Fresh most sold local 200 fish samples (spangled emperor, prang spotted, pearly goatfish, greater amberjack, and yellowstripe scad) and 200 shellfish (shrimp, oysters, crab, clam, and lobster) were purchased from local markets in four different emirates in the UAE (Al-Ain, Dubai, Fujairah, and Abu Dhabi). These emirates are housing the larger fish markets in the whole UAE. From each emirate, 50 fish and 50 shellfish samples were collected (10 samples per each above-mentioned type). The samples were purchased from June to September 2017 and transferred into sterile, sealable, labeled plastic bags. The samples were transported in dry ice to the food microbiology laboratory at the United Arab Emirates University (UAEU) for analysis.

Twenty-five grams of the flesh from fish and shellfish samples were homogenized in 225 ml alkaline peptone saline water (APSW, Hi Media®, Bombay, India) using a stomacher circular Unit 400 (Seward Ltd.®, London, United Kingdom) for 2 min at 260 rpm followed by incubation at 42°C for 8 h. Then, 10 ml of the incubated homogenate were streaked in duplicate on Thiosulfate-Citrate-Bile Salts-Sucrose Agar (TCBS Agar; Oxoid, Thermo Fischer Scientific) and Modified Cellobiose-Polymyxin B-Colistin Agar (mCPC Agar; APSW, Hi Media®, Bombay, India) followed by incubation at 37°C for 24 h. The suspected colonies were re-streaked on Tryptone Soy Agar (Oxoid, Thermo Fischer Scientific) supplemented with 3% Sodium Chloride (TSA + 3% NaCl) and incubated at 37°C for 24 h to obtain a pure isolate (Sujeewa et al., 2009).

The isolated bacteria were grown individually in Tryptone Soy Broth (Oxoid, Thermo Fischer Scientific) supplemented with 3% NaCl (TSB + 3% NaCl) and incubated at 37°C for 24 h. The isolates’ DNA was extracted using a QIAGEN DNA extraction kit as per the manufacturer’s instructions.

PCR assay was performed separately for general (Vibrio spp.) and specific (16S rRNA) genes of the suspected Vibrio spp. isolates. The process conditions were 35 cycles of amplification, denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 7 min. The reaction mixtures were resolved by electrophoresis in 2% agarose gel and visualized under UV light. Gel bands were compared with reference strains (V. parahaemolyticus DSM2172 and DSM19130, V. vulnificus DSM10143, and V. mimicus DSM19130). The reference strains were purchased from Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). The listed primers in Table 1 were employed to identify the Vibrio spp. and strains. A 100 bp marker was employed (Sujeewa et al., 2009).

Twenty-eight Vibrio isolates including 15 V. parahaemolyticus, 10 V. vulnificus, and 3 V. mimicus were used for antibiotic test. The isolates were inoculated into sterile TSB + 3% NaCl, which was then incubated at 37°C until turbidity (~16 h). Using a sterile cotton swab, the bacterium was inoculated on Muller Hinton Agar plates (Oxoid, Thermo Fischer Scientific). Antimicrobial susceptibility test disks (Oxoid, Thermo Fischer Scientific) of Penicillin G (10 iu), Vancomycin (2 mcg), Daptomycin (30 mcg), Ampicillin (10 mcg), Erythromycin (15 mcg), and Sulphamethoxazole/Trimethoprim (SXT; 25 mcg) were gently placed on the agar plates post which they were incubated at 37°C for 24 h. The inhibition zone was measured in millimeters (Yaashikaa et al., 2016).

Growth profile data in triplicate were subjected to the ANOVA using a general linear model (GLM). Mean comparisons were performed using Duncan’s multiple range test to compare significant differences between means for all analyses. Statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS®, Version 21). Values are expressed as mean ± SD. The contour plots were performed using Minitab v21 (Pennsylvania, United States). The regression equations used were:

For fish isolates, OD = −1.203 + 0.4149 Salt + 0.1402 pH + 0.00745 Temp.

For shellfish isolates, OD = −0.341 + 0.2859 Salt + 0.1119 pH − 0.00590 Temp.

Fish can harbor various microorganisms like Listeria monocytogenes, Yersinia spp., Salmonella spp., and Clostridium botulinum (Novoslavskij et al., 2016). The microorganisms in fish can use it as a nutrient medium and hydrolyze proteins to form biogenic amines (Lou et al., 2021). However, one notorious pathogen of great public health concern that fish can harbor is the Vibrio spp. (Novoslavskij et al., 2016). The cases of Vibriosis are on the rise even in developed countries like the United States (Baker-Austin et al., 2018).

In this study, eighth respect to fish and shellfish samples, the results showed that a total of 129 (64.5%), 184 (92%) were Vibrio spp., positive, respectively. The high prevalence of Vibrio spp. could be attributed to ecological contamination like that of the feed or to the water salinity and temperatures (Blanch et al., 2009; Baker-Austin et al., 2018). The UAE follows strict practices to prevent marine pollution; thereby contamination through this means is highly unlikely. High seawater rising temperatures attributable to the worldwide phenomenon of global warming could be a possible explanation (Baker-Austin et al., 2018). However, the current prevalence rates observed in this study may not necessarily be a cause of concern as the country has not witnessed any major outbreak of Cholera or Vibriosis (to the best of our knowledge; SCAD, 2020). Vibrio spp. is indigenous to marine waters (Hsiao and Zhu, 2020). Furthermore, not all variants of Vibrio spp., are considered as pathogenic (Lopez-Joven et al., 2015; Song et al., 2017).

The comparative higher prevalence in shellfish than fish is understandable because actively growing clear shellfish particles from water at rates ranging from 1 to 4 L/h (Rice, 2001; CDC, 2019a). It is thereby possible that during this filtration step, the Vibrio spp., present in the water body, is retained within the shellfish body (Rice, 2001; CDC, 2019a). A meta-analysis did indicate a general trend of higher Vibrio spp., contamination in oysters and clams compared to fish (Odeyemi, 2016).

Moreover, Fish is usually consumed after applying a heat treatment, which is expected to destroy any pathogenic Vibrio spp. (USDA, 2020). Further analysis regarding the prevalence of virulent strains is needed for better comprehension.

The prevalence of Vibrio spp., isolates in shellfish from Egypt, was observed to be 33% (Abd-Elghany and Sallam, 2013). A study in Iran reported a 17.1% prevalence of Vibrio spp., in shrimp samples (Asgarpoor et al., 2018). A higher population of Vibrio spp., in shrimp samples purchased from wet markets (5.04–6.34 log CFU/ml) compared to supermarkets (4.21–4.43 log CFU/ml) was observed in Malaysia (Letchumanan et al., 2015). Another study conducted in Iran revealed 26.8% of the examined fish samples were Vibrio spp., positive (Raissy et al., 2014). The prevalence of Vibrio spp., varies based on water temperature, level of salinity, season, water depth, and total suspended solids (Akoachere and Mbuntcha, 2014; Kokashvili et al., 2015; Lopez-Joven et al., 2015; Williams et al., 2017).

In fish, the majority species prevalence was Vibrio parahemolyticus and V. mimicus (7.5 and 1.5%, respectively; Figure 1). A similar result was observed in shellfish samples, with 13 and 8.5% prevalence, respectively (Figure 1). Vibrio vulnificus was found in fish (5%), but it was not present in the shellfish samples. A study conducted in the Suez Canal area (Egypt) reported an overall V. parahaemolyticus prevalence of 9.3% in shellfish (Youssef et al., 2018). The entire prevalence of the above-mentioned species does not need to be pathogenic. The virulence genes present in the bacteria are a cause of concern in terms of public health (Hasan et al., 2010; Gennari et al., 2012; Lopez-Joven et al., 2015). This study is limited to the typification of the species, and further research is needed to analyze the pathogenicity of these strains. The strain pathogenicity depends on factors like the presence of enzymes, such as urease, lipase, gelatinase, and hemolysin or the adhesiveness potential (Baffone et al., 2001).

The percentage of V. parahaemolyticus in shellfish harvested from Turkey was observed to be 0.8% (Colakoglu et al., 2006). Amongst all the Vibrio spp., studied, some studies indicate that V. alginolyticus was present in the greatest amount of samples (Gopal et al., 2005; Colakoglu et al., 2006; Yücel and Balci, 2010), while others were indicating a higher prevalence of other strains (Vibrio cholerae, Vibrio communis; Adebayo-Tayo et al., 2011; Amalina et al., 2019).

PCR was used for the molecular identification of the Vibrio spp., positive isolates. The presence of Vibrio spp. was confirmed by using both general and Vibrio spp., specific sequences. In this study, the presence of Vibrio spp., in shellfish samples were atypical in different locations. 16S rRNA is present in all Vibrio spp., isolates and could be used as a marker gene for specific detection of this bacterium (Kim et al., 1999).

Various antibiotic-resistant Vibrio spp., isolates have been reported worldwide (Baker-Austin et al., 2009; Kitaoka et al., 2011; Jun et al., 2012; Kang et al., 2017; Kurdi Al-Dulaimi and Ariffin, 2019). These bacteria develop the resistance through various mechanisms, including the development of multidrug efflux pumps, horizontal gene transfer, plasmid conjugation, or just simple random mutations (Kitaoka et al., 2011). If the antibiotic-resistant bacteria get transferred to humans via the food chain, it will create difficulties in disease treatment (Igbinosa, 2016).

With respect to V. parahemolyticus and V. mimicus isolated from fish in this study, none of the studied antibiotics (penicillin g, daptomycin, vancomycin, ampicillin, and erythromycin) were effective except for SXT (40% resistance). On a similar note, in shellfish isolates, treatment with penicillin, daptomycin, and vancomycin proved to be futile (Table 2). Erythromycin and SXT were more effective against V. parahemolyticus isolates (62.0 and 8.0% were resistant, respectively) compared to V. mimicus (94 and 12.0% were resistant, respectively) in shellfish (Table 2). The difference in resistance to antibiotics in fish and shellfish isolates despite belonging to the same strain could be because the immune response varies based on fish type—those strains isolated from fish with higher immune responses may have developed mechanisms for enhanced survival compared to fish with lower immunity (Smith et al., 2019). Further studies are needed to confirm this hypothesis. Similarly, any previous exposure to antibiotics may also encourage survival (Smith et al., 2019). Other modes of treatment could decrease the prevalence of the Vibrio spp., such as ultrasound, low-temperature treatment, and the use of ozone and saline (Zhou et al., 2002). Moreover, the fish/shellfish are expected to be marinated prior to consumption. The use of sugar, vinegar, lemon juice, or citric acid has been associated with decreasing the contamination of Vibrio spp., in fish and shellfish, respectively (Borazjani et al., 2003; Ibrahim et al., 2018).

In accordance with our observation, a previous study on Vibrio spp., isolates from cockles and clams showed resistance toward Penicillin (93%), Ampicillin (70%), Cephalothin (65%), Clindamycin (66%), Vancomycin (64%), and Erythromycin (51%; Kurdi Al-Dulaimi and Ariffin, 2019). A study conducted on 44 V. parahaemolyticus isolates from oysters in Korea revealed 90.9, 86.4, and 75.0% of the isolates being resistant to Vancomycin, Ampicillin, and Streptomycin treatment, respectively (Kang et al., 2017). Another study in Korea reported all 19 isolates of V. parahaemolyticus obtained from seafood to be resistant to more than four commercial antibiotics (Jun et al., 2012). Other studies also report similar observations (Baker-Austin et al., 2009; Lopatek et al., 2015). On the other hand, an assessment of the antimicrobial susceptibility profile of V. parahaemolyticus isolated from short mackerels (Rastrelliger brachysoma) in Malaysia revealed majority of the isolates were highly susceptible to Ampicillin Sulbactam, Meropenem, Ceftazidime, and Imipenem (Tan et al., 2017). Treatment with Ampicillin/Sulbactam or Chloramphenicol was effective against around 95% of the V. parahaemolyticus strains isolated from shrimps in Malaysia (Letchumanan et al., 2015).

In contrast to antibiotic therapy, acid electrolyzed ice water decreased Vibrio spp., populations by about 3% (He et al., 2022). Meanwhile, a synergistic antimicrobial effect on the microbiota of fish was observed when it was vacuum packaged with a coating of gelatin composed of grape seed extract. This strategy may also be useful on pathogenic species like Vibrio spp. (Zhao et al., 2021).