
DMF VP22 26/05/2015 30.4 8.36 − − − 
DMF VP23 26/05/2015 30.4 8.36 − − − 
10 DMF VP24 26/05/2015 30.4 8.36 − − − 
11 DMC VP25 26/05/2015 30.4 8.41 − − − − 
12 DMF VP68 27/05/2015 29.4 8.45 − − − − 
13 QTC VP27 19/10/2015 32.4 8.2 − − − − 
14 QTC VP28 19/10/2015 32.4 8.2 − − − 
15 QTC VP29 19/10/2015 32.4 8.2 − − − − 
16 QTC VP30 19/10/2015 32.3 8.26 − − − − 
17 QTC VP31 19/10/2015 32.3 8.26 − − − − 
18 QTC VP32 19/10/2015 32.3 8.26 − − − − 
19 SEC VP26 19/10/2015 31.8 8.49 − − − − 
20 TRC VP34 19/10/2015 32.4 8.36 − − − 
21 PBJ VP36 28/10/2015 29.4 8.44 − − − 
22 MOI VP125 30/12/2015 16.9 8.76 − − − − 
23 MOI VP126 30/12/2015 16.9 8.76 − − − −- 
24 MOI VP127 30/12/2015 16.9 8.76 − − − − 
25 MOI VP128 30/12/2015 16.9 8.76 − − − − 
26 MOI VP129 30/12/2015 16.9 8.76 − − − − 
27 MOI VP130 30/12/2015 16.9 8.76 − − − − 
28 MOI VP131 30/12/2015 16.9 8.76 − − − − 
29 MOI VP132 30/12/2015 16.9 8.76 − − − − 
30 MOI VP133 30/12/2015 16.9 8.76 − − − − 
31 MOI VP134 30/12/2015 16.9 8.76 − − − − 
32 MOI VP135 30/12/2015 16.9 8.76 − − − − 
33 MOI VP136 30/12/2015 16.9 8.76 − − − − 
34 MOI VP137 30/12/2015 16.9 8.76 − − − − 
35 MOI VP138 30/12/2015 16.9 8.76 − − − − 
36 MOI VP139 30/12/2015 16.9 8.76 − − − 
37 QTC VP140 24/01/2016 21.1 8.85 − − − − 
38 QTC VP141 24/01/2016 21.1 8.85 − − − − 
39 QTC VP142 24/01/2016 21.1 8.85 − − − − 
40 QTC VP143 24/01/2016 21.1 8.85 − − − − 
41 SEC VP154 24/01/2016 21.1 8.6 − − − − 
Total      41 41 
Isolate no. Sample location Isolate code Date of isolation Water parameters
 
Urease test PCR
 
Temp (°C) pH toxtlh tdh trh toxRS 
KBC VP116 24/02/2015 19.9 8.8 − − − 
FNC VP10 07/04/2015 24.8 8.35 − − − − 
FNC VP55 07/04/2015 24.8 8.35 − − − − 
FNC VP56 07/04/2015 24.8 8.35 − − − − 
FNC VP57 07/04/2015 24.8 8.35 − − − − 
FNC VP58 07/04/2015 24.8 8.35 − − − − 
FNC VP59 07/04/2015 24.8 8.35 − − − − 
DMF VP22 26/05/2015 30.4 8.36 − − − 
DMF VP23 26/05/2015 30.4 8.36 − − − 
10 DMF VP24 26/05/2015 30.4 8.36 − − − 
11 DMC VP25 26/05/2015 30.4 8.41 − − − − 
12 DMF VP68 27/05/2015 29.4 8.45 − − − − 
13 QTC VP27 19/10/2015 32.4 8.2 − − − − 
14 QTC VP28 19/10/2015 32.4 8.2 − − − 
15 QTC VP29 19/10/2015 32.4 8.2 − − − − 
16 QTC VP30 19/10/2015 32.3 8.26 − − − − 
17 QTC VP31 19/10/2015 32.3 8.26 − − − − 
18 QTC VP32 19/10/2015 32.3 8.26 − − − − 
19 SEC VP26 19/10/2015 31.8 8.49 − − − − 
20 TRC VP34 19/10/2015 32.4 8.36 − − − 
21 PBJ VP36 28/10/2015 29.4 8.44 − − − 
22 MOI VP125 30/12/2015 16.9 8.76 − − − − 
23 MOI VP126 30/12/2015 16.9 8.76 − − − −- 
24 MOI VP127 30/12/2015 16.9 8.76 − − − − 
25 MOI VP128 30/12/2015 16.9 8.76 − − − − 
26 MOI VP129 30/12/2015 16.9 8.76 − − − − 
27 MOI VP130 30/12/2015 16.9 8.76 − − − − 
28 MOI VP131 30/12/2015 16.9 8.76 − − − − 
29 MOI VP132 30/12/2015 16.9 8.76 − − − − 
30 MOI VP133 30/12/2015 16.9 8.76 − − − − 
31 MOI VP134 30/12/2015 16.9 8.76 − − − − 
32 MOI VP135 30/12/2015 16.9 8.76 − − − − 
33 MOI VP136 30/12/2015 16.9 8.76 − − − − 
34 MOI VP137 30/12/2015 16.9 8.76 − − − − 
35 MOI VP138 30/12/2015 16.9 8.76 − − − − 
36 MOI VP139 30/12/2015 16.9 8.76 − − − 
37 QTC VP140 24/01/2016 21.1 8.85 − − − − 
38 QTC VP141 24/01/2016 21.1 8.85 − − − − 
39 QTC VP142 24/01/2016 21.1 8.85 − − − − 
40 QTC VP143 24/01/2016 21.1 8.85 − − − − 
41 SEC VP154 24/01/2016 21.1 8.6 − − − − 
Total      41 41 

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.

Table 4

Results of antibiotic susceptibility testing performed on 41 V. parahaemolyticus isolates

Antibiotic class Antibiotic name No. of resistant (%) No. of intermediate (%) No. of sensitive (%) 
Penicillins Ampicillin 36 (88) 5 (12) 0 (0) 
Ticarcillin 18 (44) 22 (54) 1 (2) 
Carbenicillin 40 (98) 0 (0) 1 (2) 
Piperacillin 1 (2) 7 (17) 33 (80) 
Amoxy/Clavulanic 1 (2) 16 (39) 24 (59) 
Pipera/Tazobactam 0 (0) 0 (0) 41 (100) 
Cephalosporins Cephalothin 31 (76) 6 (15) 4 (10) 
Cefoxitin 2 (5) 21 (51) 18 (44) 
Cefaclor 25 (61) 3 (7) 13 (32) 
Ceftizoxime 0 (0) 10 (24) 31 (76) 
Cefotaxime 0 (0) 17 (41) 24 (59) 
Ceftriaxone 0 (0) 12 (29) 29 (71) 
Ceftazidime 0 (0) 0 (0) 41 (100) 
Cefepime 0 (0) 2 (5) 39 (95) 
Phenicols Chloramphenicol 0 (0) 0 (0) 41 (100) 
Aminoglycosides Kanamycin 2 (5) 29 (71) 10 (24) 
Gentamicin 0 (0) 0 (0) 41 (100) 
Streptomycin 12 (29) 27 (66) 2 (5) 
Amikacin 5 (12) 12 (29) 24 (59) 
Tetracycline Tetracycline 0 (0) 1 (2) 40 (98) 
Carbapenems Imipenem 0 (0) 0 (0) 41 (100) 
Meropenem 0 (0) 0 (0) 41 (100) 
Quinolones Nalidixic acid 0 (0) 0 (0) 41 (100) 
Ciprofloxacin 0 (0) 8 (20) 33 (80) 
Levofloxacin 0 (0) 0 (0) 41 (100) 
Sulfonamides Sulf./Trimethoprim 0 (0) 0 (0) 41 (100) 
Monobactams Aztreonam 11 (27) 20 (49) 10 (24) 
Nitrofurans Nitrofurantoin 1 (2) 5 (12) 35 (85) 
Antibiotic class Antibiotic name No. of resistant (%) No. of intermediate (%) No. of sensitive (%) 
Penicillins Ampicillin 36 (88) 5 (12) 0 (0) 
Ticarcillin 18 (44) 22 (54) 1 (2) 
Carbenicillin 40 (98) 0 (0) 1 (2) 
Piperacillin 1 (2) 7 (17) 33 (80) 
Amoxy/Clavulanic 1 (2) 16 (39) 24 (59) 
Pipera/Tazobactam 0 (0) 0 (0) 41 (100) 
Cephalosporins Cephalothin 31 (76) 6 (15) 4 (10) 
Cefoxitin 2 (5) 21 (51) 18 (44) 
Cefaclor 25 (61) 3 (7) 13 (32) 
Ceftizoxime 0 (0) 10 (24) 31 (76) 
Cefotaxime 0 (0) 17 (41) 24 (59) 
Ceftriaxone 0 (0) 12 (29) 29 (71) 
Ceftazidime 0 (0) 0 (0) 41 (100) 
Cefepime 0 (0) 2 (5) 39 (95) 
Phenicols Chloramphenicol 0 (0) 0 (0) 41 (100) 
Aminoglycosides Kanamycin 2 (5) 29 (71) 10 (24) 
Gentamicin 0 (0) 0 (0) 41 (100) 
Streptomycin 12 (29) 27 (66) 2 (5) 
Amikacin 5 (12) 12 (29) 24 (59) 
Tetracycline Tetracycline 0 (0) 1 (2) 40 (98) 
Carbapenems Imipenem 0 (0) 0 (0) 41 (100) 
Meropenem 0 (0) 0 (0) 41 (100) 
Quinolones Nalidixic acid 0 (0) 0 (0) 41 (100) 
Ciprofloxacin 0 (0) 8 (20) 33 (80) 
Levofloxacin 0 (0) 0 (0) 41 (100) 
Sulfonamides Sulf./Trimethoprim 0 (0) 0 (0) 41 (100) 
Monobactams Aztreonam 11 (27) 20 (49) 10 (24) 
Nitrofurans Nitrofurantoin 1 (2) 5 (12) 35 (85) 

Multiple antibiotic resistance index and MDR

The values of the MAR index ranged from 0.03 to 2.8. Ten different resistance patterns had a significant MAR value >0.2. Collectively, they were exhibited by 35% of the V. parahaemolyticus isolates. The MAR index of one of the tdh+ and two of the trh+ isolates was >0.2. The present study found 15 out of 41 (37%) isolates of V. parahaemolyticus to be MDR. One of the tdh+ isolates and two of the trh+ isolates were MDR.

DISCUSSION

Physical parameters

The strong association between the occurrence of V. parahaemolyticus and the increased temperature of the environment is well documented in the literature (Blackwell & Oliver 2008; Rosec et al. 2009; Urquhart et al. 2016). In accordance with the previous reports, we were able to isolate V. parahaemolyticus from water samples with temperatures between 16.9 °C and 32 °C (Deepanjali et al. 2005). Previous studies have found that the cultivation of V. parahaemolyticus during the winter months is difficult because they enter a viable but non-culturable state (Igbinosa & Okoh 2008). In contrast, our study was able to isolate V. parahaemolyticus from the Arabian Gulf during both summer and winter seasons. This may be explained by the temperate nature of the Arabian Gulf. Six out of the eight pathogenic isolates of V. parahaemolyticus (tdh+ or trh+ ) were recovered from seawater samples of temperatures above 29 °C. Our findings contrast with a previous report that demonstrated that the ratio of pathogenic to total V. parahaemolyticus is higher when temperatures are lower (Johnson et al. 2010).

The presence of V. parahaemolyticus in the coastal water

V. parahaemolyticus inhabits marine and estuarine environments (Fabbro et al. 2010). Its presence imposes potential health risks to the public due to its association with gastroenteritis, wound infection, and septicemia (Tena et al. 2010; Zhang & Orth 2013). Since the incidence and virulence of V. parahaemolyticus on the coast of the Eastern Province of Saudi Arabia is not well documented, the goal of this study was to assay seawater samples collected from the Arabian Gulf for V. parahaemolyticus. Our study confirmed the presence of V. parahaemolyticus in 6.13% of the collected seawater samples and nine of the 17 sample locations. The highest percentage of the isolates was recovered from Almorjan Island (MOI).

In this study, two selective media were used for the isolation of V. parahaemolyticus (TCBS and CHROM Vibrio agar). In accordance with previous studies, we found that TCBS did not effectively select for Vibrio spp. and that CHROM Vibrio agar was more specific and accurate than TCBS (Fabbro et al. 2010; Di Pinto et al. 2011). In addition, our study came to an agreement with those of Fabbro et al. (2010) and Ottaviani et al. (2013) on the fact that the identity of V. parahaemolyticus may not be fully confirmed by traditional microbiological techniques. Confirming previous research, we found that the toxR PCR assay was a reliable method for the detection of V. parahaemolyticus (Kim et al. 1999). The results of tlh PCR analysis were in complete harmony with the ones achieved by toxR analysis. Although the tlh gene is similar to other genes harbored by different Vibrio spp. increasing the likelihood of a false positive PCR result (Klein et al. 2014), our study found tlh to be a useful marker for confirming the identity of V. parahaemolyticus, as reported by Rojas et al. (2011). In summary, PCR-based toxR and tlh gene detection assays are the most accurate methods of choice for confirming the presence of V. parahaemolyticus (Jun et al. 2012). The genes tdh and trh are the most commonly detected virulence factors for V. parahaemolyticus, so many clinically isolated strains of V. parahaemolyticus possess hemolytic activity that is attributed to these two genes (Ceccarelli et al. 2013). To the best of our knowledge, this study represents the first report of the isolation of tdh+ and trh+ V. parahaemolyticus isolates from the coastal environment of the Arabian Gulf of the Eastern Province of Saudi Arabia. Our study reported the isolation of tdh+ and trh+ isolates of V. parahaemolyticus at a very low prevalence rate (1%), and none of the isolates harbored both genes (tdh+/trh+ ). Our results follow the trends of globally distributed studies that have demonstrated that it is rare to isolate virulent V. parahaemolyticus strains from environmental sources (Lopez-Joven et al. 2015; Di et al. 2016). The presence of tdh+ and trh+ V. parahaemolyticus in the Arabian Gulf is a pressing concern that has several impacts. First, the fact that these isolates are potentially diarrheal entails more health facilities to assess bacterial gastroenteritis clinical samples for the presence of V. parahaemolyticus (Jun et al. 2012). Second, not only does pathogenic V. parahaemolyticus contaminate seafood and cause disease, but it also causes enormous economic losses in the seafood industry (Fuenzalida et al. 2006; Thongjun et al. 2013). Thus, the results of this study also emphasize the importance of monitoring seafood for bacterial contaminants. Third, several reports have suggested that the O3:K6 group emerged after the pathogenic strains acquired genetic elements that increased their fitness and ability to infect humans (Nasu et al. 2000; Hurley et al. 2006). Although our study did not detect any toxRS+ isolates, research to detect the emergence of any pandemic clones should continue to be performed. Importantly, some of the isolated clinical strains of V. parahaemolyticus do not contain tdh and/or trh (Raghunath 2014). While the main hemolysin genes were absent from their genome, V. parahaemolyticus was still pathogenic, indicating that there are additional virulence factors associated with V. parahaemolyticus (Raghunath et al. 2009). Likewise, early reports of infection due to urease-positive/Kanagawa-negative strains of V. parahaemolyticus have been documented (Kelly & Stroh 1989; Honda et al. 1992). Our results included two isolates with a weak urease-positive result (<1%). Suthienkul et al. (1995) suggested a correlation between the production of urease and TRH in V. parahaemolyticus. However, our study disputes this theory because one of the urease positive strains isolated was trh. This finding is consistent with the results of Devi et al. (2009) and Alaboudi et al. (2016).

Antibiotic susceptibility testing

The results of the antibiotic susceptibility tests placed carbenicillin at the top of the V. parahaemolyticus resistance scope (98%). As such, the resistance of V. parahaemolyticus to carbenicillin has been previously reported by Sudha et al. (2014) and Silvester et al. (2015). Also, similar to our study, they detected a high rate of ampicillin resistance (88% in our study). In fact, V. parahaemolyticus resistance to ampicillin is well-known in the literature (Shaw et al. 2014; He et al. 2016). Interestingly, ampicillin resistance was in 100% of the strains isolated by Devi et al. (2009) and Ottaviani et al. (2013). V. parahaemolyticus resistance to ampicillin and other penicillins is believed to be due to its chromosomally encoded β-lactamase (Devi et al. 2009). In addition, a high number of V. parahaemolyticus isolates were resistant to cephalothin (76%), which agrees with the results published by Igbinosa & Okoh (2008) (82%). In regards to streptomycin, the present study reported a 27% resistance rate. In accordance, Shaw et al. (2014), Lopatek et al. (2015) and He et al. (2016) have reported streptomycin resistance with the percentages of 4%, 45%, and 70.3%, respectively. The CDC organization recommends that severe or prolonged V. parahaemolyticus infections be treated with tetracycline or ciprofloxacin antibiotics (Elmahdi et al. 2016). The sensitivity rate to tetracycline (98%) brings our result to an agreement with the CDC's results. However, our ciprofloxacin results did not follow the same trends as previous reports; the response of 20% of the V. parahaemolyticus isolates was in the intermediate range. In contrast, all of the tested isolates were sensitive to piperacillin/tazobactam, ceftazidime, gentamicin, imipenem, meropenem, and sulfamethoxazole/trimethoprim. Those results are consistent with Shaw et al. (2014). The MAR index values ranged from 0.03 to 0.28.

Ten different resistance patterns had a significant MAR value >0.2. Collectively, they were expressed by 35% of the V. parahaemolyticus isolates. Most of them were isolated from Almorjan Island (MOI). MAR indices that exceed 0.2 are rendered from high risk sources of contamination which imposes human risks (Letchumanan et al. 2015; Tanil et al. 2005). Accordingly, the present study found that 15 out of 41 (37%) isolates of V. parahaemolyticus were MDR, most of which originated from MOI as well. MDR has previously been reported in V. parahaemolyticus environmental isolates (Silvester et al. 2015; He et al. 2016). The resistance patterns observed in our study were consistent with those of Ottaviani et al. (2013); there was no significant difference in the distribution of the resistance patterns between virulent (tdh+/trh+ ) and non-virulent isolates (tdh/trh ).

The frequency of dangerous antibiotic-resistant bacteria has been increasing over the past several decades (Fair & Tor 2014). 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. 2016). Although antimicrobial drugs are essential for food security and productivity, the effects of excessive and inappropriate antimicrobial application likely outweigh the benefits (FAO 2015). The Food and Agriculture Organization of the United Nations (FAO) have recently set an action plan to improve the awareness on antimicrobial resistance and promote prudent use of antimicrobials (FAO 2015). The increase in antimicrobial resistance is also likely caused by seawater contamination from illegal dumping of medical waste or sewage containing human-consumed antibiotics (Xu et al. 2016). The transmission of MDR genes jeopardizes the effectiveness of vibrios infection control programs and complicates the treatment of severe Vibrio spp. infections (Liu et al. 2013; Li et al. 2015). Furthermore, resistant strains present in the environment act as potential reservoirs of drug resistance genes, which may be further transferred to pathogenic bacteria through horizontal gene transfer (Silvester et al. 2015).

CONCLUSIONS

Having used highly accurate detection and identification methods (i.e., a combination of PCR-based and culture-based detection techniques), this study confirms the presence of V. parahaemolyticus in the Arabian Gulf coast of the Eastern Province of Saudi Arabia. In addition, this study represents the first evidence of the presence of potentially pathogenic (tdh+ and trh+ ) isolates of V. parahaemolyticus in this area. The presence of V. parahaemolyticus in the environment is a pressing concern because it has been associated with human infections, seafood contamination, and large disease outbreaks. The antibiotic susceptibility tests revealed high resistance of V. parahaemolyticus to clinically important antibiotics. These findings highlight the need for protecting aquatic environments from the effects of irresponsible consumption of antimicrobial agents. Moreover, this study serves as a baseline on which future studies can monitor any future changes in V. parahaemolyticus antibiotic resistance and associated human health risks.

ACKNOWLEDGEMENTS

The project is funded by National Science, Technology and Innovation Program (NSTIP), King Abdul-Aziz City for Science and Technology (KACST), Kingdom of Saudi Arabia, Award Number (10-ENV1337-46).

REFERENCES

REFERENCES
Alaboudi
,
A. R.
,
Ababneh
,
M.
,
Osaili
,
T. M.
&
Al Shloul
,
K.
2016
Detection, identification, and prevalence of pathogenic Vibrio parahaemolyticus in fish and coastal environment in Jordan
.
J. Food Sci.
81
(
1
),
M130
M134
.
doi: 10.1111/1750-3841.13151
.
PMID:26554333
Ansaruzzaman
,
M.
,
Chowdhury
,
A.
,
Bhuiyan
,
N. A.
,
Sultana
,
M.
,
Safa
,
A.
,
Lucas
,
M.
,
von Seidlein
,
L.
,
Barreto
,
A.
,
Chaignat
,
C. L.
,
Sack
,
D. A.
,
Clemens
,
J. D.
,
Nair
,
G. B.
,
Choi
,
S. Y.
,
Jeon
,
Y. S.
,
Lee
,
J. H.
,
Lee
,
H. R.
,
Chun
,
J.
&
Kim
,
D. W.
2008
Characteristics of a pandemic clone of O3:K6 and O4:K68 Vibrio parahaemolyticus isolated in Beira, Mozambique
.
J. Med. Microbiol.
57
(
Pt 12
),
1502
1507
.
doi: 10.1099/jmm.0.2008/004275-0
.
PMID:19018020
Bej
,
A. K.
,
Patterson
,
D. P.
,
Brasher
,
C. W.
,
Vickery
,
M. C. L.
,
Jones
,
D. D.
&
Kaysner
,
C. A.
1999
Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tlh, tdh and trh
.
J. Microbiol. Methods
36
(
3
),
215
225
.
doi: 10.1016/S0167-7012(99)00037-8
.
Bennett
,
J. E.
,
Dolin
,
R.
&
Blaser
,
M. J.
2015
Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases
.
Saunders
,
USA
.
Blackwell
,
K. D.
&
Oliver
,
J. D.
2008
The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina estuaries
.
J. Microbiology
46
(
2
),
146
153
.
doi: 10.1007/s12275-007-0216-2
.
Brasher
,
C. W.
,
Depaola
,
A.
,
Jones
,
D. D.
&
Bej
,
A. K.
1998
Detection of microbial pathogens in shellfish with multiplex PCR
.
Curr. Microbiol.
37
(
2
).
doi: 101-107.PMID:9662609
.
CDC
2016
National Enteric Disease Surveillance: COVIS Annual Summary, 2014
.
Centers for Disease Control and Prevention
,
Atlanta, GA
,
USA
.
Ceccarelli
,
D.
,
Hasan
,
N. A.
,
Huq
,
A.
&
Colwell
,
R. R.
2013
Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors
.
Front. Cell. Infect. Microbiol.
3
.
doi:10.3389/fcimb.2013.00097
.
De Jesús Hernández-Díaz
,
L.
,
Leon-Sicairos
,
N.
,
Velazquez-Roman
,
J.
,
Flores-Villaseñor
,
H.
,
Guadron-Llanos
,
A. M.
,
Martinez-Garcia
,
J. J.
,
Vidal
,
J. E.
&
Canizalez-Roman
,
A.
2015
A pandemic Vibrio parahaemolyticus O3:K6 clone causing most associated diarrhea cases in the Pacific Northwest coast of Mexico
.
Front. Microbiol.
6
(
221
).
doi: 10.3389/fmicb.2015.00221
.
Deepanjali
,
A.
,
Kumar
,
H. S.
,
Karunasagar
,
I.
&
Karunasagar
,
I.
2005
Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India
.
Appl. Environ. Microbiol.
71
(
7
),
3575
3580
.
doi: 10.1128/AEM.71.7.3575-3580.2005
.
PMID:16000764
Devi
,
R.
,
Surendran
,
P. K.
&
Chakraborty
,
K.
2009
Antibiotic resistance and plasmid profiling of Vibrio parahaemolyticus isolated from shrimp farms along the southwest coast of India
.
World J. Microbiol. Biotechnol.
25
(
11
),
2005
2012
.
doi: 10.1007/s11274-009-0101-8
.
Di
,
D. Y.
,
Lee
,
A.
,
Jang
,
J.
,
Han
,
D.
&
Hur
,
H. G.
2016
Season-specific occurrence of potentially pathogenic Vibrio spp. on the southern coast of South Korea
.
Appl. Environ. Microbiol.
doi: 10.1128/AEM.02680-16
.
PMID:27836844
Di Pinto
,
A.
,
Terio
,
V.
,
Novello
,
L.
&
Tantillo
,
G.
2011
Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus
.
Food Control
22
(
1
),
124
127
.
doi:10.1016/j.foodcont.2010.06.013
.
Elmahdi
,
S.
,
Dasilva
,
L. V.
&
Parveen
,
S.
2016
Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: a review
.
Food Microbiology
57
,
128
134
.
doi: 10.1016/j.fm.2016.02.008
.
Fabbro
,
C.
,
Cataletto
,
B.
&
Del Negro
,
P.
2010
Detection of pathogenic Vibrio parahaemolyticus through biochemical and molecular-based methodologies in coastal waters of the Gulf of Trieste (North Adriatic Sea)
.
FEMS Microbiol. Lett.
307
(
2
),
158
164
.
doi: 10.1111/j.1574-6968.2010.01969.x
.
PMID:20412305
Fair
,
R. J.
&
Tor
,
Y.
2014
Antibiotics and bacterial resistance in the 21st century
.
Perspect. Medicin. Chem.
6
,
25
64
.
doi: 10.4137/PMC.S14459
.
PMID:25232278
FAO
2015
The FAO Action Plan on Antimicrobial Resistance 2016–2020
.
Food and Agriculture Organization of the United Nations (FAO)
,
Rome
,
Italy
.
Fuenzalida
,
L.
,
Hernandez
,
C.
,
Toro
,
J.
,
Rioseco
,
M. L.
,
Romero
,
J.
&
Espejo
,
R. T.
2006
Vibrio parahaemolyticus in shellfish and clinical samples during two large epidemics of diarrhoea in southern Chile
.
Environ. Microbiol.
8
(
4
),
675
683
.
doi: 10.1111/j.1462-2920.2005.00946.x
.
PMID:16584479
He
,
Y.
,
Jin
,
L.
,
Sun
,
F.
,
Hu
,
Q.
&
Chen
,
L.
2016
Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China
.
Environ. Sci. Pollut. Res.
23
(
15
),
15033
15040
.
doi: 10.1007/s11356-016-6614-4
.
Hurley
,
C. C.
,
Quirke
,
A.
,
Reen
,
F. J.
&
Boyd
,
E. F.
2006
Four genomic islands that mark post-1995 pandemic Vibrio parahaemolyticus isolates
.
BMC Genomics
7
(
1
),
104
.
doi: 10.1186/1471-2164-7-104
.
Igbinosa
,
E. O.
&
Okoh
,
A. I.
2008
Emerging Vibrio species: an unending threat to public health in developing countries
.
Res. Microbiol.
159
(
7–8
),
495
506
.
doi: 10.1016/j.resmic.2008.07.001
.
PMID:18692131
Johnson
,
C. N.
,
Flowers
,
A. R.
,
Noriea
,
N. F.
,
Zimmerman
,
A. M.
,
Bowers
,
J. C.
,
DePaola
,
A.
&
Grimes
,
D. J.
2010
Relationships between environmental factors and pathogenic vibrios in the northern Gulf of Mexico
.
Appl. Environ. Microbiol.
76
(
21
),
7076
7084
.
doi: 10.1128/AEM.00697-10
.
Jun
,
J. W.
,
Kim
,
J. H.
,
Choresca
,
C. H.
Jr
,
Shin
,
S. P.
,
Han
,
J. E.
,
Han
,
S. Y.
,
Chai
,
J. Y.
&
Park
,
S. C.
2012
Isolation, molecular characterization, and antibiotic susceptibility of Vibrio parahaemolyticus in Korean seafood
.
Foodborne Pathog. Dis.
9
(
3
),
224
231
.
doi: 10.1089/fpd.2011.1018
.
PMID:22216989
Kaysner
,
C. A.
&
DePaola
,
A.
2004
Vibrio. Bacteriological Analytical Manual Online
.
US Food and Drug Administration
,
Silver Springs, MD
,
USA
.
Kelly
,
M. T.
&
Stroh
,
E. M.
1989
Urease-positive, Kanagawa-negative Vibrio parahaemolyticus from patients and the environment in the Pacific Northwest
.
J. Clin. Microbiol.
27
(
12
),
2820
2822
.
PMID:2592543
Kim
,
Y. B.
,
Okuda
,
J.
,
Matsumoto
,
C.
,
Takahashi
,
N.
,
Hashimoto
,
S.
&
Nishibuchi
,
M.
1999
Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene
.
J. Clin. Microbiol.
37
(
4
),
1173
1177
.
Klein
,
S. L.
,
Gutierrez West
,
C. K.
,
Mejia
,
D. M.
&
Lovell
,
C. R.
2014
Genes similar to the Vibrio parahaemolyticus virulence-related genes tdh, tlh, and vscc2 occur in other vibrionaceae species isolated from a pristine estuary
.
Appl. Environ. Microbiol.
80
(
2
),
595
602
.
doi: 10.1128/AEM.02895-13
.
PMID:24212573
Krumperman
,
P. H.
1983
Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods
.
Appl. Environ. Microbiol.
46
(
1
),
165
170
.
PMID:6351743
Letchumanan
,
V.
,
Yin
,
W. F.
,
Lee
,
L. H.
&
Chan
,
K. G.
2015
Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia
.
Front. Microbiol.
6
(
33
).
doi: 10.3389/fmicb.2015.00033
.
PMID:25688239
Li
,
R.
,
Lin
,
D.
,
Chen
,
K.
,
Yin Wong
,
M. H.
&
Chen
,
S.
2015
First detection of AmpC beta-lactamase blaCMY-2 on a conjugative IncA/C plasmid in a Vibrio parahaemolyticus isolate of food origin
.
Antimicrob. Agent. Chemother.
59
(
7
),
4106
4111
.
doi: 10.1128/AAC.05008-14
.
Li
,
J.
,
Xue
,
F.
,
Yang
,
Z.
,
Zhang
,
X.
,
Zeng
,
D.
,
Chao
,
G.
,
Jiang
,
Y.
&
Li
,
B.
2016
Vibrio parahaemolyticus strains of pandemic serotypes identified from clinical and environmental samples from Jiangsu, China
.
Front. Microbiol.
7
,
787
.
doi: 10.3389/fmicb.2016.00787
.
PMID:27303379
Liu
,
M.
,
Wong
,
M. H. Y.
&
Chen
,
S.
2013
Molecular characterisation of a multidrug resistance conjugative plasmid from Vibrio parahaemolyticus
.
Int. J. Antimicrob. Agent.
42
(
6
),
575
579
.
doi: 10.1016/j.ijantimicag.2013.08.014
.
Lopatek
,
M.
,
Wieczorek
,
K.
&
Osek
,
J.
2015
Prevalence and antimicrobial resistance of Vibrio parahaemolyticus isolated from raw shellfish in Poland
.
J. Food Prot.
78
(
5
),
1029
1033
.
doi: 10.4315/0362-028X.JFP-14-437
.
PMID:25951402
Lopez-Joven
,
C.
,
De Blas
,
I.
,
Furones
,
M. D.
&
Roque
,
A.
2015
Prevalences of pathogenic and non-pathogenic Vibrio parahaemolyticus in mollusks from the Spanish Mediterranean Coast
.
Front. Microbiol.
6
,
736
.
doi: 10.3389/fmicb.2015.00736
.
PMID:26284033
Magiorakos
,
A. P.
,
Srinivasan
,
A.
,
Carey
,
R. B.
,
Carmeli
,
Y.
,
Falagas
,
M. E.
,
Giske
,
C. G.
,
Harbarth
,
S.
,
Hindler
,
J. F.
,
Kahlmeter
,
G.
,
Olsson-Liljequist
,
B.
,
Paterson
,
D. L.
,
Rice
,
L. B.
,
Stelling
,
J.
,
Struelens
,
M. J.
,
Vatopoulos
,
A.
,
Weber
,
J. T.
&
Monnet
,
D. L.
2012
Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance
.
Clin. Microbiol. Infect.
18
(
3
),
268
281
.
doi: 10.1111/j.1469-0691.2011.03570.x
.
PMID:21793988
Mahmoud
,
H. M.
,
Al-Hasan
,
R. H.
,
Sorkhoh
,
N. A.
,
Eliyas
,
M.
&
Radwan
,
S. S.
2009
Attenuation of oil pollutants in the Arabian Gulf water by bacteria naturally associated with live fish
.
Int. Biodeterior. & Biodegrad.
63
(
5
),
615
620
.
doi:http://dx.doi.org/10.1016/j.ibiod.2009.04.005
Martinez-Urtaza
,
J.
,
Simental
,
L.
,
Velasco
,
D.
,
Depaola
,
A.
,
Ishibashi
,
M.
,
Nakaguchi
,
Y.
,
Nishibuchi
,
M.
,
Carrera-Flores
,
D.
,
Rey-Alvarez
,
C.
&
Pousa
,
A.
2005
Pandemic Vibrio parahaemolyticus O3:K6, Europe
.
Emerg. Infect. Dis.
11
(
8
),
1319
1320
.
doi: 10.3201/eid1108.050322
.
PMID:16110585
Matsumoto
,
C.
,
Okuda
,
J.
,
Ishibashi
,
M.
,
Iwanaga
,
M.
,
Garg
,
P.
,
Rammamurthy
,
T.
,
Wong
,
H. C.
,
Depaola
,
A.
,
Kim
,
Y. B.
,
Albert
,
M. J.
&
Nishibuchi
,
M.
2000
Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses
.
J. Clin. Microbiol.
38
(
2
),
578
585
.
PMID:10655349
Naser
,
H. A.
2013
Assessment and management of heavy metal pollution in the marine environment of the Arabian Gulf: a review
.
Mar. Pollut. Bull.
72
(
1
),
6
13
.
doi: 10.1016/j.marpolbul.2013.04.030
.
PMID:23711845
Nasu
,
H.
,
Iida
,
T.
,
Sugahara
,
T.
,
Yamaichi
,
Y.
,
Park
,
K. S.
,
Yokoyama
,
K.
,
Makino
,
K.
,
Shinagawa
,
H.
&
Honda
,
T.
2000
A filamentous phage associated with recent pandemic Vibrio parahaemolyticus O3:K6 strains
.
J. Clin. Microbiol.
38
(
6
),
2156
2161
.
PMID:10834969
Okuda
,
J.
,
Ishibashi
,
M.
,
Hayakawa
,
E.
,
Nishino
,
T.
,
Takeda
,
Y.
,
Mukhopadhyay
,
A. K.
,
Garg
,
S.
,
Bhattacharya
,
S. K.
,
Nair
,
G. B.
&
Nishibuchi
,
M.
1997
Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan
.
J. Clin. Microbiol.
35
(
12
),
3150
3155
.
PMID:9399511
Ottaviani
,
D.
,
Leoni
,
F.
,
Talevi
,
G.
,
Masini
,
L.
,
Santarelli
,
S.
,
Rocchegiani
,
E.
,
Susini
,
F.
,
Montagna
,
C.
,
Monno
,
R.
,
D'Annibale
,
L.
,
Manso
,
E.
,
Oliva
,
M.
&
Pazzani
,
C.
2013
Extensive investigation of antimicrobial resistance in Vibrio parahaemolyticus from shellfish and clinical sources, Italy
.
Int. J. Antimicrob. Agents
42
(
2
),
191
193
.
doi: 10.1016/j.ijantimicag.2013.05.003
.
PMID:23796895
Quilici
,
M.-L.
,
Robert-Pillot
,
A.
,
Picart
,
J.
&
Fournier
,
J.-M.
2005
Pandemic Vibrio parahaemolyticus O3:K6 spread, France
.
Emerg. Infect. Dis.
11
(
7
),
1148
1149
.
doi: 10.3201/eid1107.041008
.
Raghunath
,
P.
2014
Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus
.
Front. Microbiol.
5
,
805
.
doi: 10.3389/fmicb.2014.00805
.
PMID:25657643
Raghunath
,
P.
,
Karunasagar
,
I.
&
Karunasagar
,
I.
2009
Improved isolation and detection of pathogenic Vibrio parahaemolyticus from seafood using a new enrichment broth
.
Int. J. Food Microbiol.
129
(
2
),
200
203
.
doi: 10.1016/j.ijfoodmicro.2008.11.026
.
Rojas
,
M. V.
,
Matte
,
M. H.
,
Dropa
,
M.
,
Silva
,
M. L.
&
Matte
,
G. R.
2011
Characterization of Vibrio parahaemolyticus isolated from oysters and mussels in Sao Paulo, Brazil
.
Rev. Inst. Med. Trop. Sao Paulo
53
(
4
),
201
205
.
PMID:21915463
Roque
,
A.
,
Lopez-Joven
,
C.
,
Lacuesta
,
B.
,
Elandaloussi
,
L.
,
Wagley
,
S.
,
Furones
,
M. D.
,
Ruiz-Zarzuela
,
I.
,
de Blas
,
I.
,
Rangdale
,
R.
&
Gomez-Gil
,
B.
2009
Detection and identification of tdh- and trh-positive Vibrio parahaemolyticus strains from four species of cultured bivalve molluscs on the Spanish Mediterranean Coast
.
Appl. Environ. Microbiol.
75
(
23
),
7574
7577
.
doi: 10.1128/AEM.00772-09
.
PMID:19801467
Rosec
,
J.-P.
,
Simon
,
M.
,
Causse
,
V.
&
Boudjemaa
,
M.
2009
Detection of total and pathogenic Vibrio parahaemolyticus in shellfish: comparison of PCR protocols using pr72 h or toxR targets with a culture method
.
Int. J. Food Microbiol.
129
(
2
),
136
145
.
doi: 10.1016/j.ijfoodmicro.2008.11.017
.
Shaw
,
K. S.
,
Rosenberg Goldstein
,
R. E.
,
He
,
X.
,
Jacobs
,
J. M.
,
Crump
,
B. C.
&
Sapkota
,
A. R.
2014
Antimicrobial susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus recovered from recreational and commercial areas of Chesapeake Bay and Maryland Coastal Bays
.
PLoS One
9
(
2
),
e89616
.
doi: 10.1371/journal.pone.0089616
.
PMID:24586914
Sheppard
,
C.
,
Al-Husiani
,
M.
,
Al-Jamali
,
F.
,
Al-Yamani
,
F.
,
Baldwin
,
R.
,
Bishop
,
J.
,
Benzoni
,
F.
,
Dutrieux
,
E.
,
Dulvy
,
N. K.
,
Durvasula
,
S. R.
,
Jones
,
D. A.
,
Loughland
,
R.
,
Medio
,
D.
,
Nithyanandan
,
M.
,
Pilling
,
G. M.
,
Polikarpov
,
I.
,
Price
,
A. R.
,
Purkis
,
S.
,
Riegl
,
B.
,
Saburova
,
M.
,
Namin
,
K. S.
,
Taylor
,
O.
,
Wilson
,
S.
&
Zainal
,
K.
2010
The Gulf: a young sea in decline
.
Mar. Pollut. Bull.
60
(
1
),
13
38
.
doi: 10.1016/j.marpolbul.2009.10.017
.
PMID:20005533
Sudha
,
S.
,
Mridula
,
C.
,
Silvester
,
R.
&
Hatha
,
A. A. M.
2014
Prevalence and antibiotic resistance of pathogenic Vibrios in shellfishes from Cochin market
.
Indian J. Geo-Mar. Sci.
43
(
5
),
815
824
.
Suthienkul
,
O.
,
Ishibashi
,
M.
,
Iida
,
T.
,
Nettip
,
N.
,
Supavej
,
S.
,
Eampokalap
,
B.
,
Makino
,
M.
&
Honda
,
T.
1995
Urease production correlates with possession of the trh gene in Vibrio parahaemolyticus strains isolated in Thailand
.
J. Infect. Dis.
172
(
5
),
1405
1408
.
PMID:7594689
Tada
,
J.
,
Ohashi
,
T.
,
Nishimura
,
N.
,
Shirasaki
,
Y.
,
Ozaki
,
H.
,
Fukushima
,
S.
,
Takano
,
J.
,
Nishibuchi
,
M.
&
Takeda
,
Y.
1992
Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction
.
Mol. Cell. Probes
6
(
6
),
477
487
.
PMID:1480187
Tanil
,
G. B.
,
Radu
,
S.
,
Nishibuchi
,
M.
,
Rahim
,
R. A.
,
Napis
,
S.
,
Maurice
,
L.
&
Gunsalam
,
J. W.
2005
Characterization of vibrio parahaemolyticus isolated from coastal seawater in peninsular Malaysia
.
Southeast Asian J. Trop. Med. Public Health
36
(
4
),
940
945
.
PMID:16295549
Tena
,
D.
,
Arias
,
M.
,
Alvarez
,
B. T.
,
Mauleon
,
C.
,
Jimenez
,
M. P.
&
Bisquert
,
J.
2010
Fulminant necrotizing fasciitis due to Vibrio parahaemolyticus
.
J. Med. Microbiol.
59
(
2
),
235
238
.
doi: 10.1099/jmm.0.014654-0
.
Thongjun
,
J.
,
Mittraparp-Arthorn
,
P.
,
Yingkajorn
,
M.
,
Kongreung
,
J.
,
Nishibuchi
,
M.
&
Vuddhakul
,
V.
2013
The trend of Vibrio parahaemolyticus infections in Southern Thailand from 2006 to 2010
.
Trop. Med. Health
41
(
4
),
151
156
.
doi: 10.2149/tmh.2013-06
.
PMID:24478592
Urquhart
,
E. A.
,
Jones
,
S. H.
,
Yu
,
J. W.
,
Schuster
,
B. M.
,
Marcinkiewicz
,
A. L.
,
Whistler
,
C. A.
&
Cooper
,
V. S.
2016
Environmental conditions associated with elevated Vibrio parahaemolyticus concentrations in Great Bay Estuary, New Hampshire
.
PLoS One
11
(
5
),
e0155018
.
doi: 10.1371/journal.pone.0155018
.
PMID:27144925
Velazquez-Roman
,
J.
,
Leon-Sicairos
,
N.
,
De Jesus Hernandez-Diaz
,
L.
&
Canizalez-Roman
,
A.
2014
Pandemic Vibrio parahaemolyticus O3:K6 on the American continent
.
Front. Cell. Infect. Microbiol.
3
,
110
.
doi: 10.3389/fcimb.2013.00110
.
PMID:24427744
Xu
,
X.
,
Cheng
,
J.
,
Wu
,
Q.
,
Zhang
,
J.
&
Xie
,
T.
2016
Prevalence, characterization, and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China
.
BMC Microbiol.
16
(
32
).
doi: 10.1186/s12866-016-0650-6
.
PMID:26955871
Zhang
,
L.
&
Orth
,
K.
2013
Virulence determinants for Vibrio parahaemolyticus infection
.
Curr. Opin. Microbiol.
16
(
1
),
70
77
.
doi: 10.1016/j.mib.2013.02.002
.
PMID:23433802