Shewanella spp. are Gram-negative, facultatively anaerobic, singly flagellated, oxidase and catalase positive, rod-shaped bacteria that can produce H₂S (Goyal et al., 2011). Members of the family Shewanellaceae are naturally occurring marine bacteria, distributed worldwide (Hau and Gralnick, 2007; Lemaire et al., 2020). Over 70 species of Shewanellaceae have been identified (Lemaire et al., 2020); however, there is still uncertainty of exactly how many species are truly human pathogens. Currently, five species have been associated with human infection: S. algae, S. putrefaciens (Hau and Gralnick, 2007), S. xiamensis (Zong, 2011), S. haliotis (Poovorawan et al., 2013; Zhang et al., 2018; Liu et al., 2019) and S. upenei (Yousfi et al., 2017). One recently identified species, S. khirikhana, has been associated with disease in cultured shrimp (Prachumwat et al., 2020). Shewanella is a genus that contains species that are human and shellfish pathogens as well as species that are widely recognized as seafood spoilage organisms.

Shewanella isolates can often exhibit hemolytic activity on blood agar plates, which is indicative of their ability to lyse red blood cells. Beta-hemolytic activity is a common feature associated with pathogenicity in various bacterial species. For instance, in Staphylococcus aureus, beta hemolysins are key virulence factors that contribute to tissue invasion and immune evasion, and their activity can be significantly reduced by inhibiting the ClpP protease, central regulator of virulence factors (Böttcher and Sieber, 2008). Similarly, Shewanella species such as S. algae and S. baltica have been shown to possess beta-hemolytic activity, and this trait may be predictive of their potential virulence, especially given their ability to grow at human body temperature (Richards et al., 2008). Moreover, the presence of genes encoding hemolysins, like those found in the S. algae STY3 genome, further supports the potential for these species to be pathogenic under the right conditions. These genes include those similar to hlyA, which encodes a toxin capable of causing cell lysis, and other genes involved in a hemolysin operon (Wu et al., 2018; Tamez et al., 2020). While S. baltica has not been commonly reported as a cause of human infections (Myung et al., 2009), the increasing number of illnesses linked to Shewanella infections in the last decade underscores the importance of considering these bacteria as emerging pathogens (Ng et al., 2022). These findings highlight the need for further research to elucidate the specific virulence mechanisms in Shewanella and their implications for human health.

Alpha hemolysins function by partially breaking down hemoglobin into biliverdin, resulting in a characteristic green discoloration around colonies on blood agar (Tasnim, 2017). This activity can damage host cells through mechanisms such as pore formation in cell membranes, as seen with pneumolysin in Streptococcus pneumoniae (Tasnim, 2017), which disrupts cellular integrity and can lead to cell death. Alpha hemolysins can interfere with host cell signaling pathways, suppressing immune responses and promoting bacterial survival. For example, pneumolysin produced by S. pneumoniae has been shown to impair host immune cells, allowing the bacteria to colonize and spread more effectively (Tasnim, 2017). In Shewanella, genomic analyses suggest the presence of genes encoding toxins similar to alpha hemolysins found in other bacteria, such as RTX-like toxins (Alav et al., 2021). These RTX toxins, like pneumolysin in S. pneumoniae, play roles in immune evasion and persistence within the host by targeting cell membranes and disrupting immune signaling (Tasnim, 2017). In Shewanella, RTX-like toxins may play a similar role in damaging host tissues, contributing to bacterial survival and colonization (Tasnim, 2017; Alav et al., 2021).

Pathogenic species reported most frequently in the literature are S. algae and S. putrefaciens, however, in the past, there have been problems identifying the microbes at the species level using conventional microbiological culture and biochemical analyses (Gram et al., 1999). More recently, this problem has been resolved by incorporating 16S rRNA gene sequencing, as the DNA sequences provide an opportunity for more definitive identification (Hau and Gralnick, 2007; Zong, 2011; Liu et al., 2013). Humans can become infected from certain Shewanella species by exposing cuts and abrasions in the skin to contaminated seawater, and from various water activities such as swimming, fishing, and boating. The consumption of raw or undercooked seafood has also been associated with illness (Myung et al., 2009; Liu et al., 2019). Some species are pathogenic to shellfish, causing production losses in the seafood industry (Huang et al., 2018). In humans, S. algae can cause soft tissue infections; bone, joint, and ear infections; lower respiratory tract infection; and occasional gastroenteritis (Holt et al., 2005; Dey et al., 2015; Takata et al., 2017; Kitaoka et al., 2019). It can also cause necrotizing fasciitis and typically attacks individuals who are immunocompromised, or have underlying conditions such as renal failure, diabetes mellitus, liver disease, and malignancies (Holt et al., 2005; Dey et al., 2015; Takata et al., 2017; Kitaoka et al., 2019).

Shewanella spp. can be readily isolated from seafood and environmental samples (Richards et al., 2008; Kang et al., 2013; Calvert County Health Department, 2017). Potentially pathogenic species were identified in the Delaware Bay with high numbers present in oysters and seawater during the summer months (Richards et al., 2008). Kang et al. (2013) isolated antibiotic resistant S. putrefaciens from shellfish in the West Sea in Korea. In 2017, cases of Shewanella infections have also been reported around the Chesapeake Bay area (Calvert County Health Department, 2017). In addition, one case has been reported during 2018 in New York involved an 87-year-old patient diagnosed with an infection caused by Shewanella haliotis (Liu et al., 2019). These cases underscore the potential pathogenicity of Shewanella species, particularly those isolated from marine environments. Furthermore, information is limited concerning the prevalence of this bacterium in oysters and seawater from the Chesapeake Bay and the Maryland Coastal Bays.

The objectives of this study were to: (i) isolate, identify, and quantify species of Shewanella in the Chesapeake and Maryland Coastal Bays; (ii) determine the levels of hemolytic Shewanella spp. in seawater and oysters as a potential indicator of their pathogenic potential, (iii) evaluate the seasonal distribution of Shewanella spp. in oysters and seawater at various sampling sites, and (iv) correlate Shewanella levels in oyster and seawater samples with physicochemical parameters of the seawater.

This is the first comprehensive study that investigates the presence, diversity, influence of environmental parameters, and pathogenic potential of Shewanella species in oysters and seawater samples recovered from the Chesapeake Bay and the Maryland Coastal Bay in the Mid-Atlantic region of the USA. To maximize the range of collection conditions encountered during the study, low and high salinity oyster harvesting sites were selected. Compared to seawater, oyster samples contained more Shewanella at all sites. This was expected due to the ability of oysters to bioaccumulate large numbers of microorganisms as they filter the water (Center for Disease Control and Prevention, 2022). Our results are consistent with the finding of a previous study that reported higher levels of Photobacterium damselae and Shewanella in oysters in comparison to seawater (Richards et al., 2008). The diversity of Shewanella species identified in this study was unexpected and some of these species have not been previously reported in U.S. coastal waters. It is uncertain whether their presence was influenced by changes in climate, better identification methods, or the fact that Shewanella research has been very limited. Nearly half of the isolates identified in this study were S. khirikhana, which was only recently recommended as a new species (Prachumwat et al., 2020). It has a 16S rRNA sequence similar to that of S. amazonensis. S. khirikhana was associated with disease of cultivated shrimp in Thailand (Prachumwat et al., 2020). We were unable to identify previous reports of S. khirikhana or S. amazonensis species in the U.S. It is unclear whether S. khirikhana is responsible for disease in shrimp, or other shellfish, or fish species in U.S. aquaculture or in wild caught seafoods. Surveillance for this shellfish pathogen may be prudent because of its high prevalence in mid-Atlantic bays and the high numbers of alpha and beta hemolytic strains detected.

While the levels of total Shewanella varied among the bays, similar results were observed in the levels of total Shewanella between sample types (oyster and seawater) and the three sampling sites in the Chesapeake Bay. Although dissolved oxygen, chlorophyll-a levels, and seawater pH showed some correlation with Shewanella levels in seawater, no correlation was observed between the physicochemical parameters and Shewanella abundances in oysters. There can be many factors outside of environmental influences (Abirami et al., 2021; Marsay et al., 2023) that can lead to higher abundances of Shewanella and other bacteria. This can include host factors (Lemaire et al., 2020), nutrient availability, biofilm formation, genetic adaptations (Lemaire et al., 2020), anthropogenic influences, and enhanced metabolic activity of bacteria (Abirami et al., 2021).

The distribution of Shewanella species may be influenced by various factors, including environmental conditions and geographical location (Marsay et al., 2023). Different regions exhibit unique sets of physical and chemical parameters that shape the microbial communities thriving within them (Hanson et al., 2019). Specific environmental conditions, such as temperature, salinity, oxygen availability, and nutrient availability can shape the prevalence and adaptation of many microbial communities (Hanson et al., 2019; Marsay et al., 2023). The presence of geographical barriers, such as ocean currents or continental landmasses, can restrict the dispersal of certain species, resulting in regional variations in species composition (Hanson et al., 2019).

By comparing the Chesapeake Bay and the Maryland Coastal Bays, it was observed that isolation of S. khirikhana in the Chesapeake Bay was high, while over 90% of the Maryland Coastal Bays’ isolates were identified as S. marisflavi or S. loihica. Additionally, other studies have reported the isolation of several other Shewanella species, namely S. algae (Gram et al., 1999; Richards et al., 2008; Taherzadeh et al., 2014; Zago et al., 2020), S. putrefaciens (Richards et al., 2008; Kang and So, 2016), and S. baltica (Richards et al., 2008; Zago et al., 2020), which were also found in the Chesapeake Bay. These Shewanella species have been discussed and documented in studies conducted in the Delaware Bay (Richards et al., 2008), Danish Coastal waters (Gram et al., 1999), Persian Gulf (Taherzadeh et al., 2014), Adriatic Sea (Zago et al., 2020) and the West Sea (Kang and So, 2016).

Although all Shewanella isolates in this study were either alpha or beta hemolytic, the pathogenic potential of Shewanella species is not yet defined. If oysters become contaminated with hemolytic Shewanella, there could be potential safety concerns for shellfish consumers. The bacteria could cause health problems such as gastrointestinal illness or even more serious conditions like sepsis, particularly in individuals with weakened immune systems. Literature suggests that bacteria containing hemolysins may have higher antibiotic resistance and be more pathogenic toward humans (Jarraud et al., 2002; Nizet, 2002; Schroeder et al., 2017; Motamedi et al., 2018). We found that 45% of Shewanella isolates in this study had beta hemolytic activity. For example, S. algae is documented as pathogenic toward humans and exhibited a high percentage of beta-hemolytic activity in this study. With 45% of Shewanella isolates demonstrating beta hemolytic activity, including 20% of S. algae isolates from oysters and seawater, this finding raises concerns about potential pathogenicity.

S. algae has been increasingly recognized as an emerging human pathogen capable of causing serious infections, including those of the skin, soft tissue, bloodstream, and peritoneum (Kitaoka et al., 2019; Tamez et al., 2020). The presence of putative hemolysin genes in S. algae as discussed by Tamez et al. (2020), provides a molecular basis for its hemolytic activity and pathogenicity. Enzymes that are responsible for β-hemolysis have been established as a virulence factor for a variety of bacterial pathogens such as Enterococci (Nizet, 2002), Escherichia coli (Bhakdi et al., 1988), Listeria monocytogenes (Goebel et al., 1988; Raybourne, 2002), and Vibrio parahaemolyticus (Okuda and Nishibuchi, 1998). The presence of hemolytic activity in Shewanella has significant ramifications for the safety and quality of oysters and may well serve as an indicator of virulence in Shewanella spp. (Richards et al., 2008).

Shewanella algae demonstrates remarkable environmental adaptability and potential pathogenicity, as evidenced by its prevalence in marine ecosystems. Our findings on the prevalence and hemolytic activity of S. algae can be contextualized by comparing them with the study by Tseng et al. (2018). While Tseng et al. (2018) reported that S. algae was present in approximately 23% of shellfish samples and 28% of water samples, our study observed a higher prevalence with 67% in oysters and 33% in seawater. This difference may be attributed to geographical variations and/or sampling methods. However, the high prevalence suggests that oysters frequently harbor this bacterium, though its direct impact on oyster health has not been discussed. Notably, Tseng et al. (2018) examined hemolytic activity at both 25 and 37°C, finding that S. algae exhibited stronger hemolytic activity at human body temperature with 100% hemolysis after 72 h at 37°C compared to 50% at 25°C. In contrast, our study maintained a constant temperature of 35°C for all hemolytic testing and found beta hemolytic activity to be 20% in oysters and 67% in seawater, while alpha hemolytic activity was drastically higher in oysters at 80%, compared to 33% in seawater. Furthermore, Tseng et al. (2018) findings on S. algae’s adaptability to varying temperatures and salinities are comparable to our results; we noted a moderate correlation (r = 0.45) between water temperature and the abundance of Shewanella in our seawater samples (as noted in Table 4). This adaptability allows S. algae to survive in various marine environments, including oyster habitats, potentially affecting oyster microbiomes. These comparisons underscore the pathogenic potential of S. algae in both marine environments and human hosts, emphasizing the importance of continued monitoring for food safety implications.

A study by Richards et al. (2008) evaluated oysters and seawater from the Delaware Bay for the presence of potentially pathogenic Shewanella and Photobacterium species using the API 20E System (biMerieux Industries, Hazelwood, MO), which is a biochemical kit used to evaluate primarily human pathogens for clinical diagnoses. They found that out of 1,421 isolates tested, 12% (n = 170) of all picked colonies were identified as presumptive S. putrefaciens. The only Shewanella listed in the API database was S. putrefaciens of which some were subsequently identified as S. algae. 16S rRNA sequencing was performed and revealed a host of different Shewanella species, not present in the API database (Richards et al., 2008). Differences in assay methodologies between the Delaware study and the present study at the Maryland sites prevent direct comparison of results. Advances in scientific techniques and technologies have allowed researchers to uncover and distinguish between previously unknown or misidentified species. As a result, the number of recognized Shewanella species has expanded over time.

The levels of total Shewanella in the Chesapeake and Maryland Coastal Bays can provide important information about the potential risks associated with consuming seafood from those areas. However, it is important to note that the presence of Shewanella alone does not necessarily indicate a safety concern, as not all species of Shewanella are pathogenic and for those that are, the levels of bacteria alone may not be sufficient to cause illness. Shewanella had the highest levels observed in 2019, while the least amount was observed in 2021. In 2019–2021, S. khirikhana was the most frequently isolated species from oyster and seawater samples. It is unclear if S. khirikhana outcompetes other species or if water conditions play some role in enhancing their growth or reproduction over other species. There was no significant variation between the years for the four most frequently isolated Shewanella species (S. khirikhana, S. marisflavi, S. loihica, and S. algae).

Knowledge is limited on the extent of disease caused by Shewanella species from the Chesapeake Bay and Maryland Coastal Bay or the origins of infections, such as from the consumption of contaminated seafood, by cuts or abrasions in the skin after exposure to a contaminated marine environment, or by recreational water activities. The detection of potentially pathogenic species in this study suggests the need for continued monitoring of Shewanella occurrences and further investigation of any association this bacterium may have with disease. Studies on factors that correlate with abundances of Shewanella to physicochemical parameters should be continued and expanded to other coastal regions. Monitoring the levels of pathogenic species should be continued, and investigations on antibiotic susceptibility should be performed. Enhanced reporting of Shewanella cases should be adopted to ascertain more accurate data on disease prevalence and its association with seafood consumption, particularly during the warmer months when Shewanella levels are the highest in both seawater and oysters.

However, the limitations of this study include limited sample size from the Maryland Coastal Bays due to logistical challenges, such as inclement weather and boating issues. In addition, the COVID-19 lockdown in 2020 caused the omission of critical sampling months (March–June). Furthermore, while hemolytic activity was assessed to infer pathogenic potential, direct evidence linking Shewanella species to seafood-related illnesses was not evaluated. Lastly, while correlations with physicochemical parameters were explored, the potential role of other environmental factors, such as pollution or nutrient inputs, remains to be evaluated, limited the number of samples collected from Maryland Coastal Bays, which may have impacted regional comparisons. Addressing these limitations in future studies will enhance our understanding of the ecological and clinical significance of Shewanella species in estuarine environments.

Shewanella species are of significant interest due to their pathogenic potential to humans and marine organisms, with multiple transmission routes including marine water, seafood, and wound infections. This study is the first comprehensive investigation into the diversity and pathogenic potential of Shewanella species recovered from oysters and seawater in the Chesapeake Bay and Maryland Coastal Bays. By filling critical data gaps on the abundance, diversity, and pathogenic potential of Shewanella, this research raises awareness of this emerging pathogen and lays the groundwork for developing intervention technologies to prevent or mitigate Shewanella-associated illnesses. Continued research in this area will be crucial to better understand the environmental factors driving the emergence of these pathogens and for informing public health strategies.