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.
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.
Total number of seawater samples collected from each location
Location . | Number of samples . |
---|---|
Alaziziyah Beach (AZB) | 25 |
Corniche Tiba Jubail (CTJ) | 5 |
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) | 5 |
Palm Beach Jubail (PBJ) | 30 |
Qatif Corniche (QTC) | 20 |
Ras Tanura Corniche (RTC) | 5 |
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) | 5 |
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) | 5 |
Palm Beach Jubail (PBJ) | 30 |
Qatif Corniche (QTC) | 20 |
Ras Tanura Corniche (RTC) | 5 |
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.
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 | 0 | 0 | 0 |
CTJ | 5 | 6 | 0 | 0 | 0 |
DMC | 10 | 2 | 1 | 1 | 1 |
DMF | 20 | 8 | 4 | 4 | 2 |
FNC | 35 | 35 | 6 | 6 | 6 |
HMF | 50 | 24 | 0 | 0 | 0 |
KBC | 35 | 40 | 1 | 1 | 1 |
KBF | 15 | 16 | 0 | 0 | 0 |
LAK | 40 | 50 | 0 | 0 | 0 |
MOI | 5 | 15 | 15 | 15 | 5 |
PBJ | 30 | 53 | 1 | 1 | 1 |
QTC | 20 | 12 | 10 | 10 | 4 |
RTC | 5 | 4 | 0 | 0 | 0 |
SEC | 15 | 4 | 2 | 2 | 2 |
SHB | 25 | 36 | 0 | 0 | 0 |
TRC | 30 | 10 | 1 | 1 | 1 |
UOD | 10 | 4 | 0 | 0 | 0 |
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 | 0 | 0 | 0 |
CTJ | 5 | 6 | 0 | 0 | 0 |
DMC | 10 | 2 | 1 | 1 | 1 |
DMF | 20 | 8 | 4 | 4 | 2 |
FNC | 35 | 35 | 6 | 6 | 6 |
HMF | 50 | 24 | 0 | 0 | 0 |
KBC | 35 | 40 | 1 | 1 | 1 |
KBF | 15 | 16 | 0 | 0 | 0 |
LAK | 40 | 50 | 0 | 0 | 0 |
MOI | 5 | 15 | 15 | 15 | 5 |
PBJ | 30 | 53 | 1 | 1 | 1 |
QTC | 20 | 12 | 10 | 10 | 4 |
RTC | 5 | 4 | 0 | 0 | 0 |
SEC | 15 | 4 | 2 | 2 | 2 |
SHB | 25 | 36 | 0 | 0 | 0 |
TRC | 30 | 10 | 1 | 1 | 1 |
UOD | 10 | 4 | 0 | 0 | 0 |
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.
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 . | toxR . | tlh . | tdh . | trh . | toxRS . | |||||
1 | KBC | VP116 | 24/02/2015 | 19.9 | 8.8 | − | + | + | + | − | − |
2 | FNC | VP10 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
3 | FNC | VP55 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
4 | FNC | VP56 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
5 | FNC | VP57 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
6 | FNC | VP58 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
7 | FNC | VP59 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
8 | DMF | VP22 | 26/05/2015 | 30.4 | 8.36 | − | + | + | − | + | − |
9 | 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 | 0 | 41 | 41 | 4 | 4 | 0 |
Isolate no. . | Sample location . | Isolate code . | Date of isolation . | Water parameters . | Urease test . | PCR . | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
Temp (°C) . | pH . | toxR . | tlh . | tdh . | trh . | toxRS . | |||||
1 | KBC | VP116 | 24/02/2015 | 19.9 | 8.8 | − | + | + | + | − | − |
2 | FNC | VP10 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
3 | FNC | VP55 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
4 | FNC | VP56 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
5 | FNC | VP57 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
6 | FNC | VP58 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
7 | FNC | VP59 | 07/04/2015 | 24.8 | 8.35 | − | + | + | − | − | − |
8 | DMF | VP22 | 26/05/2015 | 30.4 | 8.36 | − | + | + | − | + | − |
9 | 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 | 0 | 41 | 41 | 4 | 4 | 0 |
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.
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).