Molecular characterization, antibiotic resistance pattern and biofilm formation of Vibrio parahaemolyticus and V. cholerae isolated from crustaceans and humans

International Journal of Food Microbiology 274 (2018) 31–37

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Molecular characterization, antibiotic resistance pattern and biofilm formation of Vibrio parahaemolyticus and V. cholerae isolated from crustaceans and humans

T

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Heba A. Ahmeda, , Rasha M. El Bayomib, Mohamed A. Husseinb, Mariam H.E. Khedrc, Etab M. Abo Remelad,e, Ahmed M.M. El-Ashramf a

Department of Zoonoses, Faculty of Veterinary Medicine, Zagazig University, 44511, Sharkia Governorate, Egypt Department of Food Control, Faculty of Veterinary Medicine, Zagazig University, 44511 Sharkia Governorate, Egypt Department of Veterinary Public Health, Faculty of Veterinary Medicine, Zagazig University, 44511 Sharkia Governorate, Egypt d Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt e Department of Biology, Faculty of Science, Taibah University, Al Madina Al Munawarah, Saudi Arabia f Department of Fish Diseases and Health, Faculty of Fish Resources, Suez University, Egypt b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Shrimp Crab Vibrio Biofilm Antibiotic resistance

Human infection with pathogenic vibrios is associated with contaminated seafood consumption. In the present study, we examined 225 crustaceans collected from retail markets in Egypt. Stool samples from gastroenteritis patients were also examined. Bacteriological and molecular examinations revealed 34 (15.1%) V. parahaemolyticus and 2 (0.9%) V. cholerae from crustaceans, while V. parahaemolyticus isolates were identified in 3 (3%) of the human samples. The virulence-associated genes tdh and/or trh were detected in 5.9% and 100% of the crustacean and human samples, respectively, whereas the two V. cholerae isolates were positive for the ctx and hlyA genes. Antibiotic sensitivity revealed high resistance of the isolates to the used antibiotics and an average MAR index of 0.77. Biofilm formation at different temperatures indicated significantly higher biofilm formation at 37 °C and 25 °C compared with 4 °C. Frequent monitoring of seafood for Vibrio species and their antibiotic, molecular and biofilm characteristics is essential to improve seafood safety.

1. Introduction

(FDA) is 1–104 organisms/mL in raw shellfish (FDA, 2001). Human infection with V. parahaemolyticus is characterized by gastroenteritis with diarrhea (bloody or watery), low-grade fever, headache, abdominal pain, and vomiting (Honda and Iida, 1993). These symptoms are usually self-limiting; however, septicemia may develop in people with chronic debilitating conditions such as liver diseases, immune disorders and diabetes (Potasman et al., 2002). V. cholerae have been reported to cause cholera epidemics, especially in developing countries (del Refugio Castañeda Chávez et al., 2005). Out of 200 V. cholerae serogroups, most epidemics are caused by the toxigenic V. cholerae serotypes O1 and O139 (Nishibuchi and DePaola, 2005). NonO1/non-O139 strains are natural inhabitants of water environments and have recently been shown to cause sporadic cases of diarrhea due to the ingestion of contaminated seafood (Robert-Pillot et al., 2014). V. parahaemolyticus originating from seafood or the environment are mostly nonpathogenic; the pathogenicity of this species is determined by the production of thermostable toxin (TDH) and/or TDH-thermostable hemolysin (TRH), encoded by the tdh and trh genes, respectively

Seafood is a nutritious element of healthy diets in many countries, but it is also a potential source of a wide range of foodborne pathogens (Lund, 2013). Vibrio species are naturally occurring autochthonous microbes that are abundant in marine and estuarine water worldwide. Several foodborne outbreaks caused by the consumption of shellfish and fish contaminated with Vibrio spp. have been reported in different countries (Jones and Oliver, 2009; Kirs et al., 2011). Infection with Vibrio spp. occurs more frequently during summer and early fall because higher water temperatures favor the growth of these organisms (Colwell, 1984). The most important Vibrio spp. are V. parahaemolyticus and V. vulnificus, which are most frequently associated with the consumption of raw or partially cooked shellfish, whereas V. cholerae infection is mainly associated with waterborne outbreaks and, to a lesser extent, can be transmitted via shellfish consumption (BakerAustin et al., 2010; Caburlotto et al., 2016). The maximum level of V. parahaemolyticus recommended by the Food and Drug Administration

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Corresponding author. E-mail address: [email protected] (H.A. Ahmed).

https://doi.org/10.1016/j.ijfoodmicro.2018.03.013 Received 16 November 2017; Received in revised form 6 March 2018; Accepted 16 March 2018 Available online 21 March 2018 0168-1605/ © 2018 Elsevier B.V. All rights reserved.

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were then purified and identified using different biochemical tests.

(Dileep et al., 2003; Honda and Iida, 1993). The two genes are associated with hemolysis and cytotoxic activity of V. parahaemolyticus in the host cell. In V. cholerae, cholera toxin (CT) is encoded by the ctxAB gene; some strains that lack ctx gene can cause less severe and rarely life-threatening illness (Anderson et al., 2004; Karunasagar et al., 1995). Antibiotics are used intensively in aquaculture for therapy and prophylaxis (Jerbi et al., 2011), resulting in the selection of resistant strains and an increase in antibiotic resistance among Vibrio species (Tendencia and de la Peña, 2001). This has a potential risk to human health due to the direct transmission of resistant bacteria to consumers via food or the transfer of resistance genes to other human pathogens by mobile genetic elements (Duran and Marshall, 2005; Guglielmetti et al., 2009). Biofilms are complex assemblies of bacteria on biotic or abiotic surfaces and embedded within a matrix of extracellular polymeric substances that allow the organisms to survive as self-organized, threedimensional structures (Han et al., 2016; Mizan et al., 2015). The Centers for Disease Control and Prevention estimate that nearly 65% of all reported infections are caused by bacterial biofilms (Lewis, 2007). Vibrio spp. are capable of producing adherence factors that allow them to adhere to surfaces and initiate biofilm formation (Donlan, 2002). The effect of temperature on modulating virulence factors and biofilm formation in different micro-organisms has been reported (Han et al., 2016). Bacterial biofilms have heightened resistance to disinfectants, antibodies and antibiotics (Elexson et al., 2014a; Sharma et al., 2010). In Mansoura, Egypt, the prevalence of Vibrio spp. in retail shellfish has been reported, however, biofilm formation ability of the isolates was not addressed (Abd-Elghany and Sallam, 2013). To the best of our knowledge, no studies are available on the biofilm formation ability of Vibrio spp. isolated from seafood in Egypt. The correct identification and classification of Vibrio spp. in seafood sold at outlets is of utmost importance due to the associated burden on human health and the consequent economic loss for aquaculture (Chen et al., 2012; Goarant et al., 1999). Therefore, this study was designed to estimate the prevalence of Vibrio spp. in shrimp and crab sold at fish markets in Sharkia Governorate, Egypt. Diarrheal stool samples from gastroenteritis patients were also examined. The virulence determinants, antibiotic resistance profile and biofilm formation of Vibrio isolates were investigated.

2.3. Molecular identification Biochemically suspected colonies were confirmed with PCR using 16S rRNA primers specific to Vibrio species (Tarr et al., 2007). Bacterial DNA was extracted using the QIAamp DNA Mini kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's guidelines. Specific primers targeting the toxR gene were used to identify V. parahaemolyticus (Kim et al., 1999) and V. cholerae (Miller et al., 1987). To serotype V. cholerae isolates, PCR amplifying O1-rfb and O139-rfb genes were used (Hoshino et al., 1998). Molecular identification of tdh and trh virulence-associated genes in V. parahaemolyticus isolates was performed using SYBR green real-time PCR (Rizvi and Bej, 2010). Virulence-associated genes in V. cholerae isolates were identified using primers targeting the ctx gene (Mousavi et al., 2009) and the hlyA gene (Shangkuan et al., 1995). Positive controls for V. parahaemolyticus and V. cholerae were run alongside the tested isolates and were generously supplied by the Biotechnology Unit, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Giza, Egypt. Isolates identified as V. cholerae were further confirmed by sequencing of the ctx gene. QIAquick Gel Extraction Kits (Qiagen, S. A. Courtaboeuf, France) were used for amplicon extraction from gel according to the manufacturer's guidelines. DNASTAR software (Lasergene version 7.2; DNASTAR, Madison, WI) was used to analyze two sequences that were then submitted to the GenBank, which provided the two accession numbers KY228382.1 and KY228383. The identity of the two isolates with other V. cholerae isolates in the GenBank was determined. 2.4. Kanagawa phenomenon Isolates identified as V. parahaemolyticus and V. cholerae were examined for the phenotypic determination of thermostable direct hemolysin (TDH) virulence factor. For each isolate, a drop of Tryptone soya broth culture with 3% NaCl was spotted on duplicate plates of Wagatsuma agar (HiMedia, M626) containing fresh human red blood cells and then incubated at 37 °C for 24 h. Positive results were indicated by a β-hemolysis zone. Plates containing positive V. parahaemolyticus isolate and negative controls were included in the test. The positive V. parahaemolyticus isolate was generously supplied by the Microbiology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt.

2. Material and methods 2.1. Samples

2.5. Antibiotic susceptibility testing

Two hundred twenty-five crustacean samples comprising 132 shrimps (Penaeus semisulcatus) and 93 crabs (Portunus pelagicus) were aseptically collected from fish markets in Sharkia Governorate, Egypt, during June–September 2016. The shrimp samples originated from the brackish water of Gulf of Suez, while the crab samples were caught off the coast of the Mediterranean Sea at Damietta City. In addition, 100 stool samples were collected from diarrheic patients attending the Outpatient Clinic at Al-Ahrar Hospital. Informed verbal/written consent was obtained from the human participants, and the study was approved by the Committee of Animal Welfare and Research Ethics, Faculty of Veterinary Medicine, Zagazig University, Egypt.

The antibiotic susceptibility of the Vibrio isolates was determined with the Kirby–Bauer disc diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS), and the zones of inhibition were measured according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2010) when available; for ampicillin/sulbactam, kanamycin and nalidixic acid, the interpretation criteria for Enterobacteriaceae were used (Table S1). The 12 antibiotic discs (Oxoid) used were ampicillin (AMP, 10 μg), nalidixic acid (NA 30 μg), kanamycin (K, 30 μg), ciprofloxacin (CIP, 5 μg), chloramphenicol (C, 30 μg), amikacin (AK, 30 μg), gentamicin (CN, 10 μg), tetracycline (TE, 30 μg), trimethoprim/sulfamethoxazole (SXT, 25 μg), cefotaxime (CTX, 30 μg), ampicillin/sulbactam (SAM, 20 μg), and ceftazidime (CAZ, 30 μg). E. coli ATCC 25922 was used as the quality control organism. The multiple antibiotic resistance (MAR) index was determined as the ratio of the number of antibiotics to which the Vibrio isolates displayed resistance to the number of drugs to which the Vibrio isolates were exposed (Krumperman, 1983). Multidrug resistance (MDR) was defined as the resistance of an isolate to at least one agent in three or more antibiotic classes (Magiorakos et al., 2012).

2.2. Isolation and biochemical identification Isolation of Vibrio spp. was performed following the FDA's Bacteriological Analytical Manual (BAM) instructions (FDA, 2001). Ten grams of crustacean flesh were homogenized in 90 mL of sterile alkaline peptone water (HiMedia, M618) and incubated at 35 ± 2 °C for 24–48 h (ISO-TS-21872-1, 2007). A loopful of the enriched culture was streaked onto Vibrio chromogenic agar (Condalab, Pronadisa, 2054), and the plates were incubated at 37 °C for 24 h. Presumptive colonies 32

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Table 1 Proportion of Vibrio species isolated from shrimp, crab and human samples.

Table 3 Results of antibiotic susceptibility tests of Vibrio isolates.

Samples

Number examined

Vibrio spp.a

V. parahaemolyticusb

V. cholerab

Antibiotic class

Antibiotic

S

I

R

Shrimp Crab Total Humans

132 93 225 100

26 (19.7%) 10 (10.8%) 36 (16%) 3 (3%)

24 (18.2%) 10 (10.8%) 34 (15.1%) 3 (3%)

2 (1.5%) 0 2 (0.9%) 0

Penicillin

Ampicillin Ampicillin-sulbactam Cefotaxime Ceftazidime Amikacin Gentamicin Kanamycin Tetracycline Nalidixic acid Ciprofloxacin Trimethoprimsulfamethoxazole Chloramphenicol

0 0 0 1 (2.8%) 25 (69.4%) 18 (50%) 6 (16.7%) 0 8 (22.2%) 2 (5.6%) 9 (25%)

0 0 3 0 0 0 4 0 3 1 0

36 36 33 35 11 18 26 36 25 33 27

6 (16.7%)

8 (22.2%)

Cephalosporin Aminoglycosides

a b

The isolates were identified using PCR targeting 16S rRNA specific to Vibrio spp. The isolates were confirmed with species-specific PCR. Tetracyclines Quinolones

2.6. Quantification of biofilm

Sulfonamides

Biofilm formation by V. parahaemolyticus and V. cholerae at 37 °C, 25 °C and 4 °C was determined as previously described (Djordjevic et al., 2002). The experiment was performed in triplicate, and the data are represented as the mean ± standard deviation. The degrees of biofilm were determined according to O'Toole and Kolter (1998). Differences in the degree of biofilm formation at the three temperatures were examined using the Kruskal-Wallis H one-way analysis of variance (ANOVA) and post hoc Bonferroni correction. The test results were calculated using SPSS version 22 (IBM Corp. 2013, Armonk, NY). Data are presented as the mean ± SD, and significance was considered at P ˂ 0.05.

Phenolics

From June to September 2016, a qualitative analysis of 225 crustacean and 100 human samples was performed for the presence of Vibrio species. Thirty-six (16%) crustaceans were positive for vibrios. V. parahaemolyticus and V. cholerae were identified in 15.1% and 0.9% of the examined crustacean samples, respectively (Table 1). The isolation rate of V. parahaemolyticus from shrimps was significantly higher than V. cholerae, whereas V. parahaemolyticus was the only species identified in crab and human stool samples. Regarding the distribution of tdh and trh virulence-associated genes in V. parahaemolyticus isolates (Table 2), only 2 (8.4%) isolates from shrimp samples were positive; one carried the trh gene (4.2%) only, and the other harbored both genes (4.2%). Of the three isolates from humans, 2 (66.7%) were positive for the tdh gene, and 1 (33.3%) was positive for both genes. The two V. cholerae isolates harbored both the ctx and hlyA genes. To confirm the ctx gene, sequencing was performed, and 100% identity was observed with other V. cholerae isolates on the GenBank. The phenotypic characterization of hemolysis using the Kanagawa reaction revealed a positive reaction, indicated by the appearance of βhemolysis in 4 (10.8%) V. parahaemolyticus isolates (3 from humans and one from shrimp). None of the V. cholerae isolates showed the reaction. The antibiotic resistance profiles of Vibrio isolates in terms of the 12 antibiotics are shown in Table 3. All the isolates showed multiple antimicrobial resistances to at least three drugs. The MAR indices ranged from 0.58–1, with an average of 0.77 (Table 4). Four isolates exhibited resistance to all the examined antimicrobials.

Number positivea

tdh

trh

tdh/trh

Shrimp Crab Total Humans

24 10 34 3

2 (8.4%) 0 2 (5.9%) 3 (100%)

0 0 0 2 (66.7%)

1 (4.2%) 0 (0%) 1 (2.9%) 0

1 (4.2%) 0 1 (2.9%) 1 (33.3%)

(8.3%) (2.8%)

22 (61.1%)

4. Discussion 4.1. Prevalence of Vibrio spp. in crustaceans and human samples Vibrio spp. are foodborne pathogens of aquatic origin that are frequently reported in different seafoods and in gastroenteritis outbreaks worldwide. The source of Vibrio spp. in seafood could be the marine ecosystem or fecal contamination of the environment from domestic sewage (Robert-Pillot et al., 2014). In this study, 15.1% V. parahaemolyticus were identified in crustaceans. A higher isolation rate was reported for V. parahaemolyticus (33.3%) in seafood samples from Egypt (Abd-Elghany and Sallam, 2013). Higher rates have been reported in other studies, including 28% in crustaceans in Italy (Caburlotto et al., 2016), 32.3% in shrimp in Senegal (Coly et al., 2013), 37% in crabs in Brazil (Carvalho et al., 2016) and 80.8% in shrimp in Ecuador (Sperling et al., 2015). In China, contamination rates of 37.7% (Xu et al., 2014), 47.9% (Xie et al., 2017) and 81.7% in shrimp have been reported (Xie et al., 2015). Lower isolation rates of V. parahaemolyticus from shrimp were documented in Morocco (Kriem et al., 2015), Iran (Rahimi et al., 2010) and Cote d'Ivoire (Traore et al., 2012). In the present study, V. cholerae was isolated from 1.5% of the shrimp samples. This is comparable with the 2% isolation rate from shrimp in Morocco (Kriem et al., 2015). Higher percentages of 9.4% in India (Saravanan et al., 2007) and 11.4% in Ecuador (Sperling et al., 2015) were reported for shrimp. None of the examined crab samples were contaminated with V. cholerae; in contrast, other studies have reported the isolation of this pathogen from crab samples (Carvalho et al., 2016; Traore et al., 2012). Different factors caused the variation in the prevalence of Vibrio species: type of sample, isolation and identification methods, geographical origin, season, salinity, environmental parameters, storage and transportation could influence the presence of Vibrio in seafood (Abd-Elghany and Sallam, 2013; Caburlotto et al., 2016). The isolation of Vibrio spp. from the examined samples indicates a lack of hygiene, cross contamination, improper handling, or alterations

Table 2 Distribution of the virulence-associated genes tdh and trh in V. parahaemolyticus isolated from shrimp, crab and human samples. V. parahaemolyticus

(11.1%)

The results presented in Table 5 show the percentages and degrees of biofilm production by Vibrio isolates. At the three tested temperatures (37 °C, 25 °C, and 4 °C), 84.6%, 74.4%, and 43.6% of the isolates were biofilm producers, respectively. The Kruskal-Wallis H test showed a statistically significant difference in the three temperatures' effect on the ability of the Vibrio isolates to produce biofilms (P < 0.001). No significant difference between the two temperatures (25 °C and 37 °C) was observed (P = 0.364). At refrigeration temperature (4 °C), isolates were classified as weak and moderate producers, while 56.4% were unable to produce biofilm. The production of biofilm by Vibrio isolates was significantly different at 4 °C compared with 25 °C and 37 °C (P < 0.001).

3. Results

Samples

(8.3%)

(100%) (100%) (91.7%) (97.2%) (30.6%) (50%) (72.2%) (100%) (69.4%) (91.7%) (75%)

a The number positive is the number of V. parahaemolyticus isolates positive for one or both of the virulence-associated genes.

33

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Table 4 Antibiotic resistance pattern and MAR index of Vibrio spp. Resistance pattern

Resistance profile

Number of isolates

Number of antibiotics

MAR

I II III IV V VI VII VIII IX X XI XII XIII XIIII XV XVI XVII XVIII IXX XX XXI

AMP-SAM-CAZ-AK-CN-K-TE-NA-CIP-SXT-C-CTX AMP-SAM-CAZ-AK-K-TE-NA-CIP-SXT-C-CTX AMP-SAM-CAZ-CN-K-TE-NA-CIP-SXT-C-CTX AMP-SAM-CAZ-AK-K-TE-NA-CIP-SXT-CTX AMP-SAM-CAZ-AK-K-TE-NA-CIP-C-CTX AMP-SAM-CAZ-CN-TE-NA-CIP-SXT-C-CTX AMP-SAM-CAZ-CN-K-TE-CIP-SXT-C-CTX AMP-SAM-CAZ-K-TE-NA-CIP-SXT-C-CTX AMP-SAM-CAZ-K-TE-NA-CIP-SXT-C-CTX AMP-SAM-CAZ-K-TE-CIP-SXT-C-CTX AMP-SAM-CAZ-CN-TE-NA-SXT-C-CTX AMP-SAM-CAZ-CN-TE-NA-CIP-C-CTX AMP-SAM-CAZ-CN-TE-CIP-SXT-C-CTX AMP-SAM-CAZ-K-TE-NA-SXT-C-CTX AMP-SAM-CAZ-K-TE-NA-CIP-SXT-CTX AMP-SAM-CAZ-AK-K-TE-CIP-C-CTX AMP-SAM-AK-K-TE-NA-CIP-C-CTX AMP-SAM-CAZ-CN-TE-NA-CIP-CTX AMP-SAM-CAZ-K-TE-CIP-C-CTX AMP-SAM-CAZ-TE-NA-CIP-C-CTX AMP-SAM-CAZ-K-TE-CIP-CTX

4 3 8 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1a 1a 1b 2b

12 11 11 10 10 10 10 10 10 9 9 9 9 9 9 9 9 8 8 8 7

1 0.9 0.9 0.83 0.83 0.83 0.83 0.83 0.83 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.66 0.66 0.66 0.58

Average MAR index = 0.77. a V. cholerae isolates. b Clinical isolates. Table 5 Biofilm formation in Salmonella species at 4 °C, 25 °C and 37 °C. Temperature

4 °C 25 °C 37 °C

Non-producer

22 (56.4%, 0.054 ± 0.03) 10 (25.6%, 0.067 ± 0.04) 6 (15.4%, 0.072 ± 0.01)

Degree of biofilm production (%, average OD ± SD)

Overall biofilm producers

Weak

Moderate

Strong

8 (20.5%, 0.148 ± 0.03) 10 (25.6%, 0.186 ± 0.04) 7 (17.9%, 0.154 ± 0.02)

9 (23.1%, 0.291 ± 0.05) 9 (23.1%, 0.304 ± 0.05) 15 (38.5%, 0.279 ± 0.05)

0 10 (25.6%, 0.722 ± 0.06) 11 (28.2%, 0.733 ± 0.11)

17 (43.6%) 29 (74.4%) 33 (84.6%)

the tdh gene. In Egypt, 7.4% of V. parahaemolyticus isolates from seafood samples were positive for tdh, and two from shrimp harbored the trh gene (AbdElghany and Sallam, 2013). In China, 28.4% and 2.1% of V. parahaemolyticus isolates from seafood carried the trh and tdh genes, respectively, while none of the isolates harbored the two genes simultaneously (Xie et al., 2017). Another study in China reported that 45.9% of V. parahaemolyticus isolates from shrimp were positive for the trh gene, and none harbored the tdh gene (Xie et al., 2015). Other studies documented that none of the V. parahaemolyticus isolates from crustaceans harbored the tdh and/or trh genes (Coly et al., 2013; Kriem et al., 2015; Sperling et al., 2015; Traore et al., 2012). The three clinical isolates in this study were positive for the tdh and trh genes (2 harbored the tdh gene, and one carried the two genes simultaneously). In comparison, all the clinical V. parahaemolyticus isolates in China carried the tdh gene, and 77.4% carried the trh gene (Xie et al., 2017). Clinical isolates with no hemolysin genes were also found to be pathogenic, indicating that other pathogenicity determinants might be present (Mahoney et al., 2010). The pathogenicity of V. cholerae strains is associated with the cholera toxin, which is encoded by the ctxA and ctxB genes; non-O1/nonO139 strains rarely possess these genes, but they can cause less severe human illness (Rivera et al., 2001). The two V. cholerae isolates were from non-O1 and non-O139 serogroups and were positive for the ctx and hlyA genes. Most environmental V. cholerae isolates are non-toxigenic, but they can potentially cause mild gastroenteritis due to the presence of the hlyA gene (Rivera et al., 2001; Saravanan et al., 2007). In France, non-toxigenic V. cholerae non-O1/non-O139 was detected in seafood samples (Robert-Pillot et al., 2014). The ctx gene was identified in V. cholerae isolates in Iran (Mousavi et al., 2009).

in the transportation and storage temperature of the samples (Letchumanan et al., 2015). Although Vibrio spp. in raw seafood can be inactivated with heat, the possibility of cross-contamination of other food due contact with contaminated seafood has been documented (Rahimi et al., 2010). V. parahaemolyticus was isolated from 3% of the stool samples from gastroenteritis patients. In Spain, 64 patients with gastroenteritis were analyzed, and all were positive for V. parahaemolyticus (Lozano-Leon et al., 2003). Revillo et al. (2000) reported eight human cases of gastroenteritis due to V. parahaemolyticus associated with the consumption of contaminated fish and shellfish. 4.2. Virulence determinants in Vibrio isolates According to studies conducted in different regions, 0.2–3% of environmental V. parahaemolyticus isolates are potentially pathogenic due to the presence of tdh and/or trh genes (Nordstrom et al., 2007). Of the 34 examined V. parahaemolyticus isolates from crustaceans, 14.7% were positive for the tdh and/or trh genes. Phenotypic identification of potentially virulent Vibrio spp. using Kanagawa reaction revealed that only one V. parahaemolyticus isolate of shrimp origin (2.9%) and two of the clinical isolates produced β-hemolysis (66.7%). This phenomenon was also reported in 1.7% of V. parahaemolyticus isolates from seafood in Egypt (Abd-Elghany and Sallam, 2013) and 46.7% in China (Hongping et al., 2011). The detection of the tdh gene in V. parahaemolyticus isolates showed higher specificity than the Kanagawa reaction for identifying pathogenic isolates (Hongping et al., 2011). This was supported by Nishibuchi et al. (1985), who applied DNA colony hybridization to Kanagawa-negative strains; the results revealed that 16% of the isolates were positive for 34

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ranging from 4 to 37 °C was evaluated by Han et al. (2016). They reported that the isolates were able to produce biofilm at all temperatures; however, significantly stronger production was observed at 15 and 37 °C than at 4 and 10 °C. This was in accordance with the results of the current study. Mizan et al. (2016) reported that V. parahaemolyticus produced a strong biofilm at 30 °C. Thus, temperatures of 25–37 °C are considered optimum for biofilm formation by V. parahaemolyticus isolates. Another study documented that biofilm formation by V. parahaemolyticus was strongest at 37 °C, while at 4 °C, 91.7% of the isolates were weak producers, and no strong producers were observed, indicating a temperature influence (Elexson et al., 2014b). Clear temperature dependency was also observed for V. parahaemolyticus biofilm production at 25 °C compared with at 15 °C and 37 °C (Song et al., 2017). Temperature has an adverse effect on biofilm formation by affecting flagellar motility, which enhances the movement of bacteria towards the biofilm surface (Bonsaglia et al., 2014). Thus, V. parahaemolyticus can produce biofilm at markets where temperature abuse occurs, resulting in potential risks to consumers. The inhibition of biofilm formation at low temperatures (4 °C) strengthens need for strict precautions to store seafood under refrigeration conditions.

4.3. Antibiotic resistance profile The worldwide increase in the prevalence of antibiotic-resistant bacteria is a major concern for the human and veterinary sectors (Xie et al., 2015; Xie et al., 2017). Vibrio strains are exposed to antibiotics over time via the environment and thus acquire antimicrobial resistance through mobile genetic elements and horizontal gene transfer (Kümmerer, 2009). Most of the tested antibiotics, such as ciprofloxacin, chloramphenicol, gentamicin, tetracycline and trimethoprim/sulfamethoxazole, are recommended as treatments for Vibrio infection (Shaw et al., 2014). The ampicillin resistance (100%) reported in this study coincides with the CLSI standards (CLSI, 2010), which reported intrinsic resistance of Vibrio spp. to ampicillin. This is consistent with other studies (Elexson et al., 2014a; Xie et al., 2017). Although the penicillin group of antibiotics is one of the most valuable groups for primary care, the emergence of resistant bacteria in recent years has limited its usefulness (Miller et al., 2002). The clinical and aquatic isolates in the present study were highly resistant to tetracycline, ceftazidime, cefotaxime, ciprofloxacin and kanamycin. This was consistent with other studies (Jiang et al., 2014; Kitiyodom et al., 2010; Xie et al., 2015; Xu et al., 2016) and indicates public health concerns because these drugs are used to treat human infections (Xie et al., 2017). All the isolates showed multiple antimicrobial resistances to at least 7 antibiotics, with an MAR range of 0.58 to 1 and an average of 0.77. Different studies have reported V. parahaemolyticus isolates with MRA indices > 0.2 (Elexson et al., 2014a; Tang et al., 2014). The variation in the MAR index could be attributed to differences in the sources of samples (Tunung et al., 2010); geographic distribution, which has differential selective pressures for the antibiotic resistance levels (Lesley et al., 2011); and test methodologies (Robert-Pillot et al., 2004). Research has shown that an MAR higher than 0.2 could be due to contamination from high-risk sources, such as humans and farm animals frequently exposed to antibiotics, resulting in potential risk to consumers. The high MAR in the current study indicated that the isolates originated from high-risk source samples; therefore, monitoring for antimicrobial resistance is essential to identify the effectiveness of new generations of antibiotics and to ensure the safety of seafood (Yu et al., 2016). The observed high multidrug resistance of the environmental V. parahaemolyticus isolates is consistent with other observations (Xie et al., 2017). High levels of multidrug resistance could be explained by the increased chance to exchange genetic resistance determinants located on plasmids among environmental isolates as a result of extensive, uncontrolled use of antimicrobials in aquaculture and for infection treatment (Krumperman, 1983; Ottaviani et al., 2013).

5. Conclusion The present study demonstrated the presence of potentially pathogenic Vibrio species in some crustaceans for sale at markets in Egypt. These findings provide important insights onto hygienic measures that should be applied to aquatic products. The high MAR index of the isolates and their ability to form biofilm at high temperatures constitute public health hazards. Monitoring the prevalence and antibiotic resistance profile of Vibrio spp. in retail seafood is necessary to improve seafood safety. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijfoodmicro.2018.03.013. References Abd-Elghany, S.M., Sallam, K.I., 2013. Occurrence and molecular identification of Vibrio parahaemolyticus in retail shellfish in Mansoura, Egypt. Food Control 33, 399–405. Anderson, A.M., Varkey, J.B., Petti, C.A., Liddle, R.A., Frothingham, R., Woods, C.W., 2004. Non-O1 Vibrio cholerae septicemia: case report, discussion of literature, and relevance to bioterrorism. Diagn. Microbiol. Infect. Dis. 49, 295–297. Baker-Austin, C., Stockley, L., Rangdale, R., Martinez-Urtaza, J., 2010. Environmental occurrence and clinical impact of Vibrio vulnificus and Vibrio parahaemolyticus: a European perspective. Environ. Microbiol. Rep. 2, 7–18. Bonsaglia, E.C.R., Silva, N.C.C., Fernades Júnior, A., Araújo Júnior, J.P., Tsunemi, M.H., Rall, V.L.M., 2014. Production of biofilm by Listeria monocytogenes in different materials and temperatures. Food Control 35, 386–391. Caburlotto, G., Suffredini, E., Toson, M., Fasolato, L., Antonetti, P., Zambon, M., Manfrin, A., 2016. Occurrence and molecular characterisation of Vibrio parahaemolyticus in crustaceans commercialised in Venice area, Italy. Int. J. Food Microbiol. 220, 39–49. Carvalho, M.C.N., Jayme, M.M., Arenazio, G.S., Araújo, F.V., Leite, S.G.F., Del Aguila, E.M., 2016. Microbiological quality assessment by PCR and its antibiotic susceptibility in mangrove crabs (Ucides cordatus) from Guanabara Bay, Rio de Janeiro, Brazil. Int. J. Microbiol. 2016, 9. Chen, W., Xie, Y., Xu, J., Wang, Q., Gu, M., Yang, J., Zhou, M., Wang, D., Shi, C., Shi, X., 2012. Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China. Int. J. Food Microbiol. 153, 402–412. CLSI, 2010. Performance Standards for Antimicrobial Susceptibility Testing. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline, 3rd edn. (Austin, TX. M45-A2). Colwell, R.R., 1984. Vibrios in the Environment. Wiley, New York. Coly, I., Sow, A.G., Seydi, M., Martinez-Urtaza, J., 2013. Vibrio cholerae and Vibrio parahaemolyticus detected in seafood products from Senegal. Foodborne Pathog. Dis. 10, 1050–1058. del Refugio Castañeda Chávez, M., Sedas, V.P., Orrantia Borunda, E., Reynoso, F.L., 2005. Influence of water temperature and salinity on seasonal occurrences of Vibrio cholerae and enteric bacteria in oyster-producing areas of Veracruz, México. Mar. Pollut. Bull. 50, 1641–1648. Dileep, V., Kumar, H.S., Kumar, Y., Nishibuchi, M., Karunasagar, I., Karunasagar, I., 2003. Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment. Lett. Appl. Microbiol. 36, 423–427. Djordjevic, D., Wiedmann, M., McLandsborough, L.A., 2002. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl. Environ. Microbiol. 68, 2950–2958.

4.4. Biofilm formation ability V. parahaemolyticus can produce a biofilm that is more resistant than planktonic cells to antibiotics and disinfectants (Song et al., 2017). Biofilm production is dependent on the nature of the bacteria and incubation temperature. The ability of V. parahaemolyticus isolates to attach to suspended particles, zooplankton, fish and shellfish was reported to be associated with their ability to produce biofilms (Elexson et al., 2014b). This study showed that the incubation temperature had a significant effect on the ability of Vibrio spp. to produce biofilm. Incubation at 25 °C and 37 °C enhanced biofilm formation significantly more than incubation at 4 °C. Time-temperature abuse in markets could result in the propagation of the bacteria to dangerous levels (Sudha et al., 2012) and the expression of different virulence-associated determinants, including biofilms (Mahoney et al., 2010; Robert-Pillot et al., 2004). Biofilm formation by V. parahaemolyticus isolates at temperatures 35

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