Blue Crab (Callinectes sapidus) Haemolymph as a Potential Reservoir of Mesophilic Shewanella Species

Simple Summary

This study was conducted to understand whether blue crabs living in the Sacca di Goro lagoon (northeast Italy) could be carriers of bacteria that may pose a risk to people or the environment. Some of these crabs had a bacterium called Shewanella in their haemolymph, especially during the warmer months of September and October. These bacteria are usually found in the sea but can occasionally cause infections in humans. Some were also resistant to certain antibiotics, which makes treatment more difficult in the event of an infection. Our findings suggest that vulnerable people, such as those with weakened immune systems, should be cautious when handling raw crabs and that crabs should always be properly cooked before consumption. These results underscore the importance of continued research into bacterial transmission dynamics at the human–animal–environment interface, with implications for both public and marine health.

Abstract

The blue crab (Callinectes sapidus) is an invasive alien species in the Mediterranean Sea, posing threats to biodiversity, fisheries, and aquaculture. Climate change has worsened these challenges, influencing the distribution of bacterial species, including Shewanella species, which are sensitive to changes in temperature and salinity. In this study, 300 blue crabs were sampled between June and October 2024 from the Sacca di Goro (Northern Adriatic Sea, Italy) to investigate the prevalence of Shewanella species in their haemolymph. The prevalence was found to be 7% (21/300), with species such as S. mesophila, S. algae, S. cowelliana, and S. baltica identified, particularly in the months of September and October. Molecular techniques, including MALDI-TOF MS and rpoB gene amplification, were used to identify isolates. Antibiotic susceptibility testing (AST) revealed a trend of resistance to beta-lactam antibiotics. A network analysis was also conducted to examine the global trends of Shewanella research in relation to humans, animals, and the marine environment. While proper cooking eliminates the risk to consumers, handling without personal protective equipment can increase exposure, particularly for vulnerable individuals such as those who are elderly or immunocompromised. Mild symptoms are observed in children. Further studies, particularly with a One Health approach, are crucial to better understand the transmission dynamics and evolving antibiotic resistance of Shewanella species.

1. Introduction

In the Mediterranean Sea, the blue crab, Callinectes sapidus Rathbun 1896 (Brachyura, Portunidae), is an invasive species threatening biodiversity, fisheries, and aquaculture, spreading since 1948 via ballast waters [1,2,3]. Its spread in the western Mediterranean Sea and its outbreak in the Northern Adriatic Sea in 2023 caused severe damage to fisheries and shellfish industries, particularly affecting burrowing bivalves, such as the Japanese carpet shell (Ruditapes philippinarum), reducing catches and revenues [4,5].

However, crustaceans are important model organisms for research on marine ecology and pathogen–host interactions, with bacterial diseases in crabs being studied since the 1970s [6]. Their haemolymph, crucial for immunity, can host bacteria, influenced by environmental stressors, with counts of up to 6.7 × 10⁵ CFU mL⁻¹ [7,8]. Blue crabs are constantly exposed to pathogens in seawater and sediments, carrying bacterial loads of up to 10⁹ CFU mL⁻¹ [9]. Diseases in crustaceans pose health risks to both crabs and humans, with concerns about contaminants and improper cooking [10,11,12].

Nowadays, climate change is a major challenge, altering ecosystems and threatening biodiversity [13,14]. Rising temperatures and salinity levels, as projected under climate change scenarios, may lead to an increase in bacterial abundance and contamination levels in seafood, posing potential risks to food safety and public health [12]. Among the bacterial taxa influenced by these environmental changes, Shewanella MacDonell and Colwell 1986 species are particularly affected, as their spread is closely linked to variations in temperature and salinity. In particular, these bacteria are also known to thrive in extreme conditions, such as low-temperature, high-pressure, and elevated-salinity environments, further highlighting their adaptability [15].

In addition to their environmental resilience, Shewanella species are increasingly recognised as emerging multidrug-resistant pathogens. They have been found to carry β-lactam and quinolone resistance genes located on mobile genetic elements, including plasmids and integrons, posing significant challenges for antibiotic efficacy and treatment [16,17].

Infections caused by Shewanella species generally occur in warm climates or during unusually warm summers in temperate regions, where elevated temperatures create favourable conditions for their proliferation [18].

Thus, the Mediterranean is an environmental hotspot, as it is warming 20% faster than the global average, putting additional stress on ecosystems, economies, and societies [19]. Coastal areas face higher risks of flooding, erosion, and salinisation, with water temperatures expected to rise by +1.8 °C to +3.5 °C by 2100, especially in Spain and the Eastern Mediterranean [19].

Shewanella was initially isolated in 1931 from putrefied butter and was named variously as Achromobacter putrefaciens, Pseudomonas putrefaciens, and Alteromonas putrefaciens, before being classified in its current genus, which now includes more than 70 identified species [20,21,22]. Shewanella species are Gram-negative, oxidase-positive, facultatively anaerobic bacteria with a single polar flagellum that are widely recognised for their presence in marine ecosystems worldwide [23,24] (Table S1). Although they are primarily saprophytic and opportunistic pathogens, certain species like Shewanella algae, Shewanella putrefaciens, and Shewanella xiamenensis have been rarely implicated in a range of clinical infections [16,18,24,25,26,27], including skin and soft tissue conditions, E.N.T.-related diseases, abdominal and chest infections, bacteraemia, and even cardiovascular and neurological complications. These infections have raised significant global health concerns [28].

Also, Shewanella species have been often linked to diarrhoeal diseases, with isolates obtained from food poisoning cases and patients with diarrhoea [29,30,31]. Traditional identification methods are challenging due to phenotypic similarity among species [32] and limitations in 16S rRNA gene analysis [33]. Thus, advanced approaches like multilocus sequence typing [34,35] and whole-genome sequencing enhance the understanding of their taxonomy, drug resistance, and virulence [36].

Such bacteria, including S. algae, have been isolated from haemolymph samples of blue crab in the Goro lagoon (Northern Adriatic Sea, Italy). The aim of this preliminary study is to highlight the presence of these microorganisms at the species level in the haemolymph of an invasive alien species, which has seen significant spread since 2023 [37]. The enumeration of CFUs per millilitre of haemolymph was not performed, as the primary objective of this study was not to quantify bacterial load but rather to characterise the presence of specific bacterial groups. Given that blue crabs were not infected and the bacterial community detected likely represents their normal microbiome, the focus was placed on identifying taxa rather than absolute abundances. Moreover, variations in haemolymph volume and bacterial distribution could introduce biases in CFU quantification, making qualitative approaches more suitable for addressing the research question.

2. Materials and Methods

2.1. Shewanella Isolation: Sampling Site and Procedures

Between June and October 2024, sampling was carried out at a fixed point within the Sacca di Goro, Italy (Northern Adriatic Sea; 44°48′57″ N, 12°19′09″ E; Figure 1).

The sampling months were selected to ensure representation of the biological cycle, considering that after spring, smaller size classes become less available, requiring a targeted approach for meaningful comparison. A total of 300 blue crabs were sampled using fish-baited traps set at depths between −0.5 and −1.0 m, with 60 crabs collected each month. The crabs were then divided into two size categories: small (n = 30; ≤99 mm) and large (n = 30; ≥100 mm), and classified by sex. Morphological measurements were taken with a digital calliper (0.1 mm resolution), and body wet weights were recorded using a VEVOR Digital Balance (0.01 g precision).

Following standard fishing and storage procedures, Callinectes sapidus, a species not included among those protected under Legislative Decree No. 26 of 4 March 2014 [38] (implementing Directive 2010/63/EU [39] on the protection of animals used for scientific purposes), underwent a pre-cooling phase (approximately 30 min) carried out by fishermen at temperatures between 4 °C and 8 °C. After this phase, haemolymph was collected on site under sterile conditions to minimise any impact on haemolymph viscosity and ensure sample integrity. A 1 mL sterile syringe with a 26Ga needle was used to withdraw haemolymph from the leg joints or between the carapace and the abdomen, after swabbing external surfaces with 70% ethanol to prevent contamination. Since crustaceans are not covered by the cited decree, no specific ethical authorisation was required. Also, given that the blue crab is listed as an invasive alien species (IAS) under Regulation (EU) No. 1143/2014 [40] and Legislative Decree No. 230 of 15 December 2017 [41], handling practices were conducted in accordance with regulations aimed at preventing the spread of IAS. After sampling, the crabs were refrigerated at −4 °C for 24 h and subsequently frozen at −20 °C, following standard post-harvest handling procedures and respecting animal welfare standards.

After haemolymph collection, two drops of each sample were streaked onto Marine Agar 2216 (Zobell Marine Agar) plates (Sigma-Aldrich, St. Louis, MO, USA) using sterile loops to isolate heterotrophic marine bacteria. The plates were incubated at 22 ± 2 °C for 72 h [42], with daily checks. Each sample was also streaked onto laboratory-prepared thiosulphate–citrate–bile salts–sucrose (TCBS) agar and CHROMagar™ Vibrio (CHROMagar Microbiology, Paris, France). Dominant colonies and presumptive Shewanella colonies, characterised by specific colony morphology on both media, were subcultured and identified via MALDI-TOF MS. In cases where species-level identification was inconclusive for the class of Gammaproteobacteria, molecular analysis was performed.

2.2. Seawater Parameter Collection

The main physico-chemical variables of the seawater, including temperature (°C), salinity (g/L), and pH were directly measured during each sampling event using a multi-parameter probe (HI98194, HANNA^® instruments, Padova, Italy) at a depth of −0.5 m. These in situ measurements allowed for an accurate characterisation of the environmental conditions at the time of sampling, providing context for the occurrence and distribution of bacterial isolates (Figure S1).

2.3. Antimicrobial Susceptibility Testing

Antibiotic susceptibility testing (AST) was conducted using the disc diffusion method (Kirby–Bauer test) to evaluate bacterial resistance. Since no specific breakpoints are available for Shewanella, the inhibition zones were measured and classified as susceptible (S), intermediate (I), or resistant (R) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results were interpreted based on the criteria outlined in VET03 [43] and VET04 [44] for aquatic animal pathogens. The study used antimicrobial susceptibility discs (Biolab Inc., Budapest, Hungary) for a range of antibiotics, including amoxicillin (AX-25 µg), ampicillin (AM-10 µg), enrofloxacin (ENR-5 µg), florfenicol (FFC-30 µg), flumequine (UB-30 µg), oxolinic acid (OA-10 µg), oxytetracycline (T-30 µg), trimethoprim/sulphamethoxazole (SXT-1.25/23.75 µg), tetracycline (TE-30 µg), and thiamphenicol (TP-30 µg). Bacterial isolates were streaked onto Mueller–Hinton agar supplemented with 2% NaCl in three directions, rotating the plate 60° after each streak. After allowing the surface to dry for 15–20 min, antimicrobial discs were placed on the agar using sterile forceps. The plates were then incubated overnight at 22 ± 2 °C.

2.4. Genetic Characterisation and Identification

DNA extraction was performed using the boiling and freeze–thawing method [45]. To identify bacteria species, the RNA polymerase beta subunit (rpoB) gene was amplified using specific primers developed by Ki et al. [46]. The amplicons were separated on a 2% agarose gel stained with GelGreen (Biotium, Fremont, CA, USA) and visualised under UV light, with a 50–2000 kb ladder (Amplisize Molecular Ruler, Bio-Rad, Hercules, CA, USA) used as the molecular marker. Amplicons were purified using the ExtractMe DNA Clean-Up and Gel-Out Kit (Blirt, Gdańsk, Poland), and bidirectional sequencing was performed using Big Dye 3.1 (Applied Biosystems, Waltham, MA, USA) chemistry and the same primers. Sequencing products were purified with the Dye Ex 2.0 Spin Kit (QIAGEN, Hilden, Germany) and analysed on an SeqStudio Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). DNA sequences were assembled into contigs using Lasergene Software 18.0.1 (DNASTAR, Madison, WI, USA) and compared to GenBank sequences via BLAST+ 2.16.0 (NCBI, Bethesda, MD, USA).

2.5. PubMed Database Analysis of Shewanella Species

The research was conducted using the “Advanced Search Builder” function of PubMed, one of the leading biomedical databases. The keywords used for the investigation were (Shewanella) AND (Human) AND (Marine), with studies filtered for English language and only focusing specifically on marine coastal environments to align with the ecological habitat of the blue crab. These specific keywords were chosen to avoid narrowing the number of relevant studies and to focus on the primary species of interest in humans, linked to the coastal marine environment. This approach highlighted an increase in clinical infection cases caused by these species, affecting both aquatic organisms and humans. The dataset was saved in PubMed format (.txt), and uploaded to VOSviewer (v. 1.6.20) for analysis [47]. A co-occurrence analysis was conducted to assess the relationships between items by examining the frequency with which they appeared together in the same documents. To achieve a more consistent perspective less influenced by variations in terminology and to enhance data aggregation and comparability, “MeSH keywords” were used as the unit of analysis. Keywords with a minimum occurrence threshold of 5 were included in the analysis.

2.6. Statistical Analysis

Statistical analysis was conducted using R software (version 4.3.2; R Core Team, 2024). Also, to minimise any unnecessary use of animals, the sample size was carefully estimated using G*Power software (version 3.1.9.7) to ensure statistical validity while avoiding excessive sampling.

The normality of the data was evaluated with the Shapiro–Wilk test (shapiro.test() function from the “stats” package), while Levene’s test (leveneTest() function from the “car” package) was used to assess homoscedasticity. To investigate the factors influencing the presence or absence of Shewanella species, two logistic regression models were tested, both assuming a binomial distribution for the dependent variable with a logit link function:

$$\mathrm{MODEL} 1:\log \left({\displaystyle \frac{\mathrm{P}(\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{S}\mathrm{h}\mathrm{e}\mathrm{w}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{a})}{1-\mathrm{P}(\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{S}\mathrm{h}\mathrm{e}\mathrm{w}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{a})}}\right)=\mathsf{\beta }0+\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}+\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}+\mathrm{S}\mathrm{e}\mathrm{x}+\mathrm{M}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{h}$$

$$\mathrm{MODEL} 2:\log \left({\displaystyle \frac{\mathrm{P}(\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{S}\mathrm{h}\mathrm{e}\mathrm{w}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{a})}{1-\mathrm{P}(\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{S}\mathrm{h}\mathrm{e}\mathrm{w}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{a})}}\right)=\mathsf{\beta }0+\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}+\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}+\mathrm{S}\mathrm{e}\mathrm{x}+\mathrm{M}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{h}+\mathrm{T}\mathrm{e}\mathrm{m}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}+\mathrm{S}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{y}+\mathrm{p}\mathrm{H}$$

where

• The fraction represents the log-odds of bacteria presence, i.e., the logarithm of the ratio between the probability of absence and the probability of presence of Shewanella spp. (binary dependent variable: 1 = presence, 0 = absence).
• “β0” is the intercept of the model, representing the reference value of the log-odds when all other variables are equal to zero.
• “Length” is the effect of length on the log-odds of Shewanella presence.
• “Weight” is the effect of weight on the log-odds of Shewanella presence.
• “Sex” (coded as a binary variable: 0 = male, 1 = female) is the effect of sex on the log-odds of Shewanella presence.
• “Month” (sampling month) represents the effect of each month on the log-odds of Shewanella presence compared to a reference month.
• “Temperature”, “Salinity”, and “pH” represent the monthly environmental conditions. Each physico-chemical variable captures the effect of the respective environmental factor on the log-odds of Shewanella presence.

To assess the collinearity among the independent variables in the model, the Variance Inflation Factor (VIF) was calculated (vif() function from the “car” package). Additionally, to examine the influence of each predictor on the dependent variable, a deviance analysis was performed with a Chi-square test (anova() with test = “Chisq”) to test the effect of each term added to the model. A stepwise regression approach (both directions) based on the Akaike Information Criterion (AIC) was applied, moreover, to the initial logistic model.

Also, to assess the stability and precision of the model, a bootstrap analysis was performed (boot() function from the “boot” package). The logistic regression model was applied to resampled data (1000 iterations), with the coefficients being extracted and the 95% confidence intervals calculated using the “boot.ci” function. These confidence intervals for each coefficient were derived from the bootstrap results, providing a robust measure of uncertainty in the estimates of the model. A statistical significance level of p < 0.05 was set.

3. Results

3.1. Blue Crab Morphology and Seawater Physico-Chemical Variables

The specimens analysed, considering the average across all the months (mean ± standard deviation), showed the following statistics: large males had a weight of 177.42 ± 65.66 g and a length of 133.38 ± 16.54 mm. Large females had a weight of 184.69 ± 73.59 g and a length of 138.82 ± 21.09 mm. Regarding the smaller specimens, males had a weight of 45.26 ± 14.38 g and a length of 87.10 ± 11.93 mm, while small females had a weight of 46.01 ± 14.97 g and a length of 87.83 ± 11.27 mm. In

Table 1. Morphometric characteristics of blue crabs (length in mm, weight in g) and identification of Shewanella species, including MALDI-TOF MS log-scores (protein profile similarity) and sequence match percentages (GenBank reference data).

Specimens	Size	Length	Weight	Sex	Month	MALDI-TOF MS	Log-Score	Molecular Analysis	% Match	Bacteria
4	Large	188	364.98	Female	June	Shewanella mesophila	2.27	Shewanella mesophila	0	S. mesophila
11	Large	114	124.75	Female	August	Shewanella algae	2.35	0	0	S. algae
1	Small	96	69.37	Female	August	S. algae	2.36	0	0	S. algae
20	Small	95	53.99	Male	August	S. algae	2.36	0	0	S. algae
6	Large	119	205.74	Male	September	S. algae	2.36	0	0	S. algae
20	Large	137	141.77	Female	September	S. algae	2.31	0	0	S. algae
22	Large	110	113.04	Male	September	Shewanella cowelliana	2.34	0	0	S. cowelliana
30	Large	135	139.97	Female	September	S. cowelliana	2.36	0	0	S. cowelliana
16	Small	56	11.34	Female	September	S. cowelliana	2.28	0	0	S. cowelliana
21	Small	88	45.00	Female	September	S. cowelliana	2.12	0	0	S. cowelliana
2	Large	154	203.22	Male	October	S. cowelliana	2.13	0	0	S. cowelliana
3	Large	133	156.86	Male	October	S. cowelliana	2.23	0	0	S. cowelliana
15	Large	133	157.89	Female	October	Shewanella spp.	1.78	S. baltica	99.58	S. baltica
16	Large	132	155.67	Male	October	S. cowelliana	2.24	0	0	S. cowelliana
18	Large	130	151.45	Male	October	Shewanella baltica	2.32	0	0	S. baltica
8	Small	91	57.88	Female	October	Shewanella spp.	1.95	S. baltica	99.58	S. baltica
18	Small	99	63.89	Female	October	S. algae	2.36	0	0	S. algae
18	Small	99	63.89	Female	October	S. cowelliana	2.35	0	0	S. cowelliana
19	Small	91	58.32	Female	October	S. cowelliana	2.34	0	0	S. cowelliana
21	Small	99	65.78	Female	October	S. cowelliana	2.33	0	0	S. cowelliana
23	Small	98	63.87	Male	October	S. cowelliana	2.32	0	0	S. cowelliana

, the data for the specimens from which Shewanella species samples were isolated are presented.

The environmental data collected from June to October 2024 revealed fluctuations in temperature, salinity, and pH. Temperature ranged from 19.79 °C in October to 29.33 °C in August, with an average of 25.11 ± 3.81 °C. Salinity varied between 15.54 and 20.44, with an average of 18.55 ± 1.94, reaching its highest value in September at 20.44. pH levels remained relatively stable, ranging from 7.86 to 8.04, with a mean of 7.94 ± 0.07.

3.2. Prevalence and Factors Influencing Shewanella Species Presence in Blue Crab Haemolymph

The prevalence of Shewanella species, considering all isolates, in the blue crab haemolymph was found to be 4.75% (confidence interval (C.I.): 3.04–7.29%), with a total of 21 isolates from 300 specimens analysed (7%). Although 442 isolates were analysed, the number of unique samples was 300, as some specimens produced more than one isolate. Specifically, the prevalence of the different species followed this decreasing trend: S. cowelliana 2.49% (C.I.: 1.31–4.54%), S. algae 1.36% (C.I.: 0.55–3.08%), S. mesophila 0.23% (C.I.: 0.01–1.46%), and S. baltica 0.68% (C.I.: 0.18–2.14%) (Figure 2).

The results of the logistic regression models suggest that none of the variables analysed have a significant impact on the presence of the Shewanella genus. The variables “Length”, “Weight”, “Sex”, “Month” (with the categories June, September, and October), “Temperature”, “Salinity”, and “pH” are not significant predictors for the presence of these bacteria. The estimated coefficients for all variables are extremely low, with p-values equal to 1, indicating that these variables do not statistically significantly influence the probability of observing Shewanella species in the analysed sample. In particular, the intercept is very high but also not significant. Overall, the models demonstrate a poor fit to the data. The VIF analysis revealed strong multicollinearity for certain variables, with particularly high values for temperature (2230.71) and pH (2517.59), while length, weight, and salinity also showed elevated VIFs above 10. Furthermore, the deviance analysis showed no significant effects for any of the predictors, suggesting they do not contribute to explaining the variation in the presence or absence of Shewanella species in the models. Also, the stepwise regression analysis did not identify any significant predictors among the examined variables for the presence of Shewanella spp.

Based on the results and considering the variables involved, it was decided to proceed with Model 1 for further analysis. The non-parametric bootstrap analysis of Model 1 was performed to estimate the uncertainty of the coefficients in the logistic regression model with 1000 repetitions in order to obtain more robust estimates and calculate confidence intervals without making parametric assumptions (Figure 3). This approach allows for the consideration of data variability and provides a more reliable assessment of the coefficients, especially in the presence of uncertainty or poor statistical significance of the predictors in the logit model. Furthermore, the bootstrap allows for testing the stability of the estimates, identifying potential biases or large variations in the coefficients derived from the analysed samples. Therefore, this analysis complements the logit model, enhancing the robustness of the conclusions and increasing confidence in the obtained estimates.

In detail, the results of Model 1 showed that the estimated coefficients for the independent variables (length, weight, sex, and month) exhibited some degree of variability. In particular, the original estimate of the intercept (t1) was −1.594, with a standard error (S.E.) of 0.917. The 95% C.I. for this coefficient spanned from −3.457 to 0.126, suggesting the possibility of a non-significant effect for the intercept, as the interval includes zero.

The coefficient for the length variable (t2) was positive (0.026), with a precise estimate (SE: 0.011). The C.I. was between 0.004 and 0.049, indicating a positive relationship between length and the likelihood of the presence of total bacteria. This effect is likely to be statistically significant as the interval does not include zero. The coefficient for weight (t3) was negative (−0.005), with a small effect size and a low S.E. (0.004) and a C.I. ranging from −0.013 to 0.003, suggesting that weight might have a negligible or non-significant impact on the outcome. The coefficient for sex (t4) was positive (0.134), but with a larger S.E. (0.216) and a C.I. ranging from −0.292 to 0.574, indicating some imprecision in the estimate and suggesting that sex might not significantly influence the likelihood of bacteria presence. The coefficients for the months (t5-t8) varied from negative to positive values. Specifically, the coefficients for July (t5), June (t6), and October (t7) were negative (−1.014, −1.486, and −0.843, respectively), with the C.I.s suggesting significant negative effects. However, they had relatively high S.E.s (0.342, 0.370, and 0.306, respectively), indicating that the estimates for these months were less precise compared to other variables. The coefficient for September (t8) was positive (0.451), with a moderate S.E. (0.327), and its C.I. spanned from −0.157 to 1.093, suggesting a potential positive effect, though the interval includes zero, indicating that it may not be significant.

Overall, the biases for all coefficients were small, indicating that the model estimates were not significantly affected by the bootstrap procedure. However, the higher standard errors for some variables, such as months and sex, suggest that these coefficients may be less stable and more susceptible to variability in the data.

3.3. Antimicrobial Susceptibility Testing

Figure 4 summarises the antibiotic resistance profiles of the Shewanella species isolated from blue crab haemolymph, selected based on their potential implications for human health as highlighted in the literature. The findings revealed similar resistance and susceptibility patterns across the species.

Shewanella cowelliana, S. baltica, S. algae, and S. mesophila are all resistant to ampicillin and amoxicillin. For the other antibiotics tested, sensitivity varies. Shewanella cowelliana and S. algae show intermediate resistance to enrofloxacin and tetracycline, while the other two species are sensitive. Furthermore, all species are sensitive to flumequine, florfenicol, oxolinic acid, trimethoprim/sulphamethoxazole, oxytetracycline, and thiamphenicol. Overall, resistance to the most commonly used classes of antibiotics is limited in these Shewanella species, except for ampicillin and amoxicillin.

3.4. Network Visualisation of Shewanella Species

The applied filter identified 114 articles published in English between 1996 and 2024. During the period from 1996 to 2006, scientific output was limited, averaging one paper per year, with two articles published in 1998 and 2005. From 2007 onwards, the number of publications showed an exponential increase, peaking in 2024 with 12 studies, the highest recorded within this timeframe.

The network plot generated by VOSviewer highlights thematic clusters of keywords related to Shewanella research, illustrating the diverse areas of study, primarily focusing on S. algae and S. putrefaciens, species commonly associated with marine environments and human infections of varying severity (Figure 5). A central hub is formed around the keyword “Shewanella”, which connects various domains, reflecting its significance across multiple fields. The analysis reveals a strong focus on human health, with terms such as “humans”, “Gram-negative bacterial infections”, and “soft tissue infections” dominating one cluster, underscoring the clinical implications and pathogenic potential of Shewanella species, particularly S. putrefaciens. Another cluster emphasises environmental and ecological aspects, with keywords like “phylogeny”, “microbiota”, “biofilms”, and “water microbiology” pointing to the role of the genus in marine and aquatic environments. Additionally, molecular and antimicrobial studies are represented by terms such as “microbial sensitivity tests”, “beta-lactamases”, and “genome, bacterial”, reflecting research on resistance mechanisms and genomic characterisation. The overlap between clusters indicates interdisciplinary connections, particularly between environmental microbiology and human health, highlighting the broad relevance of Shewanella research.

4. Discussion

Among the more than 100 published studies, most focus primarily on two clinically significant Shewanellaceae species, Shewanella putrefaciens and S. algae, documenting cases of human infections with varying degrees of severity [18,28]. The first study, published in 1996, reports the initial cases of S. algae bacteraemia in patients with chronic leg ulcers, linked to a shared marine exposure during a warm summer, with varying clinical out-comes [48]. In 1998, a second study examined a co-infection caused by S. putrefaciens and Mycobacterium marinum, contracted at the beach [49]. Afterwards, other studies explored the presence of S. algae in European coastal waters and the impact of water temperature on its survival [50]. Between 1996 and 2006, the number of published studies remained relatively stable, averaging one publication per year. From 2008 onwards, scientific production increased, maintaining a steady rate of approximately two publications per year until 2011. In 2014, Diaz [51,52] published studies focusing on skin and soft tissue infections, as well as other infections resulting from marine injuries or exposures. More recently, Ibrahim et al. [53] conducted an analysis characterising potentially pathogenic strains of S. algae isolated from ballast water.

Shewanella infections caused by S. algae and S. putrefaciens generally present with clinical symptoms similar to those of other marine halophilic Vibrio species [54], although S. putrefaciens was identified in only 3% of clinical isolates, with no clear evidence of its pathogenic role [18].

In Denmark, S. algae is the primary cause of Shewanella-related ear infections, including both acute cases and exacerbations of chronic otitis media [18]. A study of 67 ear infections found S. algae responsible for all Shewanella cases, isolating it in 50% of the cases, while reports outside Denmark are rare and date back to the 1970s [18]. Vignier et al. [24] analysed 16 Shewanella human infections in Martinique and 239 global cases since 1997, involving soft tissue, ear, and abdominal infections, with an 87% recovery rate and 13% mortality. Skin and soft tissue infections, typically resulting from ulcers or trauma, are the most common manifestation, with bacteraemia being generally benign [18].

Two cases of S. algae bacteraemia, including one with myonecrosis in patients with chronic ulcers, have also been reported, along with primary bacteraemia linked to severe hepatobiliary disease and neoplasms, often progressing rapidly [55,56,57]. Also, Shewanella infections have been reported in 19 underweight neonates in South Africa, with bacteraemia linked to respiratory distress at birth, indicating intrapartum infection [18]. Respiratory colonisation and bacteraemia were also seen in patients with peritoneal dialysis, near-drowning, tuberculosis, and pleural empyema [18].

Shewanella species are commonly found in seawater with salinities of 15–20, particularly when temperatures exceed 13 °C [43], which may explain the increased incidence of infections in warmer climates or during hot summers in temperate regions [16]. Our findings from blue crab (Callinectes sapidus) haemolymph align with these environmental conditions, with observed temperatures ranging from 19.79 to 23.84 °C and salinities from 18.09 to 20.44.

Also, these environmental conditions may promote the production of potent neurotoxins by Shewanella species, such as tetrodotoxin and its analogues (TTXs) [58,59,60].

Several studies have demonstrated the ability of Shewanella species to produce TTXs, potentially contributing to clinical infections and ecological shifts. Some strains of Shewanella have been isolated from the intestines of food-poisoning patients and from contaminated food, with TTX production confirmed through mouse bioassays, suggesting that these bacteria can survive in the human gut and contribute to foodborne outbreaks [60]. In addition, S. alga was identified among TTX-producing bacteria isolated from marine organisms, further highlighting the ecological role of this genus in the marine environment [58]. Moreover, S. putrefaciens has been associated with TTX accumulation in pufferfish, including Lunartail puffer Lagocephalus lunaris, with its toxin production influenced by environmental conditions, particularly seawater temperature, indicating that ecological factors may regulate the toxigenic potential of Shewanella species [59].

Recently, advancements in pathogen identification and characterisation techniques have led to an increasing frequency of isolated strains from the Shewanellaceae family in aquatic organisms.

Regarding crustaceans, a multi-resistant S. putrefaciens isolate has been identified as the causative agent of hepato-pancreas necrosis in the Chinese mitten crab (Eriocheir sinensis), with an LD50 of 2.20 × 10⁵ CFU mL⁻¹ [61].

A recent study also highlighted the presence of S. algae in blue crab from the coastal and lagoon areas of the northwestern Adriatic Sea [62]. In our study, we detected S. algae in the same geographical areas; however, there is a discrepancy between the number of specimens analysed and the detected percentages, with our results showing a lower prevalence (21/300, 7%) compared to those reported by Rubini et al. [62] (18/72, 25%). These differences in percentages could be due to differences in the number of specimens analysed, sampling methods, diagnostic techniques, or geographical areas examined, all of which may influence the detection of S. algae. Conversely, our results are similar to those found by Richards et al. [63], where from 276 water and shellfish samples analysed, 1,421 bacterial isolates were selected, with 170 (12.0%) identified as Shewanella spp., the majority being S. putrefaciens. However, our study is a preliminary investigation. It would be beneficial to improve the models by extending the sampling period to better capture the prevalence of Shewanella species throughout the year. The low number of isolates (21) limits the ability to achieve statistical significance, and the high correlation between temperature, salinity, pH, and month may hinder the capacity of the models to differentiate their individual effects. Increasing the sample size and refining the model would provide a more accurate understanding of the variables influencing the presence of Shewanella species in the blue crab haemolymph.

Several Shewanella species have also been isolated from both freshwater and marine fishes. For example, S. putrefaciens was first isolated from diseased Marbled spinefoot (Siganus rivulatus) in 1985 [64], with few reports of fish infections. Shewanella putrefaciens was isolated from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) [65], while S. algae strains were isolated in 2011 from Italian aquaculture farms along the northern and southern Adriatic Sea, specifically from European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), and shi drum (Umbrina cirrosa) [66] (Table S1). The study of Zago et al. [67] identified beta-lactam resistance genes in marine strains from Italian aquaculture [66]. Multidrug-resistant (MDR) S. algae clones were traced along the Adriatic coast, showing point mutations and phylogenetic links [67]. The rise of community-acquired infections caused by antibiotic-resistant bacteria has highlighted the presence of antimicrobial resistance genes (ARGs) in natural environments, including seawater. Resistance to beta-lactams, widely used in medicine, has significantly increased [68,69]. However, limited data are available in Italy on the prevalence of ARGs in emerging pathogens, such as Shewanella species, which are responsible for human infections. Most studies focus on resistance to beta-lactams like penicillins and cephalosporins [70] and other antibiotics such as tetracycline and sulphonamides [66]. A recent study by Torri et al. [71] reported S. algae infections showing susceptibility to third-generation cephalosporins and gentamicin, while amikacin, carbapenems, and piperacillin/tazobactam proved effective. These bacteria are emerging as MDR pathogens, harbouring resistance genes to beta-lactams and quinolones, often carried by mobile genetic elements [16]. Findings have highlighted the role of Shewanella in spreading antibiotic resistance, particularly through plasmids, transposons, and integrons [16]. Resistance mechanisms include beta-lactamases, altered penicillin-binding proteins (PBPs), and efflux systems, with countermeasures including stable beta-lactams and agents targeting resistant PBPs [68]. Additionally, beta-lactam antibiotics degrade under environmental conditions, with degradation rates influenced by pH, temperature, and functional group reactivity, leading to reduced antimicrobial activity [72]. Pangenome analysis of S. xiamenensis has also revealed genetic traits associated with its diversity, pathogenicity, and evolving antibiotic resistance, highlighting recent changes in its resistance spectrum due to acquired genes [17]. Our results align with the current literature, confirming the trends of beta-lactam antibiotic resistance observed in previous studies.

5. Conclusions

Shewanella species are environmental bacteria commonly found in marine and coastal environments. Previous studies have highlighted the widespread presence of these bacteria in both seawater and aquatic organisms, such as fish, crustaceans, and bivalve molluscs. A significant presence of potentially harmful Shewanella strains has also been identified in the marine coastal waters of the Adriatic Sea in Italy, with occasional human infections reported between 2013 and 2016, primarily caused by S. algae. These infections are strongly associated with marine water exposure during the warmer months.

Climate change, with rising temperatures and salinity levels, may further facilitate the spread and persistence of these pathogens, increasing the likelihood of contact with both aquatic organisms and humans year-round. However, proper cooking of blue crabs and other aquatic species eliminates the risk to consumers. The risk may increase during handling, especially in aquatic environments without proper personal protective equipment (PPE; e.g., gloves), as this can expose individuals to the bacteria. The most vulnerable individuals are immunocompromised patients and elderly people (over 60 years old), with severe cases or fatalities being rare and often linked to other complications. In contrast, children (aged 9 years and under) tend to experience mild symptoms, such as otitis, which is not a cause for concern.

Further research is needed to better understand the presence of Shewanella during different months and its transmission through aquatic species such as blue crabs. Increasing the sample size in future studies is therefore recommended to enhance detection power. Moreover, this study highlights the need to verify the virulence of these motile Gram-negative bacilli, as the current study does not provide conclusive evidence of their pathogenicity. Accordingly, future investigations are required to assess the potential risks posed by these bacteria and their role in disease transmission. A One Health approach, integrating human and veterinary medicine, will be crucial in addressing this issue. Additionally, the growing concern regarding antibiotic resistance in this pathogen, which may lead to more severe clinical complications, underscores the importance of continued investigation, particularly in the context of the increasing prevalence of resistant strains.