Pathogenic Vibrio Parahaemolyticus Research Articles

Use of Direct‐Fed Microbes to Enhance Shrimp Resistance to a Vibrio Parahaemolyticus Strain Causing Early Morality Syndrome (EMS)

ABSTRACTEarly mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPND) is an epizootic bacterial infection of shrimp that has had significant negative impact on the global shrimp aquaculture industry. This disease is largely attributed to pathogenic Vibrio parahaemolyticus EMS strains (VP‐EMS). Application of probiotics was examined for control of pathogenic VP‐EMS infections in Pacific white shrimp (Litopenaeus vannamei) under laboratory‐scale conditions. Two probiotic products, a single strain (O14VRQ) of Bacillus subtilis and a blend (Plus10) of five strains of Bacillus from four different species (Bacillus amyloliquifaciens, Bacillus megaterium, Bacillus brevis, and two distinct strains of Bacillus subtilis), were evaluated at various concentrations as feed additives (applied as a top coat on commercial feed) or dosed directly into the culture water. Two trials were conducted in which shrimp were fed either a control feed (no probiotics) or probiotic‐coated feed for 7 days prior to be being challenged with VP‐EMS. Shrimp were observed for clinical signs of disease and mortalities during the disease challenge study. These experiments demonstrated that while both Bacillus probiotic products were shown to significantly (p < 0.05) improve shrimp survival, the O14VRQ strain provided the most consistent protection across the trials in top‐coated feed. Overall, better efficacy was observed with probiotic‐coated feeds. Findings were directly and positively correlated with concentration for top‐coated and even more so for direct in‐tank applications. Collectively, these findings contribute to our understanding about how probiotic strains could be applied to enhance shrimp health in aquaculture.

Sous vide and chitooligosaccharide-catechin conjugate for inactivation of Vibrio parahaemolyticus and Shewanella algae in refrigerated Asian green mussel

Impacts of sous vide (SV) combined with chitooligosaccharide-catechin conjugate (COSC) on inactivation of pathogenic Vibrio parahaemolyticus and Shewanella algae in vitro and those inoculated in half-shelled Asian green mussel (H-AGM) were investigated. SV at varying temperatures (45 and 65 °C) and times (15, 30, 60, 90 and 120 min) was employed for treatment of H-AGM. SV process at 45 °C for 30 min rendered the yield of edible portion by 25.61%, maintained texture properties and inactivated vegetative microflora. COSC showed MIC/MBC toward V. parahaemolyticus and S. algae at 128/512 mg/L and 1,024/4,096 mg/L, respectively. For morphology, both bacterial cells had the pore and shrinkage when treated with COSC. In vitro study revealed that combining COSC (500 mg/L) with SV at 45 °C was optimal for inactivating V. parahaemolyticus and S. algae by >6 log10 CFU/mL within 30–90 min. However, the combination of COSC and SV at 45 °C for 30 min completely inactivated V. parahaemolyticus and S. algae inoculated in H-AGM during storage at 4 °C up to 9 days. Additionally, the virulence characteristics (tdh+ and β-hemolysis positive) of inoculated pathogenic V. parahaemolyticus were not detected during the 9–day storage period. Therefore, COSC along with mild SV process was effective in preserving the quality and inhibiting microbial growth in AGM products.

The effectiveness of gliding arc discharge plasma in sterilizing artificial seawater contaminated with Vibrio parahaemolyticus

The rapid proliferation of the halophilic pathogen Vibrio parahaemolyticus poses a severe health hazard to halobios and significantly impedes intensive mariculture. This study aimed to evaluate the potential application of gliding arc discharge plasma (GADP) to control the infection of Vibrio parahaemolyticus in mariculture. This study investigated the inactivation ability of GADP against Vibrio parahaemolyticus in artificial seawater (ASW), changes in the water quality of GADP-treated ASW, and possible inactivation mechanisms of GADP against Vibrio parahaemolyticus in ASW. The results indicate that GADP effectively inactivated Vibrio parahaemolyticus in ASW. As the volume of ASW increased, the time required for GADP sterilization also increased. However, the complete sterilization of 5000 mL of ASW containing Vibrio parahaemolyticus of approximately 1.0 × 104 CFU/mL was achieved within 20 min. Water quality tests of the GADP-treated ASW demonstrated that there were no significant changes in salinity or temperature when Vibrio parahaemolyticus (1.0 ×104 CFU/mL) was completely inactivated. In contrast to the acidification observed in plasma-activated water (PAW) in most studies, the pH of ASW did not decrease after treatment with GADP. The H2O2 concentration in the GADP-treated ASW decreased after post-treatment. The NO2-concentration in the GADP-treated ASW remained unchanged after post-treatment. Further analysis revealed that GADP induced oxidative stress in Vibrio parahaemolyticus, which increased cell membrane permeability and intracellular ROS levels of Vibrio parahaemolyticus. This study provides a viable solution for infection with the halophilic pathogen Vibrio parahaemolyticus and demonstrates the potential of GADP in mariculture.

Investigation of the Protective Effect of Probiotic Lactobacillus plantarum Ep-M17 on the Hepatopancreas of Penaeus vannamei

Infection with the pathogenic bacterium Vibrio parahaemolyticus typically causes severe hepatopancreatic damage in Penaeus vannamei, often resulting in acute shrimp mortality. Therefore, protecting the shrimp’s hepatopancreas is crucial for enhancing their disease resistance. Previous research has demonstrated that the probiotic strain Lactobacillus plantarum Ep-M17 inhibits the growth of V. parahaemolyticus E1 in vitro. However, it remains uncertain whether Ep-M17 can provide protective benefits to the shrimp’s hepatopancreas. To address this knowledge gap, our present study investigated the histological changes, enzyme activity, gene transcription, and metabolite levels in the hepatopancreas of shrimp after a 4-week diet supplemented with Ep-M17. The results revealed that incorporating Ep-M17 into the shrimp’s diet alleviated the damage by V. parahaemolyticus E1 infection in hepatopancreatic cells. In addition, the inclusion of Ep-M17 notably boosted the effectiveness of immunodigestive enzymes such as SOD, AKP, and CAT. Furthermore, Ep-M17 stimulated gene transcription in crucial immune response-related signalling pathways like the mitogen-activated protein kinase signaling pathway and the antigen processing and presentation pathway. Moreover, the incorporation of Ep-M17 into shrimp diets increased the levels of β-alanine, and histidine in the hepatopancreas, enhancing anti-inflammatory capacity and improving the shrimp’s immune response. Overall, the results indicate that incorporating Ep-M17 into the diet can enhance shrimp disease resistance by bolstering both immune response and metabolic activity within the hepatopancreas. These results underscore the importance of probiotics in controlling aquatic animal diseases and highlight Ep-M17 as a promising dietary supplement for enhancing shrimp health and immunity in aquaculture.

The effect of supplementation of Lactococcus lactis strain as probiotic on the growth and survival of Litopenaeus vannamei

Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) is a bacterial disease in shrimps caused by pathogen Vibrio parahaemolyticus. It has culminated in huge loss in global shrimp production due to mass mortality. A probiotic strain Lactococcus lactis strain FA1 was recently isolated from shark intestine, showing inhibition towards the growth of the pathogen. Due to inhibitory potential, the effect of probiotics strain on growth performance of shrimp infected with V. parahaemolyticus was evaluated. The probiotic strain was incorporated into feed for juvenile shrimps Litopanaeus vannamei for 3 weeks before which they were then challenged with pathogen Vibro parahaemolyticus. The study compares 4 shrimp groups: Control (Without any treatment); Group A (Probiotic treated, uninfected); Group B (Probiotic treated, infected) and Group C (No probiotic, infected). The survival and growth performance (weight and length gain) of shrimps were evaluated in the following 30 days. Statistical analyses (ANOVA; Post Hoc Tukey) were used to compare between shrimp groups. In general, infected shrimp demonstrated some of the key symptoms of AHPND (pale or white hepatopancreas), transparent body and erratic swimming behaviour. The supplementation of probiotics resulted in an improved survivability (65±1%) compared to infected shrimp (45±1%). The probiotic treated shrimp group showed to have better % body weight gain, in which weight gain between group B (treated/infected) and group C (untreated/infected) were significantly different (at P=0.046); and so, to group A (treated/uninfected) when compared with group C (P=0.047). There is however no significant difference in % length gain between the groups. Meanwhile, some of the infected shrimps were able to show recovery from the infection. Pre-infection probiotic treatment showed better performance compared to post-infection probiotic treatment. This indicated that the strain Lactococcus lactis is highly suitable for use as the future probiotic in shrimp aquaculture.

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

Detection of pathogenic Vibrio spp. in foods: polymerase chain reaction-based screening strategy to rapidly detect pathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus in bivalve mollusks and preliminary results.

The majority of human diseases attributed to seafood are caused by Vibrio spp., and the most commonly reported species are Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. The conventional methods for the detection of Vibrio species involve the use of selective media, which are inexpensive and simple but time-consuming. The present work aimed to develop a rapid method based on the use of multiplex real-time polymerase chain reaction (PCR) to detect V. parahaemolyticus, V. vulnificus, and V. cholerae in bivalve mollusks. 30 aliquots of bivalve mollusks (Mytilus galloprovincialis) were experimentally inoculated with two levels of V. parahaemolyticus, V. vulnificus, and V. cholerae. ISO 21872-1:2017 was used in parallel for qualitative analysis. The limit of detection of 50% was 7.67 CFU/g for V. cholerae, 0.024 CFU/g for V. vulnificus, and 1.36 CFU/g for V. parahaemolyticus. For V. vulnificus and V. cholerae, the real-time PCR protocol was demonstrated to amplify the pathogens in samples seeded with the lowest and highest levels. The molecular method evaluated showed a concordance rate of 100% with the reference microbiological method. V. parahaemolyticus was never detected in samples contaminated with the lowest level, and it was detected in 14 samples (93.33%) seeded with the highest concentration. In conclusion, the developed multiplex real-time PCR proved to be reliable for V. vulnificus and V. cholerae. Results for V. parahaemolyticus are promising, but further analysis is needed. The proposed method could represent a quick monitoring tool and, if used, would allow the implementation of food safety.

End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases, early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO), a pH sensitive dye, to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh, trh1, and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Following the standard LAMP protocol, LAMP reaction temperature for rpoD, tdh, trh1, and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix, betaine, MgSO4, Bst 2.0 WarmStart DNA polymerase, reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP, PCR, and quantitative PCR (qPCR) assays. Subsequently, the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Under optimal conditions (65°C for 75 min), rpoD-LAMP-XO and tdh-LAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction, or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP, comparable to that of the gold standard qPCR, and 10-100 times higher than that of PCR. In spiked samples, rpoD-LAMP-XO, tdh-LAMP-XO, and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples, LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100%, respectively. To detect total and pathogenic V. parahaemolyticus, at least rpoD-LAMP-XO and trh2-LAMP-XO should be used, as both showed 100% sensitivity, specificity, and accuracy. With short turnaround time, ease, and reliability, LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V.parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest.

Detection of multi-antibiotic resistant Vibrio parahaemolyticus isolated from fresh produce in Dhaka, Bangladesh

The demand for fresh produce is rising daily as it's an excellent way to maintain a healthy lifestyle. The current work fixed the target employing culture-based and molecular techniques for isolating and identifying the pathogenic Vibrio parahaemolyticus from fresh produce. The colonial appearance on TCBS agar and CHROMagar culture plates revealed the prevalence of Vibrio spp. and V. parahaemolyticus in all examined samples, which were 45/112 (40.2%) and 40/112 (35.7%), with the highest load of up to 2.0x105cfu/g and 6.4x103cfu/g, respectively. Sixty isolates in all were chosen for further molecular characterization. Multiplex PCR results exhibited that all tested isolates were positive for tlh gene and six for tdh gene but none of isolate for trh gene. According to obtained data on antimicrobial susceptibility, the major portion of tested strains was found to be resistant to ampicillin (90%), tetracycline (80%), and streptomycin (70%). The range of the multiple antibiotic index was 0-0.33. This study finding revealed that the existence of potential pathogenic V. parahaemolyticus in tested fresh produce samples constitutes a risk to public health and consumer safety thus urging continued surveillance.

Detection of Vibrio parahaemolyticus Based on Magnetic and Upconversion Nanoparticles Combined with Aptamers.

Vibrio parahaemolyticus is a halophilic and heat-labile gram-negative bacterium and is the most prevalent foodborne bacterium in seafood. In order to develop a rapid and sensitive method for detecting the foodborne pathogenic bacterium Vibrio parahaemolyticus, an aptamer-modified magnetic nanoparticle and an aptamer-modified upconversion nanoparticle were synthesised and used as a capture probe and a signal probe, respectively. The aptamer-modified magnetic nanoparticle, V. parahaemolyticus cell, and aptamer-modified upconversion nanoparticle formed a sandwich-like complex, which was rapidly separated from a complex matrix using a magnetic force, and the bacterial concentration was determined by fluorescence intensity analysis. The results showed that the fluorescence intensity signal correlated positively with the concentration of V. parahaemolyticus in the range of 3.2 × 102 to 3.2 × 105 CFU/mL, with a linear equation of y = 296.40x - 217.67 and a correlation coefficient of R2 = 0.9610. The detection limit of the developed method was 4.4 CFU/mL. There was no cross-reactivity with other tested foodborne pathogens. This method is highly specific and sensitive for the detection of V. parahaemolyticus, and can achieve the qualitative detection of this bacterium in a complex matrix.

Effects of Piper betle and Phyllanthus emblica leaf extracts on the growth and resistance of black tiger shrimp, Penaeus monodon, against pathogenic Vibrio parahaemolyticus

Effects of Piper betle and Phyllanthus emblica leaf extracts on the growth and resistance of black tiger shrimp, Penaeus monodon, against pathogenic Vibrio parahaemolyticus

Impact of rising seawater temperature on a phagocytic cell population during V. parahaemolyticus infection in the sea anemone E. pallida.

Climate change is increasing ocean temperatures and consequently impacts marine life (e.g., bacterial communities). In this context, studying host-pathogen interactions in marine organisms is becoming increasingly important, not only for ecological conservation, but also to reduce economic loss due to mass mortalities in cultured species. In this study, we used Exaiptasia pallida (E. pallida), an anemone, as an emerging marine model to better understand the effect of rising temperatures on the infection induced by the pathogenic marine bacterium Vibrio parahaemolyticus. The effect of temperature on E. pallida was examined at 6, 24, or 30 h after bath inoculation with 108 CFU of V. parahaemolyticus expressing GFP (Vp-GFP) at 27°C (husbandry temperature) or 31°C (heat stress). Morphological observations of E. pallida and their Hsps expression demonstrated heat stress induced increasing damage to anemones. The kinetics of the infections revealed that Vp-GFP were localized on the surface of the ectoderm and in the mucus during the first hours of infection and in the mesenterial filaments thereafter. To better identify the E. pallida cells targeted by Vp-GFP infection, we used spectral flow cytometry. E. pallida cell types were identified based on their autofluorescent properties. corresponding to different cell types (algae and cnidocytes). We identified an AF10 population whose autofluorescent spectrum was identical to that of human monocytes/macrophage, suggesting that this spectral print could be the hallmark of phagocytic cells called "amebocytes''. AF10 autofluorescent cells had a high capacity to phagocytize Vp-GFP, suggesting their possible role in fighting infection. This was confirmed by microscopy using sorted AF10 and GFP-positive cells (AF10+/GFP+). The number of AF10+/GFP+ cells were reduced at 31°C, demonstrating that increased temperature not only damages tissue but also affects the immune response of E. pallida. In conclusion, our study provides a springboard for more comprehensive studies of immune defense in marine organisms and paves the way for future studies of the dynamics, activation patterns, and functional responses of immune cells when encountering pathogens.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

Specific gut bacterial taxa inhabited in healthy shrimp (Penaeus vannamei) confer protection against Vibrio parahaemolyticus challenge

Host gut bacterial community has strong resistance to the invasion of pathogens. However, the specific gut taxa that could confer the resistance against pathogens in shrimp are largely unknown. Here, we demonstrated that the shrimp in the same tank showed different resistance after pathogenic Vibrio parahaemolyticus challenge. Subsequently, the gut bacterial communities of healthy, diseased, and moribund shrimp were analyzed. The results indicated that the gut bacterial community in the healthy shrimp still kept a high stability after challenge, while they were gradually disordered with the severity of disease in the diseased, and moribund shrimp. Certain specific taxa belonging to Rhodobacteraceae (e.g., Ruegeria, Paracoccus, Sulfitobacter, and Phaeobacter), Flavobacteriaceae (e.g., Tenacibaculum) and Demequinaceae (e.g., Demequina) were mainly depleted in the moribund shrimp, compared to that in healthy, and diseased shrimp. Seven corresponding strains of above specific taxa including five Rhodobacteraceae, one Tenacibaculum and one Demequina were isolated from the shrimp gut. Then, four different synthetic communities (SynComs) were constructed by using these isolates: SynComA, all five strains from Rhodobacteraceae; SynComB, SynComA + T. lutimaris HBG23; SynComC, SynComA + D. globuliformis TCG4; and SynComD, SynComA + T. lutimaris HBG23 + D. globuliformis TCG4. The addition of these SynComs could not only improve the fitness of shrimp, but also protect them against subsequent V. parahaemolyticus challenge, especially the addition of SynComD. Our study revealed that specific gut bacteria inhabited in healthy shrimp could improve shrimp resistance against pathogenic microbes.

Gut microbiota of Pacific white shrimp (Litopenaeus vannamei) exhibits distinct responses to pathogenic and non-pathogenic Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), a high-mortality-rate shrimp disease, is caused by specific Vibrio parahaemolyticus (Vp) strains with a plasmid encoding the PirABVp toxins. As a bacterial pathogen, the invasion of AHPND-causing Vp might impose pressure on commensal microbiota in the shrimp gut, while the relationship between the pathogenesis of AHPND and the dysbiosis of gut bacterial communities remains unclear. Here we explored the temporal changes of shrimp gut microbiota in response to AHPND-causing and non-AHPND-causing Vp strains, with the non-infected controls as a baseline of the shrimp gut microbiota. The diversity and composition of bacterial communities from 168 gut samples (covering three treatments at seven time points with eight individuals per set) were investigated using 16S rRNA gene metabarcoding with high-throughput sequencing. The results showed that (i) species diversity of gut bacterial communities declined in Vp-infected shrimp, independent of the strain pathogenicity; (ii) taxonomic compositions of gut bacterial communities were significantly different between shrimp infected by AHPND-causing and non-AHPND-causing Vp strains; (iii) short-term (within 6 hours) compositional shifts in the gut microbiota were found only in AHPND-causing Vp-infected shrimp; (iv) the gut microbiota of AHPND-causing Vp-infected shrimp was enriched with genera Photobacterium and Vibrio, with a decline in Candidatus Bacilliplasma; and (v) functional predictions suggested the loss of normal metabolism due to compositional shifts in the gut microbiota. Our work reveals distinct features of community dynamics in shrimp gut microbiota, associated with pathogenic versus non-pathogenic Vibrio infections, providing a new perspective of the pathogenesis of AHPND. IMPORTANCE Shrimp production is continually threatened by newly emerging diseases, such as AHPND, which is caused by specific Vp strains. Previous studies on the pathogenesis of AHPND have mainly focused on the histopathology and immune responses of the host. However, more attention needs to be paid to the gut microbiota, which acts as the first barrier to pathogen colonization. In this study, we revealed that shrimp gut microbiota responded differently to pathogenic and non-pathogenic Vp strains, with bacterial genera Photobacterium and Vibrio enriched in pathogenic Vp-infected shrimp, and Candidatus Bacilliplasma enriched in non-pathogenic Vp-infected shrimp. Moreover, functional predictions suggested that changes in taxonomic compositions would further affect normal metabolic functions, emphasizing the importance of sustaining an equilibrium in the gut microbiota. Several biomarkers associated with specific microbial taxa and functional pathways were identified in our data sets, which help predict the incidence of disease outcomes.

Exploring the potential of algae-bacteria interactions in the biocontrol of the marine pathogen Vibrio parahaemolyticus

The marine bacterium Vibrio parahaemolyticus is the leading cause of seafood-related food poisoning worldwide and a pathogen of marine species harvested in aquaculture. An outbreak of pathogenic V. parahaemolyticus within crustacean and bi-valve aquaculture facilities often results in significant loss of farmed product and in the spread of the pathogen into the human population. Evidence shows several marine bacteria and microalgae species have antibacterial effects against marine pathogens, including V. parahaemolyticus. This study explored the potential of combining possible mutualistic species as a consortium to enhance antibacterial properties against V. parahaemolyticus. Marine bacteria and microalgae were screened with the aim of identifying those able to successfully coexist while demonstrating growth suppression of multiple V. parahaemolyticus strains. This trial involved four screening phases to find effective inhibitor species and to gain insight into species-specific influences on cell growth. The combination of Tetraselmis sp. and Pseudoalteromonas peptidolytica achieved the greatest inhibition rate of V. parahaemolyticus of all combinations. This pairing resulted in the significant reduction of up to 24 ± 15% in Vibrio sp. copy number mL−1 day−1 in two of the five examined V. parahaemolyticus strains within five days, compared to control cultures absent of probiotic bacteria. Applying probiotic consortia such as this has the potential for use as a biocontrol technique within mariculture, but it will require additional research into the self-sustainability of successful consortia in natural aquatic conditions.

Trend of Pathogenic Vibrio parahaemolyticus Occurrences in Bivalve Molluscs Harvested in Sardinian Coastal Environments Between 2011 and 2018

The aim of the present study was to evaluate Vibrio parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2013 and 2015. The prevalence of pathogenic V. parahaemolyticus isolates is based on the detection of the two major virulence genes thermostable direct hemolysin (tdh) and thermolabile hemolysin (trh) To assess changes between 2011 and 2018 in the prevalence of V. parahaemolyticus in bivalve molluscs, we compared our results with those of previous investigations. In total, 2,933 samples were collected: 1,079 in 2013, 1,288 in 2014, and 566 in 2015. The mean prevalence of V. parahaemolyticus in shellfish was 3.5% in 2013, 1.7% in 2014, and 3.5% in 2015. The highest percentage of positive samples in 2013 and 2014 was observed in clams (3.5% and 2.7%, respectively), whereas in 2015, it was reported in oysters (15.1%). By comparing the sampling period of 2011–2014 with that of 2015–2018, an increase in the prevalence of V. parahaemolyticus was observed in shellfish (p < 0.05). In parallel, 208 potentially enteropathogenic V. parahaemolyticus strains were identified through the years 2011–2018 and, in particular, 10 trh+ and six tdh+ isolates. Our present study provides information regarding trends of V. parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2011 and 2018 suggesting that the prevalence varies depending on the sampling period and shellfish species.

Evaluation of Biofloc-Based Probiotic Isolates on Growth Performance and Physiological Responses in Litopenaeus vannamei

A comparison of the growth performance of Penaeus vannamei was ascertained by supplementing the potential probiotics isolated from a biofloc system incorporated through feed. Post-larvae shrimp (0.045 ± 0.005 g) were stocked at a density of 500/m3 in FRP tanks (500 L) in triplicates for a period of 60 days. A total of 40 bacterial strains were isolated from previous biofloc culture trials and tested for their antimicrobial activity against the pathogen Vibrio parahaemolyticus. Among these, Bacillus megaterium, Exiguobacterium profundum, Pseudomonas balearica, and Pseudomonas stutzeri showed higher antimicrobial activity. The treatment groups included clear water with no probiotics (CW), clear water + isolated probiotic (CW + IP), biofloc alone (BFT), and biofloc + isolated probiotic (BFT + IP), in triplicates. Distillery spent wash was used as a carbon source for biofloc development and maintenance. A probiotic concentration of 1 × 109 cfu/g was supplemented throughout the trial. The recorded water quality parameters (pH, alkalinity, calcium, and magnesium) were observed to be significant among the experimental groups (p ≤ 0.05). The highest weight gain (2.43 g), SGR, PER, and lower FCR values were recorded in BFT + IP. The lowest values of total Vibrio were found in BFT. The histology analysis revealed that there was a mild increase in the B and R cell vacuoles in the hepatopancreas of CW and BFT + IP, whereas mild degeneration was found in the intestine of CW and CW + IP. Microbiome analysis of the shrimp gut revealed that Proteobacteria was the most abundant phylum in all experimental groups. P. balearica, K. pneumoniae, P. stutzeri, and E. profundum were present in the gut of C, whereas P. balearica, K. pneumonia, and P. stutzeri were present in the gut of CW + IP and BFT + IP. The results proved that the probiotics isolated from biofloc colonized in shrimp gut could play a promising role in aquaculture.

Predator-Prey Interactions between Halobacteriovorax and Pathogenic Vibrio parahaemolyticus Strains: Geographical Considerations and Influence of Vibrio Hemolysins.

Halobacteriovorax is a genus of naturally occurring marine predatory bacteria that attack, replicate within, and lyse vibrios and other bacteria. This study evaluated the specificity of four Halobacteriovorax strains against important sequence types (STs) of clinically relevant Vibrio parahaemolyticus, including pandemic strains ST3 and ST36. The Halobacteriovorax bacteria were previously isolated from seawater from the Mid-Atlantic, Gulf of Mexico, and Hawaiian coasts of the United States. Specificity screening was performed using a double agar plaque assay technique on 23 well-characterized and genomically sequenced V. parahaemolyticus strains isolated from infected individuals from widely varying geographic locations within the United States. With few exceptions, results showed that Halobacteriovorax bacteria were excellent predators of the V. parahaemolyticus strains regardless of the origins of the predator or prey. Sequence types and serotypes of V. parahaemolyticus did not influence host specificity, nor did the presence or absence of genes for the thermostable direct hemolysin (TDH) or the TDH-related hemolysin, although faint (cloudy) plaques were present when one or both hemolysins were absent in three of the Vibrio strains. Plaque sizes varied depending on both the Halobacteriovorax and Vibrio strains evaluated, suggesting differences in Halobacteriovorax replication and/or growth rates. The very broad infectivity of Halobacteriovorax toward pathogenic strains of V. parahaemolyticus makes Halobacteriovorax a strong candidate for use in commercial processing applications to enhance the safety of seafoods. IMPORTANCE Vibrio parahaemolyticus is a formidable obstacle to seafood safety. Strains pathogenic to humans are numerous and difficult to control, especially within molluscan shellfish. The pandemic spread of ST3 and ST36 has caused considerable concern, but many other STs are also problematic. The present study demonstrates broad predatory activity of Halobacteriovorax strains obtained along U.S. coastal waters from the Mid-Atlantic, Gulf Coast, and Hawaii toward strains of pathogenic V. parahaemolyticus. This broad activity against clinically relevant V. parahaemolyticus strains suggests a role for Halobacteriovorax in mediating pathogenic V. parahaemolyticus levels in seafoods and their environment as well as the potential application of these predators in the development of new disinfection technologies to reduce pathogenic vibrios in molluscan shellfish and other seafoods.

Protective effects of indigenous lactic acid bacteria in Artemia salina challenged with Vibrio parahaemolyticus: an in vitro and in silico approach

ABSTRACT Aquaculture is one of the fastest-growing sectors of the food industry and vibriosis is a major disease affecting finfish and shellfish production capabilities. In recent years, probiotics specifically lactic acid bacteria (LAB) have proven to be an environmentally friendly alternative to antibiotics to maintain aquatic animal health. In this study, five strains of indigenous LAB isolated from traditional and non-traditional sources were evaluated for their potency in the prevention of vibriosis in brine shrimp, Artemia salina as a model organism. The LAB were well tolerated at all doses, and no negative effects on the hatching ability of brine shrimps were observed when exposed to Lactiplantibacillus plantarum KCFe63 and Limosilactobacillus fermentum NCCu21. Furthermore, all the tested LAB were able to protect the brine shrimp from the pathogen Vibrio parahaemolyticus under co-exposure and pre-exposure conditions. Molecular docking analysis revealed a high binding affinity of common probiotic metabolites lactic, butyric and propionic acids to the PirAvp and PirBvp proteins of V. parahaemolyticus which can prevent toxin formation and thereby acute hepatopancreatic necrosis disease (AHPND). Overall results suggest that two strains, L. plantarum KCFe63 and L. fermentum NCCu21, are suitable candidates to reduce the incidence of vibriosis and AHPND during brine shrimp cultivation.