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This study developed a postbiotic fermentation solution for fresh oyster preservation with the use of food waste soy whey. Lactiplantibacillus plantarum 299V was able to proliferate in soy whey within 24 h without any supplementation. Pacific oysters (Magallana gigas) were immersed in the postbiotic fermentation solution and stored at 4 °C for 12 days. Pathogenic bacteria Vibrio parahaemolyticus and Salmonella enterica introduced by bioaccumulation were suppressed to levels below the detection limit (<2 log CFU/g) within 4 days. The spoilage-related microbial parameters and chemical parameters were maintained at low levels across the 12 days. Sensory evaluation revealed that the product had a positive effect on most of the participants (>60%). Overall, the postbiotic fermentation solution reported in this study enhanced the shelf life and safety of oysters in a sustainable way and could also be recognized as an innovative probiotic vehicle with potential implications for human health promotion. [ABSTRACT FROM AUTHOR]

Taura syndrome in shrimp, caused by Taura syndrome virus (TSV), is a disease listed by the World Organisation for Animal Health (OIE) that has been identified in five continents, causing serious economic losses in years. Due to the lack of specific drugs available to treat Taura syndrome, an effective TSV detection method is urgently needed to ensure pathogen prediction and disease control. In this study, we developed and evaluated an isothermal enzymatic recombinase amplification–based assay (TSV-ERA) that was able to diagnose TSV within 20 min at an optimal temperature of 42 °C. In the reaction system, the optimal concentrations of the primer and the probe were 500 nM and 120 nM, respectively. When the plasmid standards were used as a detection template, the check-out time of TSV-ERA was 12 min (21.76 ± 0.60 Ct), and the limit of detection (LOD) was 3.6 × 101 copies/µL. Compared to the sensitivity of the TSV detection method recommended by the OIE, TSV-ERA was equal to real-time PCR (qPCR) and higher than one-step PCR. This assay was highly specific for TSV and had no cross-reactivity with Enterocytozoon hepatopenaei (EHP), white spot syndrome virus (WSSV), Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (VpAHPND), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and the genomic DNA of specific pathogen-free (SPF) shrimp. Compared with the common detection methods, the TSV-ERA assay was able to shorten the detection time and decrease the detection temperature while sustaining accurate sensitivity. Based on these results, our TSV-ERA detection method is simple, rapid, and effective, and has great potential in the detection of TSV. [ABSTRACT FROM AUTHOR]

The underlying evolutionary mechanisms driving global expansions of pathogen strains are poorly understood. Vibrio parahaemolyticus is one of only two marine pathogens where variants have emerged in distinct climates globally. The success of a Vibrio parahaemolyticus clone (VpST3) in Latin America- the first spread identified outside its endemic region of tropical Asia- provided an invaluable opportunity to investigate mechanisms of VpST3 expansion into a distinct marine climate. A global collection of VpST3 isolates and novel Latin American isolates were used for evolutionary population genomics, pangenome analysis and combined with oceanic climate data. We found a VpST3 population (LatAm-VpST3) introduced in Latin America well before the emergence of this clone in India, previously considered the onset of the VpST3 epidemic. LatAm-VpST3 underwent successful adaptation to local conditions over its evolutionary divergence from Asian VpST3 isolates, to become dominant in Latin America. Selection signatures were found in genes providing resilience to the distinct marine climate. Core genome mutations and accessory gene presences that promoted survival over long dispersals or increased environmental fitness were associated with environmental conditions. These results provide novel insights into the global expansion of this successful V. parahaemolyticus clone into regions with different climate scenarios. Vibrio parahaemolyticus Sequence Type 3 was first reported in India and expanded globally to areas with distinct climates including Latin America. In this study, the authors investigate the evolutionary mechanisms driving emergence of the strain in Latin America using 280 publicly available genomes and 32 samples newly sequenced in this study. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus, an important food-borne pathogens found to be associated with seafoods and marine environs. It has been a topic of debate for many decades that most pathogens are known to enter a viable but nonculturable (VBNC) state under cold temperature and nutrient limited conditions. The present study examined the time required for the induction of VBNC state and the revival strategies of both the endemic O3:K6 and O1:K25 sporadic strains of V. parahaemolyticus. The results revealed that V. parahaemolyticus survived even after 55 days of incubation in nutrient starved media such as phosphate buffered saline (PBS) and Coastal Water (CW) and could be recovered by temperature upshift method, and compared the resuscitation using Dulbecco’s Modified Eagle Medium (DMEM), sheep blood serum, chitin flakes with live Artemia salina, and the results suggests that chitin plays a significant role in regulating the VBNC state. It was also confirmed by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscope (SEM) analysis that VBNC cells can alter their morphology to coccoid forms in order to survive in most extreme nutrient limited environment. Further data on the promoting factors and the exact mechanism that resuscitate VBNC V. parahaemolyticus in cold natural environments and frozen foods are needed to perform a robust risk assessment. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is associated with severe food-borne illness in humans as well as substantial financial setbacks in the fishing sector. To investigate the prevalence, pathogenicity, virulence, as well as the resistance patterns of V. parahaemolyticus retrieved from Oreochromis niloticus, 180 O. niloticus (moribund fish; n = 120 and apparently healthy; n = 60) were indiscriminately collected from Lake Manzala, Egypt. Consequently, the clinical inspection, necropsy finding, and bacteriological analysis were performed. All the retrieved isolates tested positive for the groEL species-specific gene. Herein, the prevalence of V. parahaemolyticus in the collected fish was 8.9% (16/180), whereas the liver was the organ most prominently affected. PCR revealed that the most prevalent virulence-determinant genes associated with V. parahaemolyticus were the toxR and tlh genes (100% for each), then tdh (73.3%) and trh (26.7%) genes. Furthermore, 40% (18/45) of isolated V. parahaemolyticus were extensively drug-resistant (XDR) to seven antimicrobial classes and have blaTEM, blaOXA, sul1, aadA, ermB, and tetA genes. Additionally, 17.8% (8/45) of the obtained V. parahaemolyticus were multidrug-resistant (MDR) to three antimicrobial classes and owned blaTEM, ermB, and aadA genes. The pathogenicity assay accentuated the correlation between cumulative mortality and the virulent characteristics exhibited by the inoculated strain. Definitely, 77% mortality was recorded within eight days in fish injected with a virulent V. parahaemolyticus strain. Briefly, this research accentuated the occurrence of XDR V. parahaemolyticus in fish specifying a public health concern. The emerging MDR and XDR V. parahaemolyticus frequently harbored toxR, tlh, and tdh virulence genes, and blaTEM, blaOXA, sul1, aadA, ermB, and tetA or tetB antimicrobial resistance genes. [ABSTRACT FROM AUTHOR]

The shrimp farming sector has faced significant hurdles due to acute hepatopancreatic necrosis disease (AHPND). In this study, an attempt was made to combine the enzymatic recombinase amplification (ERA) assay and CRISPR-Cas12a (ERA-Cas12a) for the rapid detection of AHPND. A set of crRNA and ssDNA with high sensitivity and good specificity was screened, and the best optimized one-tube system was obtained. Our additional analysis focused on the sensitivity and specificity of ERA-Cas12a, contrasting it with the established methodology of the World Organization for Animal Health (WOAH). The ERA-Cas12a could deliver the desired outcomes in under 40 min with a detection limit of 2 × 103 copies/µL and showed no interaction with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), Taura syndrome virus (TSV), covert mortality nodavirus (CMNV), and decapod iridescent virus 1 (DIV1). Our actual sample detection results showed that the ERA-Cas12a system has the advantage of being more intuitive and fast without losing too much sensitivity compared with the nested PCR and quantitative real-time PCR (qPCR). Moreover, the detection results could be directly observed under blue light irradiation. All these results indicate that ERA-Cas12a provides a rapid and specific diagnosis of AHPND, which could further reduce the dependence on the instrument and effectively reduce aerosol pollution. It could be helpful in the prevention of shrimp disease and in reducing the economic loss of shrimp aquaculture. [ABSTRACT FROM AUTHOR]

Sexual dimorphism is a common feature in all crustaceans. However, little information is available on the relationship between sex and disease resistance in Litopenaeus vannamei. This study aimed to evaluate the differences in innate immunity characteristics between male and female L. vannamei under stimulation with Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND) or lipopolysaccharide (LPS). We found that the survival rates of female shrimp were significantly higher than those of males during VpAHPND stimulation (P < 0.05). In transcriptome analysis, we discovered obvious differences in the pathways activated for immune and metabolic responses between male and female shrimp after VpAHPND stimulation. Genes associated with immunity were downregulated in the VM (male stimulation group) vs. BM (male control group) comparison, whereas they were upregulated in the VF (female stimulation group) vs. BF (female control group) comparison. Furthermore, the transcriptome results showed that changes in processes such as carbohydrate metabolism and amino acid metabolism were also different between females and males. After LPS stimulation, the contents of semi-granular cells (SGCs) significantly decreased while those of granular cells (GCs) significantly increased in male shrimp, while the number of hyaline cells (HCs) decreased in female shrimp. The phagocytosis rate of hemocytes in male shrimp significantly decreased, while the catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and glutathione S-transferase (GSH-ST) contents in the plasma of female shrimp were higher than those in males. These results collectively indicate that female shrimp have superior disease resistance compared to males, which provides an important reference for subsequent breeding efforts aimed at enhancing disease resistance in L. vannamei. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease (AHPND) is an emerging disease that has recently severely damaged global shrimp aquaculture industries. Aside from antibiotics, using a probiotic, Lactobacillus spp., can become an alternative for AHPND prevention in shrimp aquaculture. Therefore, this study aims to screen the most suitable Lactobacillus strains for black tiger shrimp (Penaeus monodon) against AHPND. Forty-nine lactic acid bacteria were identified out of 232 isolates from fresh wild-caught fish and shrimp in the local wet market. From there, six Lactobacillus spp. strains were chosen for the hemolytic test, colony top assay, well diffusion assay, and co-culture assay. Subsequently, three strains of L. plantarum, including strains L20, KD2, and T31, were chosen for feeding trial. Bacterial dosages of 108 CFU/mL of the L. plantarum were incorporated into basal feed, respectively. A total of 600 shrimp postlarvae (PL15) were distributed into 4 treatment groups (control, L20, KD2, and T31) with triplicates. Shrimp in treatment groups were fed with different supplemented feeds for 35 days. On the 35th day, all shrimp were challenged via immersion with 6 × 105 CFU/mL of Vibrio parahaemolyticus S2-4 positive AHPND. All shrimp from the feeding trial and the challenge test were sampled for gene expression and histopathology throughout the study. Results revealed that L. plantarum strain L20 was the best probiotic among the three strains since it improved shrimp growth performance during the feeding trial, shrimp gastrointestinal microbiota, and survival of P. monodon postlarvae against V. parahaemolyticus S2-4 in the challenge test. This study demonstrated the potential allochthonous L. plantarum L20 as a probiotic in shrimp against AHPND, which causes significant losses in the aquaculture industry of Malaysia. [ABSTRACT FROM AUTHOR]

Vibrioparahaemolyticus is a gram-negative motile bacterium inhabiting marine, estuarine, and coastal environments. It is a critical aquatic pathogen in shrimp farming, and strains possessing pir A and pir B toxins can cause acute hepatopancreatic necrosis disease (AHPND) in shrimps. The study aimed to isolate V. parahaemolyticus from the gut and hepatopancreas of shrimps from the selected districts of Tamil Nadu for a duration of 6 months from December 2022 to May 2023 by random sampling, and thirty-two strains were confirmed as V. parahaemolyticus from 110 presumptive bacterial isolates. All 32 isolates used in the AP4 PCR protocol failed to detect any isolates carrying AHPND pir toxins (pir A and pir B). Forty-four percent of isolates have shown β-haemolytic activity on blood agar. Of the 32 isolates, two, eleven, and twenty-nine harboured tdh, trh, and T3SS1 genes, respectively, and none of them possessed T3SS2 gene. Isolates of V. parahaemolyticus were resistant to gentamycin, vancomycin, and erythromycin and highly susceptible to ciprofloxacin, ofloxacin, chloramphenicol, amoxyclav, trimethoprim, streptomycin, cefoxitin, and ceftazidime. AMR genes encoding qnrA, tet A, blaSHV, and aac-3-IIa were present in 6%, 16%, 16%, and 22% of the isolates. AMR genes catA1 and str B were negative for all 32 isolates. It is concluded that the prevalence and incidence of V. parahaemolyticus were 29% in the studied coastal districts of Tamil Nadu. All isolates were found to be negative for AHPND. Hence, shrimp farms in the studied area were free from infection of AHPND. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease (AHPND) is a severe condition that has caused significant damage and substantial financial losses to the global shrimp industry. Consequently, developing a convenient, sensitive, and specific analytical assay for accurately detecting AHPND is crucial for controlling the spread of the disease. The etiologic agent of AHPND is a strain of Vibrio species that carries a plasmid encoding the virulent binary toxins PirA and PirB. In this study, a pair of novel high-affinity monoclonal antibodies (MAbs) targeting the PirB toxin was generated and employed to develop a highly sensitive lateral flow immunochromatographic assay (LFIA). The LFIA results are visible to an unaided eye within 15 min of applying the bacterial colonies or pre-enrichment sample prepared in the application buffer to the test strip. This assay exhibited high specificity, successfully identifying AHPND-causing Vibrio species (Vibrio parahaemolyticus and Vibrio campbellii) without cross-reacting with non-AHPND Vibrio species or other bacterial isolates examined. The assay demonstrated a detection limit of approximately 40 ng/ml for PirB toxin, representing a 100-fold improvement over the sensitivity of the previous test strip for PirB toxin. Notably, the detection limit was further enhanced after a 6-h pre-enrichment process, enabling the identification of toxin-producing bacteria at concentrations as low as 1 colony-forming unit (CFU)/ml. Thus, it achieves a detection limit comparable to that of one-step PCR assays. Due to its high specificity, ease of use, rapid results, and no requirement for specialized equipment, the LFIA strip is an ideal tool for monitoring AHPND outbreaks and accurately detecting AHPND infections in shrimp cultures. [ABSTRACT FROM AUTHOR]

A highly sensitive and selective electrochemical DNA sensor based on a carboxyl functionalized graphene oxide/single-stranded DNA/6-mercapto-1-hexanol/gold electrode (COOH/ssDNA/MCH/Au) was developed for the rapid detection of Vibrio parahaemolyticus in shrimp samples. The sensor exhibited excellent selectivity, with peak current ratios of 8.3 and 4.1 for complementary to non-complementary and one-base mismatched targets, respectively. It also demonstrated a wide linear range from 10 femtomolar to 10 nM, with a low detection limit of 3 femtomolar. The reproducibility of the sensor was good, with a relative standard deviation of 4.8% for five independently fabricated electrodes. When applied to the detection of V. parahaemolyticus in spiked shrimp samples, the sensor showed high accuracy, with recovery rates ranging from 92.5 to 108.3% and relative standard deviation values below 5%. The proposed sensor outperformed most previously reported electrochemical DNA sensors for V. parahaemolyticus detection in terms of sensitivity and linear range. [ABSTRACT FROM AUTHOR]

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5′ end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection. [ABSTRACT FROM AUTHOR]

H. pylori infection is gaining increasing attention, but detailed investigations into its impact on gastric microbiota remain limited. We collected gastric mucosa samples from 47 individuals divided into three groups: 1. Group HP: patients with initial positive H. pylori infection (25 cases); 2. Group ck: H. pylori-negative patients (14 cases); 3. Group DiffHP: patients with refractory H. pylori infection (8 cases). The samples were analyzed using 16S rDNA sequencing and functional prediction with PICRUSt. Group HP showed differences in flora distribution and function compared to Group ck, while Group DiffHP overlapped with Group HP. The abundances of Aeromonas piscicola, Shewanella algae, Vibrio plantisponsor, Aeromonas caviae, Serratia marcescens, Vibrio parahaemolyticus, Microbacterium lacticum, and Prevotella nigrescens were significantly reduced in both Group DiffHP and Group HP compared to Group ck. Vibrio shilonii was reduced only in Group DiffHP compared to Group ck, while Clostridium perfringens and Paracoccus marinus were increased only in Group DiffHP. LEfSe analysis revealed that Clostridium perfringens and Paracoccus marinus were enriched, whereas Vibrio shilonii was reduced in Group DiffHP compared to Group ck at the species level. In individuals with refractory H. pylori infection, the gastric microbiota exhibited enrichment in various human diseases, organic systems, and metabolic pathways (amino acid metabolism, carbohydrate metabolism, transcription, replication and repair, cell cycle pathways, and apoptosis). Patients with multiple failed H. pylori eradication exhibited significant changes in the gastric microbiota. An increase in Clostridium perfringens and Paracoccus marinus and a decrease in Vibrio shilonii appears to be characteristic of refractory H. pylori infection. [ABSTRACT FROM AUTHOR]

Whiteleg shrimp Penaeus vannamei, a highly valuable commercial species in aquaculture, is known to be susceptible to environmental stressors. In this study, we examined how dietary astaxanthin impacts the survival, behavior, and molecular responses of shrimp under acute cold stress. Shrimp were split into two groups: one fed a standard diet, the other fed an astaxanthin-enriched diet. Following feeding periods of 2 and 4 weeks, the shrimp were exposed to an acute temperature stress of 10 °C. Survival rates, behavior, and gene expression (heat shock protein [HSP]70, HSP90, hepatic lectin-like, prophenoloxidase [proPO], lysozyme, hemocyanin C chain-like, catalase [CAT], superoxide dismutase [SOD]) were studied. Astaxanthin as dietary supplement significantly improved survival under cold stress, suggesting enhanced stress tolerance. Molecular analysis showed slightly lower HSP70 and HSP90 expression in the astaxanthin group across various tissues, indicating a potential mechanism by which astaxanthin aids in stress mitigation. The astaxanthin-supplemented diet positively influenced immune and antioxidant gene expression, implying a stronger immune response and defense. These findings highlight astaxanthin's potential to boost stress resilience and overall health in Whiteleg shrimp in aquaculture, resulting in enhanced immune responses and increased disease resistance. The results showed an effect on immunity, increasing their resistance to challenge and effectively protecting shrimp against Vibrio parahaemolyticus infection, resulting in increased resistance and high survival. Understanding these effects can contribute to better aquaculture practices, ensuring the well-being of this commercially important species in varied environmental conditions, especially in the temperate region. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus poses a significant risk to public health due to its high pathogenicity and mortality rates as a common seafood-borne pathogen. Therefore, rapid, accurate, and specific detection methods for V. vulnificus are crucial for ensuring human safety and minimizing economic losses. This study identified vvhA and vv08030 as promising targets for V. vulnificus detection using bioinformatics screening and PCR analysis. Based on these specific target genes, a duplex real-time PCR (qPCR) assay and a duplex droplet digital PCR (ddPCR) assay were developed and evaluated for the rapid quantitative detection of V. vulnificus. These methods showed 100% specificity to V. vulnificus and no cross-reaction with other strains. It was proved in this research that the qPCR method can detect genomic DNA as low as 31.4 fg/μL, and the quantification range of the bacterial suspension was between 8.25 × 102 and 8.25 × 107 CFU/mL. On the other hand, ddPCR exhibited greater sensitivity with genomic sensitivity reaching 3.14 fg/μL and could accurately quantify bacterial suspensions between 8.25 × 101–8.25 × 105 CFU/mL. The feasibility of the ddPCR method in detecting V. vulnificus was assessed in spiked food samples. The lowest detectable V. vulnificus in salmon was 5.74 × 101 CFU/g without any pre-enrichment. These methods demonstrated high sensitivity, accuracy and rapidity, with potential applications in V. vulnificus detection strategies. [ABSTRACT FROM AUTHOR]

The effect of betel (Piper betle) and amla (Phyllanthus emblica) leaf extracts as feed supplement was studied on the growth and immune functions of the shrimp Penaeus monodon when exposed to a virulent strain of Vibrio parahaemolyticus. Six experimental groups (PB-1, PB-2, PB-3 and PE-1, PE-2, PE-3) of shrimp were fed extract at varying concentrations, 0.05%, 0.1%, and 0.2%, before being compared to a control group that did not receive any extract. After a feeding trial of 4 weeks, each group of shrimp was challenged with V. parahaemolyticus at a concentration of 106 CFU/mL and monitored for 2 weeks. The lowest concentration of P. betle extract and the moderate concentration of P. emblica extract in the diet induced a significant increase in shrimp growth performance parameters (final body weight, weight gain, specific growth rate) and feed utilization efficiency (low feed conversion ratio, high protein efficiency ratio). The dietary extracts also exhibited a significant positive influence on the immunological parameters such as total haemocyte count, prophenoloxidase activity, and superoxide dismutase activity before and after the challenge with V. parahaemolyticus. Compared to the control group, the total Vibrio load was significantly lower in the shrimp groups fed with extracts with the highest Vibrio density found in the hepatopancreas. The cumulative mortality of shrimp fed with extracts during 14 days decreased significantly, whereas control shrimps displayed 100% mortality upon V. parahaemolyticus challenge. In summary, we propose that a diet supplemented with P. betle and P. emblica leaf extract improves the growth of shrimp, and improves their immunity as is evident from their decreased susceptibility to V. parahaemolyticus infection. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of Chlorella vulgaris on growth performance, feed consumption, body composition, and immune response of white shrimp (Litopenaeus vannamei) against Vibrio parahaemolyticus infection. Shrimps (4.00 ± 0.04 g) were divided into five groups (3 replicates/20 each) in 15 hapa (1 m3) and cultured for 56 days with five C. vulgaris experimented diets at five different concentrations (0, 15, 20, 25, and 30 g/kg feed). Growth performance, feed utilization, biomass, and body composition (protein, lipid, and ash) were analyzed and revealed significant increase (P < 0.05) in shrimp fed the high C. vulgaris diet. Additionally, the survival rate of shrimp in all experimental feeds was improved, but was not significant. Shrimp fed with C. vulgaris at different concentrations showed significantly (P < 0.05) higher antioxidant activity. The study showed that shrimp fed with a concentration of 30 g/kg C. vulgaris in the diet showed significantly (P < 0.05) highest disease resistance against the pathogen tested. Hence, dietary C. vulgaris might be used to improve growth performance, feed utilization, body composition, and immune biomarker responses, leading to disease resistance in cultured shrimp. [ABSTRACT FROM AUTHOR]

Shrimp is a popular commodity due to its fast growth and high economic value. However, stress-related disease outbreaks caused by intensifying agricultural practices and environmental factors have led to significant financial losses globally. Using chemical compounds to combat infectious pathogens has also led to antibiotic resistance and accumulation of residues in tissues and the environment, posing consumer risks. This study investigated the potential benefits of Terminalia catappa leaves (TCL) as a natural plant product-based dietary supplement for Litopenaeus vannamei postlarvae against Vibrio parahaemolyticus infection. Experimental diets with TCL extract were prepared and the feeding trial was carried out for 28 days with L. vannamei postlarvae (PL10). The growth parameters and digestive enzymes were measured. Vibrio challenge test was conducted for 48 h. Oxidative stress, immunomodulation and histopathology of hepatopancreases were analysed after vibrio challenge in PL given with/without TCL diet. The study revealed that TCL diet has an increased body weight and survival rate of the shrimp than basal diet via increasing digestive enzyme activity. TCL diet also increases the survival of shrimp against the vibrio challenge. It increases antioxidant enzymes and eliminates oxidative stress. TCL acts as an immunostimulant by increasing prophenoloxidase, crustin, lysozyme and penaeid 3a genes. Moreover, the cellular damage caused by oxidative stress to hepatopancreases of the shrimp was reduced. The findings in this study suggest that the TCL diet has a dual property of growth promoter and immunostimulant and can be a sustainable and cost-effective alternative to traditional aquaculture practices. [ABSTRACT FROM AUTHOR]

This study assessed the effects of dietary supplementation of poly-β-hydroxybutyrate (PHB) on growth performance, feed efficiency, non-specific immunity, digestive enzyme capacity, phagocytic activity, hemocyte count, intestinal morphology, and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp (Penaeus vannamei). Six diets were prepared by supplementing graded levels of PHB at 0.00, 0.25, 0.50, 1.00, 2.00, and 4.00% (Con, P0.25, P0.5, P1.0, P2.0, and P4.0, respectively). Triplicate groups of 90 shrimps (initial body weight 0.25 ± 0.01 g) per treatment were randomly assigned and fed an experimental diet for 56 days. The growth performance of shrimp was significantly improved by 1% dietary PHB supplementation. PHB-included diets fed shrimp showed significantly improved hepatopancreatic trypsin, chymotrypsin, and pepsin activities. Villus height was significantly increased with dietary PHB supplementation, and villus width was increased at a 1% inclusion level. P0.25, P0.5, and P4.0 groups significantly increased phenoloxidase activity, and the P2.0 group significantly increased anti-protease activity compared to the Con group. The survival of shrimp challenged against V. parahaemolyticus was higher in P0.5, P1.0, and P2.0 groups than in the Con diet. Dietary PHB supplementation improved weight gain, digestive enzyme activity, intestinal morphology, non-specific immunity, and disease resistance against V. parahaemolyticus of shrimp. According to the above observations, the optimal dietary PHB supplementation level for maximum weight gain would be 1% for Pacific white shrimp. [ABSTRACT FROM AUTHOR]

In shrimp aquaculture, a major economic loss due to the bacterial disease called acute hepatopancreatic necrosis disease (AHPND) infected by Vibrio parahaemolyticus and caused the highest mortality (100%). Many approaches have been addressed to control vibriosis diseases in the shrimp industry, such as water monitoring and replacements, management practices and use of antibiotics but not fully controlled. V. parahaemolyticus has important virulence factors such as thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), both are strongly directly associated with pathogenicity. Materials can be designed to specifically target PirA, PirB, TDH, and TRH, offering a targeted and effective approach to prevent infections in aquaculture. In the present study, in silico approach to unveil the potential of nanomaterials (ZnO, CuO, Se, and Fe2O3) as inhibitors against the toxins and thermostable proteins of V. parahaemolyticus has been studied via molecular docking analysis, AutoDock paired an empirical free energy force field with a Lamarckian Genetic Algorithm was used. The interaction results were visualized using molecular graphics tool Chimera. The current work is focused on predicting the ligands (ZnO, CuO, Se, and Fe2O3 nanoparticles) interact with the toxin and thermostable proteins of V. parahaemolyticus. The conformations of ligands bonded to macromolecular proteins can be assessed with the use of computational docking. These findings may be used to develop novel strategies for halting as well as controlling V. parahaemolyticus infections. [ABSTRACT FROM AUTHOR]

Climate change and disease threaten shrimp farming. Here, we studied the beneficial properties of a phytogenic formulation, Shrimp Best (SB), in whiteleg shrimp. Functional studies showed that SB dose-dependently increased shrimp body weight and decreased feed conversion ratio. We found that SB protected against Vibrio parahaemolyticus as evidenced by survival rate, bacterial load, and hepatopancreatic pathology in shrimp. Finally, we explored the likely mechanism by which SB affects growth performance and vibriosis in shrimp. The 16S rRNA sequencing data showed that SB increased 6 probiotic genera and decreased 6 genera of pathogenic bacteria in shrimp. Among these, SB increased the proportion of Lactobacillus johnsonii and decreased that of V. parahaemolyticus in shrimp guts. To dissect the relationship among SB, Lactobacillus and Vibrio, we investigated the in vitro regulation of Lactobacillus and Vibrio by SB. SB at ≥ 0.25 μg/mL promoted L. johnsonii growth. Additionally, L. johnsonii and its supernatant could inhibit V. parahaemolyticus. Furthermore, SB could up-regulate five anti-Vibrio metabolites of L. johnsonii, which caused bacterial membrane destruction. In parallel, we identified 3 fatty acids as active compounds from SB. Overall, this work demonstrated that SB improved growth performance and vibriosis protection in shrimp via the regulation of gut microbiota. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a major foodborne pathogen causing serious illnesses. In this study, a new lytic bacteriophage SSJ01 that infects V. parahaemolyticus was isolated and characterized. It had a short non-contractile tail and belonged to the Caudoviricetes class. It rapidly adsorbed onto host cells, exhibited a short latent period, and has a large burst size. It showed lytic activities under a broad range of temperature (− 18 to 60 °C), pH (5 to 11), and salinity (0 to 6%). It contained 35 open reading frames with a G + C content of 49.16% without toxic or lysogen-forming genes. The MOI of 105 phage-treated group in vitro reduced the target cells up to 3.49-log CFU/mL at 6 °C and 3.47-log CFU/mL at 25 °C, respectively. In aquatic environments (6 and 25 °C), bactericidal activities showed a significant decrease within 2 h. Therefore, the bacteriophage SSJ01 has potential as a biocontrol agent to control V. parahaemolyticus in marine culture. [ABSTRACT FROM AUTHOR]

Assessing the co-occurrence of multiple health risk factors in coastal ecosystems is challenging due to the complexity of multi-factor interactions and limited availability of simultaneously collected data. Understanding co-occurrence is particularly important for risk factors that may be associated with, or occur in similar environmental conditions. In marine ecosystems, the co-occurrence of harmful algal bloom toxins and bacterial pathogens within the genus Vibrio may impact both ecosystem and human health. This study examined the co-occurrence of Vibrio spp. and domoic acid (DA) produced by the harmful algae Pseudo-nitzschia by (1) analyzing existing California Department of Public Health monitoring data for V. parahaemolyticus and DA in oysters; and (2) conducting a 1-year seasonal monitoring of these risk factors across two Southern California embayments. Existing public health monitoring efforts in the state were robust for individual risk factors; however, it was difficult to evaluate the co-occurrence of these risk factors in oysters due to low number of co-monitoring instances between 2015 and 2020. Seasonal co-monitoring of DA and Vibrio spp. (V. vulnificus or V. parahaemolyticus) at two embayments revealed the co-occurrence of these health risk factors in 35% of sampled oysters in most seasons. Interestingly, both the overall detection frequency and co-occurrence of these risk factors were considerably less frequent in water samples. These findings may in part suggest the slow depuration of Vibrio spp. and DA in oysters as residual levels may be retained. This study expanded our understanding of the simultaneous presence of DA and Vibrio spp. in bivalves and demonstrates the feasibility of co-monitoring different risk factors from the same sample. Individual programs monitoring for different risk factors from the same sample matrix may consider combining efforts to reduce cost, streamline the process, and better understand the prevalence of co-occurring health risk factors. [ABSTRACT FROM AUTHOR]

Food-related illnesses have become a growing public concern due to their considerable socioeconomic and medical impacts. Vibrio parahaemolyticus and Staphylococcus aureus have been implicated as causative organisms of food-related infections and poisoning, and both can form biofilms which confer antibiotic resistance. Hence, the need for continuous search for compounds with antibiofilm and antivirulence properties. In this study, 22 iodinated hydrocarbons were screened for their antibiofilm activity, and of these, iodopropynyl butylcarbamate (IPBC) was found to effectively control biofilm formation of both pathogens with a MIC of 50 µg/mL which was bactericidal to V. parahaemolyticus and S. aureus. Microscopic studies confirmed IPBC inhibits biofilm formation of both bacteria and also disrupted their mixed biofilm formation. Furthermore, IPBC suppressed virulence activities such as motility and hemolytic activity of V. parahaemolyticus and the cell surface hydrophobicity of S. aureus. It exhibited a preservative potential against both pathogens in a shrimp model. IPBC disrupted the cell membrane of S. aureus and V. parahaemolyticus and differentially affected gene expressions related to biofilm formation and virulence. Additionally, it displayed broad-spectrum antibiofilm activities against other clinically relevant pathogens. These findings indicate IPBC offers a potential means of controlling infections mediated by Vibrio and Staphylococcus biofilms. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effects of black soldier fly larval (BSFL) meal as a fish meal (FM) replacement on the growth performance, survival, immune responses, and resistance to Vibrio parahaemolyticus infection in Pacific white shrimp fed a low FM diet. Four experimental diets were formulated: control (with 12% FM), 2, 5, and 10% BSFL meal. In Experiment 1, shrimp postlarvae were divided into four groups, corresponding to the four diets; they were fed four times daily for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were challenged with V. parahaemolyticus by immersion at a concentration of 105 CFU/mL and were fed with the same diets for another 7 days to evaluate the disease resistance. Results demonstrated that the 5 and 10% BSFL meal-fed shrimp had significantly increased body weight, decreased hepatopancreatic and intestinal Vibrio spp. count, and improved immune parameters. The survival rates of all BSFL meal groups after the bacterial challenge were at least 60%, which were significantly higher than the control (50%). In short, 5–10% BSFL meal can partially replace FM in the shrimp feed without compromising health conditions and disease resistance. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the different effects of dietary microencapsulated synbiotics on the growth, intestinal histology, immune responses, and gut microbiota diversity of white shrimp (Penaeus vannamei). This study used a completely randomized design with five treatments and three replications. Five different feed treatments were used to feed experimental shrimp for 8 weeks, namely, SBM (1% Bacillus NP5 108 CFU g−1 and 0.4% mannan-oligosaccharides (MOS)), SPM (1% Pseudoalteromonas piscicida 1Ub 108 CFU g−1 and 0.4% MOS), and SBPM (0.5% Bacillus NP5 108 CFU g−1, 0.5%, P. piscicida 1Ub 108 CFU g−1, and 0.4% MOS) and the controls without synbiotics administration consisted of the positive control (PC) and the negative control (NC). Pacific white shrimp from all treatments, except NC, were challenged with 106 CFU mL−1Vibrio parahaemolyticus, injected intramuscularly. In this study, an in vitro test was performed that showed that P. piscicida 1Ub could utilize MOS as a prebiotic (synergistic effects). The results showed that dietary synbiotics with different effects for 60 days resulted in higher growth performance, wider gut microbiota diversity, improved intestinal histology, and elevated immune responses compared with controls (p < 0.05). Dietary synbiotics with synergistic effects also affected the composition of intestinal microflora. Beneficial bacteria, such as Bacillus, Glycomyces, and Weissella, were more abundant in SPM and SBPM treatments than in the other groups. After the challenge test, SPM and SBPM treatments showed significantly higher levels of immune response than in the PC. SPM and SBPM treatments (synbiotics with synergistic effects) showed a significantly higher survival rate (SR) than the other treatments. [ABSTRACT FROM AUTHOR]

C-type lectin is a type of calcium-dependent sugar recognition protein, which plays an important role in recognizing invading microorganisms as non-self and assists in the clearance of pathogenic microorganisms. In this study, a C-type lectin homolog was identified from Penaeus vannamei (PvCTL2) using RACE-PCR method and functionally characterized in detail. The open reading frame (ORF) of PvCTL2 was 1143 bp, encoding 380 amino acids, which contained a conservative carbohydrate recognition domain (CRD) and a low-density lipoprotein receptor (LDLR) domain A. The predicted molecular weight was 41.79 kDa and the theoretical isoelectric point was 6.27. PvCTL2 was relatively high expressed in muscle, intestine, heart, nerve, and hepatopancreas, and Vibrio parahaemolyticus challenge could significantly upregulate the expression level of PvCTL2 in muscle. Agglutination test of recombinant protein of PvCTL2 in vitro showed that rPvCTL2 was a Ca2+-dependent C-type lectin, which could agglutinate five types of bacteria, including Gram-negative bacteria and fungi, as well as bind different concentrations of monosaccharides. RNAi-based silencing of PvCTL2 gene resulted in significantly higher mortality rate when shrimp was challenged with Vibrio parahaemolyticus, and the median lethal time was significantly shorter than the control. These results suggested that PvCTL2 could be involved in the immune response against bacterial challenge and contributed to non-self recognition as a pattern recognition receptor in the innate immune system of shrimp P. vannamei. [ABSTRACT FROM AUTHOR]

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding. [ABSTRACT FROM AUTHOR]

Siderophores are small-molecule iron chelators produced by many microorganisms that capture and uptake iron from the natural environment and host. Their biosynthesis in microorganisms is generally performed using non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) enzymes. Vibrio parahaemolyticus secretes its cognate siderophore vibrioferrin under iron-starvation conditions. Vibrioferrin is a dehydrated condensate composed of α-ketoglutarate, L-alanine, aminoethanol, and citrate, and pvsA (the gene encoding the ATP-grasp enzyme), pvsB (the gene encoding the NIS enzyme), pvsD (the gene encoding the NIS enzyme), and pvsE (the gene encoding decarboxylase) are engaged in its biosynthesis. Here, we elucidated the biosynthetic pathway of vibrioferrin through in vitro enzymatic reactions using recombinant PvsA, PvsB, PvsD, and PvsE proteins. We also found that PvsD condenses L-serine and citrate to generate O-citrylserine, and that PvsE decarboxylates O-citrylserine to form O-citrylaminoethanol. In addition, we showed that O-citrylaminoethanol is converted to alanyl-O-citrylaminoethanol by amidification with L-Ala by PvsA and that alanyl-O-citrylaminoethanol is then converted to vibrioferrin by amidification with α-ketoglutarate by PvsB. [ABSTRACT FROM AUTHOR]

ADP-activated β-d-manno-heptoses (ADP-β-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-β-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6′′ epimer, ADP-l-glycero-β-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli–derived HldEEC were thus carried out using β-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains. Key points: • Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-d-manno-heptoses. • HldEs from two Vibrio strains and E. coli could activate β-d-mannose 1-phosphate to ADP-β-d-mannose. • Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1–26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation. [ABSTRACT FROM AUTHOR]

In aquatic environments, Vibrio and cyanobacteria establish varying relationships influenced by environmental factors. To investigate their association, this study spanned 5 months at a local shrimp farm, covering the shrimp larvae stocking cycle until harvesting. A total of 32 samples were collected from pond A (n = 6), pond B (n = 6), effluent (n = 10), and influent (n = 10). Vibrio species and cyanobacteria density were observed, and canonical correspondence analysis (CCA) assessed their correlation. CCA revealed a minor correlation (p = 0.847, 0.255, 0.288, and 0.304) between Vibrio and cyanobacteria in pond A, pond B, effluent, and influent water, respectively. Notably, Vibrio showed a stronger correlation with pH (6.14–7.64), while cyanobacteria correlated with pH, salinity (17.4–24 ppt), and temperature (30.8–31.5 °C), with salinity as the most influential factor. This suggests that factors beyond cyanobacteria influence Vibrio survival. Future research could explore species-specific relationships, regional dynamics, and multidimensional landscapes to better understand Vibrio-cyanobacteria connections. Managing water parameters may prove more efficient in controlling vibriosis in shrimp farms than targeting cyanobacterial populations. [ABSTRACT FROM AUTHOR]

Cirrhinus reba (Hamilton, 1822) has gained high recognition in southern Asia for its nutritional and commercial benefits, although the carp is now facing productivity constraints. Probiotic-based captive breeding of the carp can be a promising technique to enhance the productivity and health status of the carp. The comprehensive study was performed on bacteria in the intestinal tract of C. reba. The total load of culturable bacteria in the gut of the carp was 8.34 ± 1.64 × 107 CFU/g comprising 19 different types of colonies. Most (73.7%) of the isolates were Gram-positive and rod-shaped bacteria. Among them, two intestinal isolates (PKS9 and PKS10) had shown significant antagonism against common fish pathogens namely, Staphylococcus aureus, Vibrio harveyi, and Vibrio parahaemolyticus. 16S rDNA sequencing identified the isolates PKS9 and PKS10 as Bacillus paramycoides (OM038513) and Bacillus cereus (OM033468), respectively. The isolates were determined to be biofilm formers, could persist in both acidic and alkaline environments, had high resistance to bile salts, possess surface hydrophobicity and auto-aggregation efficacy. The tests on pathogenicity and bio-safety confirmed the non-pathogenic nature of the isolates. Moreover, the strains had liberated extracellular enzymes and exhibited antioxidative properties. Hence, the isolates might be used as potential candidate probiotics for the cultivation of C. reba to enhance the nutritional and health status of the species. To the best of our knowledge, this is the first report analyzing the gut microbiota of Reba carp and establishing B. paramycoides as aquaculture probiotics. [ABSTRACT FROM AUTHOR]

In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples. [ABSTRACT FROM AUTHOR]

The effect of bacterial additives on immune response, intestinal microbiota, and survival of the white shrimp Litopenaeus vannamei was evaluated. Bacillus licheniformis BCR 4–3 and Vibrio parahaemolyticus IPNGS16 cultures were spray-dried. Inulin, probiotics, and postbiotics of Bacillus (SPB) and postbiotics of Vibrio (PVp) were added to the commercial feed. An experiment with three treatments in triplicate was performed during 19 days. The growth was not affected by the additives but survival of animals was significantly higher as compared to control group. The expression of SOD, crustin, and penaeidin4 in PVp-treated animals was significantly higher compared to the control but lysozyme expression did not change. The phyla, families, and genera that predominated in the white shrimp intestine were Proteobacteria, Bacteroidetes, Rhodobacteraceae, Vibrionaceae, Pseudoalteromonadaceae, Vibrio, Ruegeria, and Phaeobacter. The Vibrio genus decreased significantly in SPB and PVp treatments, compared to the control. The control, SPB, and PVp shared 294 OTUs. No changes were observed in the alpha and beta diversity. The metabolic activity of Vibrio and Roseobacter was higher in the control compared to SPB and PVp, whereas for Phaeobacter and Ruegeria, it decreased. Feed additives increase survival and modulated the immune response and gut microbiota of the white shrimp, so they can be used in commercial farms to increase production. [ABSTRACT FROM AUTHOR]

The shrimp immune system defends and protects against infection by its naturally expressing antimicrobial peptides. Stylicin is a proline-rich anionic antimicrobial peptide (AMP) that exhibits potent antimicrobial activity. In this study, stylicin gene was isolated from Penaeus vannamei, cloned into vector pET-28a (+), and overexpressed in Escherichia coli SHuffle T7 cells. The protein was purified and tested for its antibiofilm activity against shrimp pathogen Vibrio parahaemolyticus. It was resulted that the recombinant stylicin significantly reduced the biofilm formation of V. parahaemolyticus at a minimum inhibitory concentration (MIC) of 200 µg. Cell aggregation was observed by using scanning electron microscopy and confocal laser scanning microscopy, and it was resulted that stylicin administration significantly affects the cell structure and biofilm density of V. parahaemolyticus. In addition, real-time PCR confirmed the downregulation (p < 0.05) of genes responsible for growth and colonization. The efficacy of stylicin was tested by injecting it into shrimp challenged with V. parahaemolyticus and 7 days after infection, stylicin-treated animals recovered and survived better in both treatments (T2—100 µg stylicin, − 68.8%; T1—50 µg stylicin, 60%) than in control (7%) (p < 0.01). Comparative proteomic and mass spectrometry analysis of shrimp hemolymph resulted that the expressed proteins were involved in cell cycle, signal transduction, immune pathways, and stress-related proteins representing infection and recovery, and were significantly different in the stylicin-treated groups. The result of this study suggests that the stylicin can naturally boost immunity and can be used as a choice for treating V. parahaemolyticus infections in shrimp. [ABSTRACT FROM AUTHOR]

Phenyllactic acid (PLA) generally recognized as a natural organic acid shows against Vibrio parahaemolyticus activity. In this study, V. parahaemolyticus ATCC17802 (Vp17802) was cultured under the stress of 1/2MIC PLA, and then the antibacterial mechanisms were explored via transcriptomics. The minimum inhibitory concentration (MIC) of PLA against Vp17802 was 3.2 mg/mL, and the time-kill analysis resulted that Vp17802 was inhibited. PLA was able to destroy the bacterial membrane, leading to the leakage of intracellular substances and decline of ATP levels. The RNA-sequencing analysis results indicated that 1616 significantly differentially expressed genes were identified, among which 190 were up-regulated and 1426 were down-regulated. Down-regulation of the icd2 gene in the TCA cycle mediates blockage of tyrosine metabolic, arginine biosynthesis, and oxidative phosphorylation, causing insufficient energy supply of Vp17802. Moreover, PLA could cause amino acids, metal ions, and phosphate transporters to be blocked, affecting the acquisition of nutrients. The treatment by PLA altered the expression of genes encoding functions involved in quorum sensing, flagellar assembly, and cell chemotaxis pathway, which may be interfering with the biofilm formation in Vp17802, reducing cell motility. Overall, 1.6 mg/mL PLA inhibited the growth of Vp17802 by disrupting to uptake of nutrients, cell metabolism, and the formation of biofilms. The results suggested a new direction for exploring the activity of PLA against Vp17802 and provided a theoretical basis for bacterial pathogen control in the food industry. Key points: •RNA sequencing was carried out to indicate the antibacterial mechanism of Vp17802. •The icd2 gene in the TCA cycle mediates blockage of metabolic of Vp17802. •The biofilm formation has interfered with 1.6 mg/mL PLA, which could reduce cell motility and virulence. [ABSTRACT FROM AUTHOR]

In this study, Penaeus monodon were gave basic feed supplemented with three levels of Enterococcus faecium. Then, the expression of non-specific immunity-related genes, and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (AKP), phenol oxidase (PO) were evaluated. Meanwhile, the disease resistance test and intestinal flora determination were conducted. The results showed that the MDA levels of 2% and 5% E. faecium groups were significantly lower than that of the control group (P < 0.05). While the SOD and T-AOC and ACP and AKP of experimental groups were significantly higher (P < 0.05), the PO of experimental groups were significantly lower than that of the control group (P < 0.05). In addition, the expressions of immunity-related genes (tlr22, dorsal, lysozyme, crustin, imd, and relish) in the 2% and 5% E. faecalis groups were significantly greater than those in the control group (P < 0.05). After P. monodon was challenged with Vibrio parahaemolyticus for 7 days, the average cumulative mortality of P. monodon in the 2% and 5% groups were significantly lower than that in the 0% group (P < 0.05). With the increase of feeding time, the number of effective OTUs in each group showed a downward trend. At the 14th d, Proteobacteria, Bacteroidetes and Firmicutes, the dominant flora in the intestinal tract of P. monodon. In summary, supplied with E. faecium could increase the expression of non-specific immunity-related genes, enhance the immune capacity of P. monodon. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a significant cause of seafood-associated gastroenteritis and pestilence in aquaculture worldwide. Despite extensive research, strategies for protein depletion in this pathogen remain limited. Herein, we constructed a new CRISPR interference (CRISPRi) system for gene repression based on the combination of a shuttle vector pVv3 and the nuclease-null Cas9 variant (dead Cas9, or dCas9) from Streptococcus pyrogens. This CRISPRi is induced by adding both IPTG and arabinose. We showed that gene repression is scalable via the use of multiple sgRNAs. We also demonstrated that this gene repression can be precisely tuned by adjusting the amount of two different inducers and can be reversed by removing the inducers. This system provides a simple approach for selective gene repression on a genome-wide scale in V. parahaemolyticus. Application of this system will dramatically accelerate investigations of this bacterium, including studies of physiology, pathogenesis, and drug target discovery. [ABSTRACT FROM AUTHOR]

In order to explore new gallic acid derivatives with high antibacterial activity, we synthesize a series of new 1-(3,4,5-trihydroxybenzoyl)-4-(substituted benzoyl)thiosemicarbazide and evaluate their activities against Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in vitro. Through the experiments, we found that compound 7d has the best inhibitory activity against V. harveyi and V. cholerae, the minimum inhibitory concentration (MIC) values were 0.0078 mg/mL and 0.0156 mg/mL; compound 7h has the best inhibitory activity against V. parahaemolyticus and V. vulnificus, with MIC values of 0.0313 mg/mL and 0.0625 mg/mL. [ABSTRACT FROM AUTHOR]

Actinomycetes from marine habitats are relatively unexplored. They are reported as potential sources of novel bacterial species. In this current study, 16 strains of bacteria were isolated using Actinomycetes Isolation Agar medium. Two strains showed antibacterial activity against the shrimp pathogens, Aeromonas hydrophila and Vibrio parahaemolyticus. Among them, only 1 strain was found to have potent antibacterial properties. This strain was designated as Bacillus licheniformis VIT02. The crude bioactive compounds obtained from Bacillus licheniformis were extracted and identified using FTIR and GC–MS analyses. The data revealed that the phenolic bioactive compound, phenol,2,4-bis(1,1-dimethylethyl), and ester compound, propanoic acid, 2-Hydroxy-, Ethyl Ester, (S) have antimicrobial properties, and hence, can be used as potent antimicrobial drugs after performing further studies. The in vivo pathogenicity in Macrobrachium rosenbergii was investigated against A. hydrophila for 120 h. After 120 h of exposure, 34% of mortality was observed at the highest concentration of 106A. hydrophila. The in vivo treatment using the bioactive compound from the isolated strain B. licheniformis was performed against A. hydrophila in M. rosenbergii. The shrimp challenged with A. hydrophila when treated with 25 µL of bioactive compounds extracted from B. licheniformis showed 90% of survival up to 15 days of exposure. A survey of literature revealed that this could be the first study reporting the treatment of A. hydrophila infection in M. rosenbergii using the bioactive compounds from marine Actinomycete, Bacillus licheniformis VIT02. The histopathological analysis revealed that there was difference between the infected and the treated groups when compared with the control. The results indicated that the crude bioactive compound revealed potent disease resistance capacity against A. hydrophila in M. rosenbergii. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, undergoes wrinkly and smooth colony switching on the plate. The wrinkly spreader grew faster, had stronger motility and biofilm capacity when compared with the smooth one. However, whether the two phenotypes differ in their virulence still needs to be further investigated. In this study, the data showed that the smooth spreader had stronger virulence phenotypes, including the cytotoxicity against HeLa cells, antibacterial activity against E. coli, adhesive capacity toward HeLa cells, and lethality in zebrafish, relative to the wrinkly one. However, the colony morphology variation had no influence on the haemolytic activity. The mRNA levels of major virulence genes including T3SS1, T6SS1, and T6SS2 were significantly enhanced in the smooth colonies relative to those in the wrinkly colonies. Taken together, the presented work highlighted the different virulence profiles of the wrinkly and smooth colony phenotypes. [ABSTRACT FROM AUTHOR]

Cirrhinus reba (Hamilton, 1822) has gained high recognition in southern Asia for its nutritional and commercial benefits, although the carp is now facing productivity constraints. Probiotic-based captive breeding of the carp can be a promising technique to enhance the productivity and health status of the carp. The comprehensive study was performed on bacteria in the intestinal tract of C. reba. The total load of culturable bacteria in the gut of the carp was (8.34 ± 1.64) × 107 CFU/g comprising 19 different types of colonies. Most (73.7%) of the isolates were Gram-positive and rod-shaped bacteria. Among them, two intestinal isolates (PKS9 and PKS10) had shown significant antagonism against common fish pathogens namely, Staphylococcus aureus, Vibrio harveyi, and Vibrio parahaemolyticus. 16S rDNA sequencing identified the isolates PKS9 and PKS10 as Bacillus paramycoides (OM038513) and Bacillus cereus (OM033468), respectively. The isolates were determined to be biofilm formers, could persist in both acidic and alkaline environments, had high resistance to bile salts, possess surface hydrophobicity and auto-aggregation efficacy. The tests on pathogenicity and bio-safety confirmed the non-pathogenic nature of the isolates. Moreover, the strains had liberated extracellular enzymes and exhibited antioxidative properties. Hence, the isolates might be used as potential candidate probiotics for the cultivation of C. reba to enhance the nutritional and health status of the species. To the best of our knowledge, this is the first report analyzing the gut microbiota of Reba carp and establishing B. paramycoides as aquaculture probiotics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In Southeast Asia, the penaeid shrimp aquaculture production faces a new pandemic bacterial disease called acute hepatopancreatic necrosis disease (AHPND). The highly profitable pacific white shrimp, Litopenaeus vannamei, has become a challenging species due to severe lethal infections. Recent research has identified a critical pathogen, Vibrio parahaemolyticus, which caused significant loss in the shrimp industry. The disease pathway involves a virulence plasmid encoding binary protein toxins (PirA/B) that cause cell death. The protein toxins were inherited and conjugatively transferred to other Vibrio species through a post-segregational killing system. In this study, "in silico" (Glide, 2021) analysis identified four phytocompounds as myricetin (Myr), (+)-taxifolin (TF), (-)-epigallocatechin gallate (EGCG), and strychnine (STN) which could be most effective against both the toxins concerning its docking score and affinity. The interactions of complexes and the critical amino acids involved in docking were analyzed using the Discovery Studio (version 2016). Molecular dynamic studies showed lower root mean square deviations (RMSD) and improved stabilization of (+)-taxifolin (TF) and (-)-epigallocatechin-3-gallate (EGCG) against both the protein toxins. The antibacterial potential of all four selected compounds had tested against pathogenic strains of V. parahaemolyticus through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The best MBC results were observed at concentrations of 1 mg/mL for EGCG and 1.25 mg/mL for TF. Moreover, the complete reduction of viable cell counts in the in vitro bactericidal activity had recorded after 24 h of incubation. [ABSTRACT FROM AUTHOR]

Olive ridleys (Lepidochelys olivacea) are the most common sea turtle found in the Gulf of California. Unfortunately, the bacterial flora of nesting olive ridley turtles is still unknown. We conducted a study to identify, characterize, serotype, and determine the antibiotic resistance of potentially pathogenic bacteria isolated from olive ridley turtles nesting in northwestern Mexico. Bacteria were isolated and identified from the oral cavity and cloaca of 47 postnesting turtles. Escherichia coli and Vibrio parahaemolyticus were characterized, and antibiotic resistance testing was performed. One hundred bacteria belonging to 21 species were isolated, 53 from the oral cavity and 47 from the cloaca, the most prevalent being Pseudomonas aeruginosa, followed by Aeromonas hydrophila, Vibrio alginolyticus, Vibrio parahaemolyticus, Klebsiella pneumoniae, and E. coli, among others. Moreover, two to three different bacterial species were found co-colonizing both anatomical sites in some turtles. E. coli phylogroups B1, A, F, and unknown were identified as diarrheagenic E. coli (enteroaggregative and enteropathogenic E. coli). O1, O4, K8, K12, OUT, and KUT of V. parahaemolyticus serogroups were identified, also comprising pathogenic and nonpathogenic strains. Finally, 100% of the bacterial species tested were antibiotic resistant, and both MDR and XDR strains were found. In conclusion, olive ridley turtles are colonized by a diversity of bacterial species with a high rate of antibiotic resistance, some with pathogenic potential to turtles, representing a health risk factor for the species. [ABSTRACT FROM AUTHOR]

Vibrio species can cause foodborne infections and lead to serious gastrointestinal illnesses. The purpose of this research was to detect the Vibrio cholerae and Vibrio parahaemolyticus in raw milk, dairy products, and water samples. Also, it investigated the virulence factors, antibiotic resistance and biofilm formation in isolated bacteria. Conventional and molecular approaches were used to identify the isolates in this study. Vibrio species were detected in 5% of the samples. Vibrio cholerae and Vibrio parahaemolyticus were isolated from 1.25 and 1.5%, respectively, of the total samples. Penicillin resistance was detected in all strains of Vibrio cholerae and Vibrio parahaemolyticus, with a MAR index ranging from 0.16 to 0.5. Four isolates were moderate biofilm producer and three of them were MDR. When Vibrio cholerae was screened for virulence genes, ctxAB, hlyA, and tcpA were found in 80, 60, and 80% of isolates, respectively. However, tdh + /trh + associated-virulence genes were found in 33.3% of Vibrio parahaemolyticus isolates. [ABSTRACT FROM AUTHOR]

The global aquaculture industry has suffered significant losses due to the outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticus. Since the use of antibiotics as control agents has not been shown to be effective, an alternative anti-infective regimen, such as phage therapy, has been proposed. Here, we employed high-throughput screening for potential phages from 98 seawater samples and obtained 14 phages exhibiting diverse host specificity patterns against pathogenic VPAHPND strains. Among others, two Chimallinviridae phages, designated Eric and Ariel, exhibited the widest host spectrum against vibrios. In vitro and in vivo studies revealed that a cocktail derived from these two nucleus-forming vibriophages prolonged the bacterial regrowth of various pathogenic VPAHPND strains and reduced shrimp mortality from VPAHPND infection. This research highlights the use of high-throughput phage screening that leads to the formulation of a nucleus-forming phage cocktail applicable for bacterial infection treatment in aquaculture. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus, a potent human and aquatic pathogen, is usually found in estuaries and oceans. Human illness is associated with consuming uncooked/partially cooked contaminated seafood. The study on bivalve-associated V. parahaemolyticus revealed that the post-monsoon season had the highest bacterial abundance (9 ± 1.5 log cfu) compared to the monsoon season (8.03 ± 0.56 log cfu). Antimicrobial resistance (AMR) profiling was performed on 114 V. parahaemolyticus isolates obtained from bivalves. The highest AMR was observed against ampicillin (78%). Chloramphenicol was found to be effective against all the isolates. Multiple antibiotic resistance index values of 0.2 or higher were detected in 18% of the isolates. Molecular analysis of antimicrobial resistant genes (ARGs) revealed the high prevalence (100%) of the TEM-1 gene in the aquatic environment. After plasmid profiling and curing, 41.6% and 100% of the resistant isolates were found to be sensitive to ampicillin and cephalosporins, respectively, indicating the prevalence of plasmid-associated ARGs in the aquatic environment. A study to evaluate the antagonistic properties of Bacillus subtilis, Pseudomonas aeruginosa, and Bacillus amyloliquefaciens against V. parahaemolyticus isolates identified the potential of these bacteria to resist the growth of V. parahaemolyticus. [ABSTRACT FROM AUTHOR]