As an opportunistic pathogen, Vibrio alginolyticus is a Gram-negative bacterium that is found in marine animals and humans. Fish (Rigos and Katharios, 2010), crustaceans (Liu et al., 2004), and mollusks (Luna-González et al., 2002) are commonly associated with V. alginolyticus. Meanwhile, V. alginolyticus is a notable human enteropathogen associated with seafood-borne mortality and illness worldwide (Mizan et al., 2017).

With the gradual improvement of people’s living standards and the fast-growing industry for food production, aquaculture supplies over 50% of aquatic protein sources to fulfill human needs (Little et al., 2016; Norman et al., 2019). Approximately 6–8% of the total food-borne diseases are associated with fish, which is greater than the incidence of food illness cases from chicken and beef (Sheng and Wang, 2021). Moreover, pathogenic infections by bacteria cause serious disease outbreaks leading to great economic loss, which hampers sustainable development of the aquaculture industry globally (Ghittino et al., 2003; Stentiford et al., 2012; Yang et al., 2022). In European marine aquaculture, Vibrio alginolyticus is a common inhabitant of fish and bivalve hatcheries, and it has been linked to larval mortality when poor water quality favors its growth (Pantelis et al., 2017). The pathogen is particularly relevant to the Chinese aquaculture industry where it has been associated with severe economic damage (Yang et al., 2022).

It has been estimated that 1.9 million people die each year as a result of food- and water-borne illnesses in developing countries (Schlundt et al., 2004). Approximately one-third of the population suffers from microbiological food-borne diseases each year in developed countries (Andargie et al., 2008). V. alginolyticus can be transmitted to humans through raw or undercooked contaminated seafood or direct contact of open wounds or broken skin with contaminated salt or brackish water (Klontz et al., 1988; Dechet et al., 2008). The infection may cause severe soft tissue infections, sepsis, and other extraintestinal infections (Altekruse et al., 2000; Dechet et al., 2008; Scallan et al., 2011; Newton et al., 2012; Pickering et al., 2013).

Antibiotics are commonly used as the primary treatment against Vibrio alginolyticus infections in marine aquaculture (Cao et al., 2018). However, the extensive and sometimes inappropriate use of antibiotics can lead to the development of antibiotic resistance and residual antibiotics in the environment. The isolation of V. alginolyticus strains with multiple antibiotic resistance has been reported from several recent outbreaks (Mechri et al., 2015; Rameshkumar et al., 2017; Mohamad et al., 2019b).

Whole-genome sequencing (WGS) provides comprehensive genetic information and becomes more accessible and useful for serotyping (Yang et al., 2021). Many studies on genetic characteristics and drug resistance among food-borne pathogens in seafood and freshwater products have been performed worldwide (Campos et al., 2013; Odeyemi and Sani, 2016). Nevertheless, there is nearly no WGS information on V. alginolyticus isolates from seafood and freshwater products in China.

The prevalence of antibiotic-resistant microbes (ARMs) has become a worldwide issue in seafood, and many isolates from seafood have shown a higher degree of resistance against a wide range of antibiotics (Nguyen et al., 2014). This study aimed to present the genomic epidemiology and antimicrobial susceptibility of V. alginolyticus isolates from 23 types of fish products collected from four different retail outlets (supermarkets, wet markets, restaurants, and online shops) in Shanghai and nine provinces (Fujian, Shandong, Zhejiang, Sichuan, Guangxi, Jiangsu, Liaoning, Guangdong, and Heilongjiang) in China, focusing on microbiological contamination.

E. coli and Vibrio species, which are the most common bacterial pathogens in the world, pose a serious risk to public health (Chan et al., 2003; Bresee et al., 2012). Fish proteins are widely used in pharmaceuticals, nutraceuticals, and food applications (Xu et al., 2020). China is currently the largest fish consumer, producer, and retailer of seafood products in the world (Villasante et al., 2013). Over the past three decades, marine fish products with high values have become increasingly appreciated in China (Chai and Bi, 2022). Moreover, aquatic products are a popular food in China due to their taste and nutritional value (Chen et al., 2018).

However, annually approximately 800 million meals of potentially contaminated filter-feeding shellfish/bivalves and other seafood products are raw or lightly steamed (Shuval, 2003). The transmission of pathogenic bacteria in fish marketed for human consumption has implications for the fish market and consumer health (Chai and Bi, 2022). The residues of antibiotics in farmed fish influence consumers’ perceptions of the wholesomeness and safety of farmed fish over wild fish (Claret et al., 2014). Therefore, it is crucial to monitor Vibrio contamination for the safety of fish-derived food.

The isolation of V. alginolyticus strains with multiple antibiotic resistance has been reported in several recent outbreaks (Mechri et al., 2015; Mohamad et al., 2019b). Multiple antibiotic resistance (MAR) strains of V. alginolyticus have caused severe economic setbacks to the aquaculture industry (Mohamad et al., 2019b). Our study revealed that V. alginolyticus had a higher level of antibiotic resistance, which contrasts with previous research findings. For example, some studies have reported similar detection for antibiotic sensitivity of V. alginolyticus. The detection of V. alginolyticus in Hong Kong was 49.02% (25/51), all strains were sensitive to ceftriaxone, and almost all were sensitive to ceftazidime, chloramphenicol, and sulfamethoxazole except one or two strains. Most isolates were resistant to ampicillin (60.8%, 31/51), cefuroxime (66.7%, 34/51), and kanamycin (58.8%, 30/51) (Li et al., 1999). More than 80% of Vibrio isolates were resistant to ampicillin, and 2.5% of V. alginolyticus were resistant to ceftazidime and cefotetan in Italy (Zanetti et al., 2001). On the west coast of Korea, the detection of V. alginolyticus was 17.78% (16/90) and all V. alginolyticus were resistant to ampicillin and sensitive to gentamicin, tetracycline, and chloramphenicol (Kang et al., 2016). Some Vibrio species develop and are no longer susceptible to ampicillin, cefotaxime, tetracycline, and chloramphenicol (Li et al., 1999; Zanetti et al., 2001). In our study, two isolates (2020RZ128 and 2020RZ167) (1.56%, 2/128) of V. alginolyticus were resistant to gentamicin, two isolates (2020RZ111 and 2020RZ129) (1.56%, 2/128) were resistant to tetracycline, and one isolate (2020RZ129) (7.8%, 1/128) was resistant to chloramphenicol.

The use of antimicrobials in aquaculture may cause the potential risk of resistance, its transfer into the aquatic environment, and the selection of resistant strains (Balebona et al., 1998). Multidrug resistance of Vibrio species posed a major challenge for health practitioners and a huge threat to human health. Among 128 V. alginolyticus isolates, 8 (6.25%) isolates were demonstrated multiple resistance. Isolates collected from freshwater products had a higher level of antibiotic resistance (7.8%, 4/51) than marine foods (4%, 3/75). Variation in the prevalence of V. alginolyticus was likely attributable to the sampling sites, water salinity, seasons of the year, species of fish, postharvest practices, and hygienic standards applied during the handling, transport, and storage of seafood products (Lee et al., 2008; Jun et al., 2012; Abd-Elghany and Sallam, 2013).

Antibiotic resistance mechanisms of Vibrio and other bacterial species included mutation, acquisition of resistance-conferring plasmids/episomes, modification or degradation of target sites, altered drug uptake/altered membrane permeability, and induction or upregulation of drug efflux (Blair et al., 2015; Acosta-Smith et al., 2017; Teschler et al., 2017; Bina et al., 2018; Rajpara et al., 2018). On the other hand, the deregulation of critical cellular metabolic pathways, including carbon and energy metabolism, plays a role in or can modulate the resistance of pathogenic bacteria to small-molecule drugs, including antibiotics (Peng et al., 2015; Gutierrez et al., 2017; Su et al., 2018). Furfural and malonate, inhibitors of pyruvate dehydrogenase and succinate dehydrogenase (P cycle enzymes), increased resistance of isogenic ceftazidime-resistant V. alginolyticus to antibiotics (Liu et al., 2019).

The prevalence of antibiotic resistance among Vibrio species highlights the adaptive mechanisms bacteria have developed to overcome antimicrobial agents. Studies from various geographical regions demonstrate a wide range of resistance patterns to commonly used antibiotics. For instance, high levels of resistance to ampicillin have been observed in Vibrio isolates, indicating a significant challenge in the treatment of infections caused by these pathogens. Moreover, the emergence of resistance to other antibiotics such as ceftazidime, cefotetan, and even tetracycline underscores the dynamic nature of bacterial resistance and its implications for therapeutic strategies. This variability in resistance patterns across different locations and Vibrio strains reflects the complex interplay between bacterial genetics, antibiotic usage, and environmental factors.

In our study, 13 antibiotic-resistant genes were detected. As the most commonly used drug in poultry, the resistance of tetracycline is mediated by more than 40 acquired tetracycline-resistant genes, which encode for either efflux pumps, enzymatic inactivation, or ribosomal protection genes (Møller et al., 2016). Tet gene encoded a tetracycline efflux (van den Bogaard and Stobberingh, 2000; Reynolds et al., 2020); 97.1% (99/102) of isolates carried the tet gene, while two isolates are resistant to tetracycline. Given the observed resistance to tetracycline among Vibrio species, understanding the molecular mechanisms behind this phenomenon becomes crucial. Proteins encoded by tet genes confer tetracycline resistance to bacteria via multiple mechanisms: active efflux of tetracycline (efflux pumps) (Zhu et al., 2018), modification of the tetracycline binding site to inhibit binding (Li et al., 2013), and enzymatic deactivation of tetracycline (Jahantigh et al., 2020). These strategies ensure bacterial survival in the presence of tetracycline-class antibiotics, representing a significant facet of bacterial adaptive evolution.

Urinary tract infections (UTIs) are among the most common bacterial infections (Ortega-Lozano et al., 2023). The antibiotic Bactrim, or trimethoprim-sulfamethoxazole, is commonly used in the treatment of UTIs (DeMasi et al., 2021). It operates by sulfamethoxazole inhibiting bacterial folate synthesis (Iftikhar et al., 2023), while trimethoprim blocks the utilization of folate by bacteria (Janssen et al., 2015), collectively halting bacterial proliferation. However, the increasing reports of resistance to Bactrim have raised significant concerns. In our findings, five strains were resistant to Bactrim, while two isolates carried the sul gene. The emergence of resistance diminishes the therapeutic efficacy of Bactrim, complicating the treatment of UTIs. This not only poses a direct threat to patient health but also exacerbates the burden on healthcare resources.

It has been reported that fluoroquinolone resistance can be acquired through mutations in the quinolone resistance gene qnrS as well as mutations in the quinolone resistance determining region (QRDR) containing the gyrA, gyrB, parC, and parE genes (Chiou et al., 2014; Ingle et al., 2019). In our study, the isolate that carried the qnrS gene was resistant to ciprofloxacin. In total, 96% of isolates that carried the blaCARB gene were resistant to ampicillin. Interestingly, the isolate (2020RZ111) which carried the floR gene was sensitive to chloramphenicol, the same as two isolates (2020RZ79 and 2020RZ111) that carried aph(3″)-Ib gene and aph(6)-Id gene were sensitive to aminoglycoside.

The antimicrobial susceptibility testing in this study revealed differences in resistance among strains from various sources, highlighting the risk posed by Vibrio to food safety and public health. Our analysis showed that resistance is primarily influenced by the presence and expression levels of specific genes. Whole-genome sequencing and MLST typing demonstrated genetic similarities among strains, providing insight into their spread across different regions and food sources. This study emphasizes the importance of investigating strain diversity and distribution in the context of global food production and supply chains.

V. alginolyticus has been the second most common Vibrio species since 2007 due to increasing overall vibriosis rates (CDC 2014). Due to the significant economic losses caused by V. alginolyticus, the identification of its virulence genes has attracted increasing attention. However, most of the studies conducted on V. alginolyticus in the last 20 years focused on virulence-based genes. Type III secretion system (T3SS) is a major virulence factor that delivers effectors into the host eukaryotic cytoplasm (Lian et al., 2021). Basically, T3SS are ring-like structures embedded within a basal membrane and hollow needles formed from polymerized proteins. vscU, vscS, vscR, and vcrD genes encoded the T3SS C-ring protein. Vsco encoded T3SS protein. vscN encoded T3SS ATPase. vscI, vscF, tyeA, and vcrH encoded T3SS inner rod protein, needle protein, regulatory protein, and chaperone, respectively. vpoR encoded T3SS effector. vopD and vopB encoded T3SS translocator protein. In our study, we found that 54.4% (31/57) of isolates carried vopD, vopB, vcrH, and tyeA simultaneously. Additionally, 50.9% (29/57) of isolates carried the vscI and vscF genes, simultaneously, while 40.4% (23/57) of isolates simultaneously carried vscS, vscR, and vscN. Thermolabile hemolysin (tlh) is the primary virulence determinant of Vibrio (Meza et al., 2022). In our study, 42.1% (24/57) of isolates carried tlh gene, although tlh gene is known as a species marker for V. parahaemolyticus (Mohamad et al., 2019a). Different species of Vibrio form cohesive groups within which they easily exchange genetic elements to confer greater antibiotic resistance as well as regulate virulence (Ashrafudoulla et al., 2019).

Vibrio alginolyticus has been identified as prevalent in both freshwater products and marine foods, with certain isolates displaying resistance to multiple antibiotics alongside carrying the T3SS-related gene and the virulence gene tlh. This multifaceted antibiotic resistance highlights freshwater products and marine foods as potential vectors for V. alginolyticus infections in humans. Currently, the management of bacterial infections within aquaculture heavily relies on antibiotics, raising concerns about environmental impact and consumer health. Given the paramount importance of food safety in public health, there is an urgent need for the rational application of antibiotics in the aquaculture industry and stringent control measures against V. alginolyticus in retail meat products.

Our research highlights the significant ramifications of antibiotic resistance in V. alginolyticus on both public health and aquaculture methodologies. It advocates for intensified monitoring, judicious application of antibiotics, and stringent adherence to food safety standards to diminish the hazards posed by antibiotic-resistant organisms. This investigation delineates a strategic approach for ensuring food safety and fortifying public health in China, accentuating the imperative for a collaborative endeavor to adeptly navigate antibiotic resistance within aquacultural environments.

The data has been uploaded to the bioproject PRJNA1090258 on https://www.ncbi.nlm.nih.gov/bioproject.

YS: Data curation, Visualization, Formal analysis, Writing – original draft. YY: Data curation, Visualization, Formal analysis, Writing – original draft. SY: Data curation, Visualization, Formal analysis, Software, Writing – review & editing. FL: Resources, Investigation, Methogology, Writing – review & editing. YL: Resources, Investigation, Methogology, Writing – original draft. LY: Resources, Investigation, Methogology, Writing – original draft. DY: Resources, Investigation, Methogology, Writing – original draft. ZP: Resources, Investigation, Methogology, Writing – original draft. BY: Investigation, Methogology, Writing–original draft. JS: Investigation, Methogology, Writing – original draft. JX: Investigation, Methogology, Writing – original draft. YD: Investigation, Methogology, Writing – original draft. YB: Data curation, Conceptualization, Methodology, writing – review & editing, Funding acquisition.