ZntA maintains zinc and cadmium homeostasis and promotes oxidative stress resistance and virulence in Vibrio parahaemolyticus.

Although metals are essential for life, they are toxic to bacteria in excessive amounts. Therefore, the maintenance of metal homeostasis is critical for bacterial physiology and pathogenesis. Vibrio parahaemolyticus is a significant food-borne pathogen that mainly causes acute gastroenteritis in humans and acute hepatopancreatic necrosis disease in shrimp. Herein, we report that ZntA functions as a zinc (Zn) and cadmium (Cd) homeostasis mechanism and contributes to oxidative stress resistance and virulence in V. parahaemolyticus. zntA is remarkably induced by Zn, copper, cobalt, nickel (Ni), and Cd, while ZntA promotes V. parahaemolyticus growth under excess Zn/Ni and Cd conditions via maintaining Zn and Cd homeostasis, respectively. The growth of ΔzntA was inhibited under iron (Fe)-restricted conditions, and the inhibition was associated with Zn homeostasis disturbance. Ferrous iron supplementation improved the growth of ΔzntA under excess Zn, Ni or Cd conditions. The resistance of ΔzntA to H2O2-induced oxidative stress also decreased, and its virulence was attenuated in zebrafish models. Quantitative real-time PCR, mutagenesis, and ß-galactosidase activity assays revealed that ZntR positively regulates zntA expression by binding to its promoter. Collectively, the ZntR-regulated ZntA is crucial for Zn and Cd homeostasis and contributes to oxidative stress resistance and virulence in V. parahaemolyticus.

Morinda citrifolia fruit extract enhances the resistance of Penaeus vannamei to Vibrio parahaemolyticus infection.

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.

The Yin Yang 1 of Penaeus vannamei regulates transcription of the small subunit hemocyanin gene during Vibrio parahaemolyticus infection.

Hemocyanin is a respiratory protein, it is also a multifunctional immune molecule that plays a vital role against pathogen invasion in shrimp. However, the regulation of hemocyanin gene expression in shrimp hemocytes and the mechanisms involved during pathogen infection remains unclear. Here, we used DNA pull-down followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the Yin Yang 1 transcription factor homolog in Penaeus vannamei (PvYY1) as a key factor that modulates transcription of the small subunit hemocyanin gene of P. vannamei (PvHMCs) in hemocytes during Vibrio parahaemolyticus AHPND (VPAHPND) infection. Bioinformatics analysis revealed that the core promoter region of PvHMCs contains two YY1 motifs. Mutational and oligoprecipitation analyses confirmed that PvYY1 could bind to the YY1 motifs in the PvHMCs core promoter region, while truncation of PvYY1 revealed that the N-terminal domain of PvYY1 is essential for the transactivation of PvHMCs core promoter. Besides, the REPO domain of PvYY1 could repress the activity of the PvHMCs core promoter. Overexpression of PvYY1 significantly activates the promoter activity of PvHMCs core promoter, while PvYY1 knockdown significantly decreases the expression level of PvHMCs in shrimp hemocytes and survival rate of shrimp upon infection with VPAHPND. Our present study provides new insights into the transcriptional regulation of PvHMCs by PvYY1 in shrimp hemocytes during bacteria (VPAHPND) infection.

The effect of alginate oligosaccharides on intestine barrier function and Vibrio parahaemolyticus infections in the white shrimp Litopenaeus vannamei.

The intestine is a host-pathogen interaction site and improved intestinal barrier function help to prevent disease in shrimp. Alginate oligosaccharides (AOS) are derived from resourceful brown algae. The intestine protection properties of AOS were widely recognized, and their benefits in fish have been reported. Nevertheless, there are no reports on AOS in shrimp and other crustaceans. In the present work, we measured the effects of AOS on growth performance and disease resistance in the white shrimp Litopenaeus vannamei and investigated their effects on intestinal health. Shrimps with an initial weight of about 2 g were fed with diets supplemented with 0 (control), 0.07%, 0.2%, 0.6%, or 1.2% of AOS for 56 days and were sampled and challenged with Vibrio parahaemolyticus. Dietary AOS did not significantly influence weight gain or feed utilization (P > 0.05). However, AOS considerably decreased the seven-day cumulative mortality after the challenge at any dose (P < 0.05). Dietary AOS improved the intestinal structure, significantly boosted the intestinal villus height at 0.6% and 1.2% levels, and increased intestinal wall thickness by 0.2%, 0.6%, and 1.2%. The alkaline phosphatase and maltase activities were also increased, suggesting that AOS improved the intestinal condition. Redox homeostasis in intestinal was improved by AOS, as expressed by the enhanced total antioxidant capacity and decreased malonaldehyde content, partly due to the increased superoxide dismutase and catalase activities. Compared with the antioxidant system, AOS's stimulating effects on immunity were more significant. At any level, AOS significantly activated lysozyme activity, the expression of propo and two antimicrobial peptide genes (pen-3 and crusin). However, the lowest concentration of AOS did not stimulate the gene expression of all three assayed pattern recognition receptors (LGBP, Toll, and IMD), and only the highest concentration of AOS increased the expression of imd. These findings suggest that AOS are highly efficient immunostimulants, and various immune pathways in shrimp are differentially sensitive to AOS. Finally, our findings suggest that AOS significantly alter the gut microbiota and their relative abundance at the phylum, family, and genus levels. In conclusion, AOS significantly enhances disease resistance in L. vannamei, possibly attributed to improved intestinal development, increased intestinal immunity and altered microbiota. These findings could provide a basis for future studies on the practical use of AOS and its mechanisms of action.

Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei.

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

Peroxiredoxin-4 supplementation modulates the immune response, shapes the intestinal microbiome, and enhances AHPND resistance in Penaeus vannamei.

Peroxiredoxin-4 from Penaeus vannamei (LvPrx4) is considered a damage-associated molecular pattern (DAMP) that can activate the expression of immune-related genes through the Toll pathway. We previously demonstrated that the recombinant LvPrx4 (rLvPrx4) can enhance shrimp resistance against Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (VPAHPND), which causes great production losses in shrimp farming. Herein, we showed that the rLvPrx4 had a thermal tolerance of around 60 °C and that the ionic strength had no noticeable effect on its activity. We discovered that feeding a diet containing rLvPrx4 to shrimp for three weeks increased the expression of the immune-related genes LvPEN4 and LvVago5. Furthermore, pre-treatment with rLvPrx4 feeding could significantly prolong shrimp survival following the VPAHPND challenge. The shrimp intestinal microbiome was then characterized using PCR amplification of the 16S rRNA gene and Illumina sequencing. Three weeks of rLvPrx4 supplementation altered the bacterial community structure (beta diversity) and revealed the induction of differentially abundant families, including Cryomorphaceae, Flavobacteriaceae, Pirellulaceae, Rhodobacteraceae, and Verrucomicrobiaceae, in the rLvPrx4 group. Metagenomic predictions indicated that some amino acid metabolism pathways, such as arginine and proline metabolism, and genetic information processing were significantly elevated in the rLvPrx4 group compared to the control group. This study is the first to describe the potential use of rLvPrx4 supplementation to enhance shrimp resistance to VPAHPND and alter the composition of a beneficial bacterial community in shrimp, making rLvPrx4 a promising feed supplement as an alternative to antibiotics for controlling VPAHPND infection in shrimp aquaculture.

A comparative study on effects of dietary three strains of lactic acid bacteria on the growth performance, immune responses, disease resistance and intestinal microbiota of Pacific white shrimp, Penaeus vannamei.

The present study evaluated the growth performance, immune responses, disease resistance and intestinal microbiota in Penaeus vannamei fed diets supplemented with three strains of lactic acid bacteria (LAB). The basal diet (control, CO) supplemented with Lactobacillus plantarum W2 (LA), Pediococcus acidilactici Nj (PE), Enterococcus faecium LYB (EN) and florfenicol (FL), respectively, formed three LAB diets (1 × 1010 cfu kg-1) and a florfenicol diet (15 mg kg-1, positive control), were fed to shrimp for 42 days. Results indicated that specific growth rate, feed efficiency rate, and disease resistance of shrimp against Vibrio parahaemolyticus in the treatment groups were significantly improved versus the control (P < 0.05). Compared with the control, acid phosphatase, alkaline phosphatase, phenonoloxidase, total nitric oxide synthase, peroxidase, superoxide dismutase activities, total antioxidant capacity, and lysozyme content in the serum and the relative expression levels of SOD, LZM, proPO, LGBP, HSP70, Imd, Toll, Relish, TOR, 4E-BP, eIF4E1α and eIF4E2 genes in the hepatopancreas of LAB groups were enhanced to various extents. Intestinal microbiota analysis showed that the LA and EN groups significantly improved microbial diversity and richness, and LAB groups significantly altered intestinal microbial structure of shrimp. At the phylum level, the Verrucomicrobiota in the LA and PE groups, the Firmicutes in the EN group, and the Actinobacteriota in the PE and EN groups were enriched. Moreover, the CO group increased the proportion of potential pathogens (Vibrionaceae and Flavobacteriaceae). The potential pathogen (Vibrio) was reduced, and potential beneficial bacteria (Tenacibaculum, Ruegeria and Bdellovibrio) were enriched in response to dietary three strains of LAB. When the intestinal microbiota homeostasis of shrimp is considered, L. plantarum and E. faecium showed better effects than P. acidilactici. However, due to the concerns on the possible potential risks of E. faecium strains to human health, L. plantarum W2 is more suitable for application in aquaculture than E. faecium LYB. Considering collectively the above, Lactobacillus plantarum W2 could be applied as better probiotic to improve the growth performance, non-specific immunity, disease resistance and promote intestinal health of P. vannamei.

Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei.

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

Improving red-color performance, immune response and resistance to Vibrio parahaemolyticus on white shrimp Penaeus vannamei by an engineered astaxanthin yeast.

Astaxanthin (AST), a super antioxidant with coloring and medical properties, renders it a beneficial feed additive for shrimp. This study conducted a white shrimp feeding trial of 3S, 3'S isoform AST, which was derived from metabolic-engineered Kluyveromyces marxianus fermented broth (TB) and its extract (TE) compared to sources from two chemically synthetic ASTs (Carophyll Pink [CP] and Lucantin Pink [LP]), which contain 3S, 3'S, 3R, 3'S (3S, 3'R) and 3R, 3'R isoforms ratio of 1:2:1. The effects on red coloration, immune parameters and resistance to Vibrio infection were evaluated. Four AST sources were incorporated into the diets at concentrations of 0 (control), 100 mg kg-1 (TB100, TE100, CP100, and LP100), and 200 mg kg-1 (TB200, TE200, CP200, and LP200). Results revealed that in week 4, shrimps that received AST-supplemented feeds, especially TB100, TB200, and TE200, significantly increased redness (a*) values. Immune responses including phagocytosis activity, superoxide-anion production, phenoloxidase activity, and immune-related genes were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Generally, shrimps that received AST-supplemented feeds exhibited higher immune responses on days 7 and 14 than the control feed. Gene expression levels of superoxide dismutase and glutathione peroxidase were significantly upregulated on days 7 and 14 in shrimps that received AST-supplemented feeds, while genes of penaeidins, antilipopolysaccharide factor, and lysozyme were upregulated on days 4, 7, and 14, especially received TB200 and TE200. Furthermore, shrimps that received TB100, TE100, CP100, and LP100 7 days were then challenged with Vibrio parahaemolyticus and the result demonstrated higher survival rates especially TB100 at 168 h than the control feed. In conclusion, incorporating AST into the diets enhanced shrimp red coloration, immune parameters, and resistance against V. parahaemolyticus infection. The K. marxianus-derived AST exhibited higher performance than did chemical AST to be a potential feed additive in shrimp aquaculture.

Dietary supplementation of Salvinia cucullata in white shrimp Litopenaeus vannamei to enhance the growth, nonspecific immune responses, and disease resistance to Vibrio parahaemolyticus.

The current study investigates the effect of ethanolic extract of Salvinia cucullata (EESC) on growth, non-specific immune parameters, and disease resistance to Vibrio parahaemolyticus in Litopenaeus vannamei. The in-vitro cytotoxicity investigation was performed on shrimp hemolymph hemocytes to assess the toxicity and immunological responses with various concentrations of EESC, and no significant difference in cell viability was seen across dosages, but substantial changes in Phenol Oxidase (PO) and phagocytosis were reported. The in-vivo investigation was conducted on white shrimp for 56 days using varied amounts of 0 (control), 5 (EESC5), 10 (EESC10), and 20 (EESC20) g kg-1 containing feeds and challenged against Vibrio parahaemolyticus. The shrimp fed the EESC10 diet gained the most weight, had the highest specific growth rate (SGR) and had a better feed conversion ratio (FCR). The highest cumulative survival percentage was noted on the EESC10 diet-fed shrimps followed by EESC20 and EESC5 groups after the bacterial challenge with V. parahaemolyticus. The results of immune parameters such as total protein, total carbohydrate, coagulation time, total hemocytes count (THC), superoxide dismutase (SOD), ProPO, and phagocytosis levels were better in the EESC10 group. EESC5 and EESC20 groups were also shown better immunomodulatory effects than the control group. In conclusion, the oral administration of EESC was found to be an effective functional feed additive to improve the growth, immune parameters, and disease resistance against V. parahaemolyticus in L.vannamei.

Effect of multifactorial free and liposome-coated of bay laurel (Laurus nobilis) and rosemary (Salvia rosmarinus) extracts on the behavior of Listeria monocytogenes and Vibrio parahaemolyticus in silver carp (Hypophthalmichthys molitrix) stored at 4 °C.

In this study, the effect of bay laurel (Laurus nobilis) (LE) and rosemary (Salvia rosmarinus) (RE) extracts, in two free forms and loaded with liposome, on the behavior of Listeria monocytogenes and Vibrio parahaemolyticus in silver carp (Hypophthalmichthys molitrix) minced, were examined. After extraction, the extracts were evaluated for phenolic, flavonoid, and antibacterial compounds (determination of MIC and MBC). The treatments studied included control treatment, treatments containing 1 and 1.5% of free extracts, and treatments containing 1 and 1.5% of liposome-coated extracts of LE and RE which were examined at times of 0, 4, 8, and 12 days with 3 replications. The findings indicated that the amount of flavonoid and phenolic compounds and the results of antibacterial tests (MIC and MBC tests) in RE extract were more favorable than LE extract. The aqueous extract of rosemary had higher levels of phenolic (344.66 mg gallic acid/g extract) and flavonoid (245.33 mg Catechin/g extract) compounds compared to the bay laurel extract (257.66 mg gallic acid/g extract) and (151.26 mg Catechin/g extract) respectively. The results of the behavior of L. monocytogenes and V. parahaemolyticus in fish showed that with increasing the storage time at 4 °C, these parameters increased, but in the treatment containing the coated forms of LE and RE extracts (concentration 1.5%), changes were significantly slower than other treatments. According to the obtained results, it can be concluded that in general, adding extracts of bay laurel and rosemary in a concentration of 1.5% reduces the proliferation of bacteria that cause food poisoning.

Supplementing artemisinin positively influences growth, antioxidant capacity, immune response, gut health and disease resistance against Vibrio parahaemolyticus in Litopenaeus vannamei fed cottonseed protein concentrate meal diets.

Artemisinin (ART) is a kind of Chinese herbal medicine worth exploring, which obtains various physiological activities. In order to study the prebiotic effect of ART on Litopenaeus vannamei fed cottonseed protein concentrate meal diets, six groups of isonitrogenous and isolipid diets were prepared (including the fish meal control group, FM; cottonseed protein concentrate replacing 30% fishmeal protein and supplementing ART groups: ART0, ART0.3, ART0.6, ART0.9, and ART1.2). The feeding trials was lasted for 56 days. The results showed that the final body weight, weight gain and specific growth rate of the ART0.6 group were the highest, yet the feed coefficient rate of the ART0.6 group was the lowest significantly (P < 0.05). There was no significant difference in survival rate among treatments (P > 0.05). In serum, the content of malondialdehyde in ART0 group was the highest (P < 0.05); the activities of superoxide dismutase, catalase, phenol oxidase and lysozyme increased firstly and then decreased among the ARTs groups (P < 0.05). The activities of intestinal digestive enzymes (including the trypsin, lipase and amylase) showed an upward trend among the ARTs groups (P < 0.05). The histological sections showed that the intestinal muscle thickness, fold height and fold width in the FM group were significantly better than those in the ART0 group; while the mentioned above morphological indexes in the ART0 group were significantly lowest among the ARTs groups (P < 0.05). Sequencing of intestinal microbiota suggested that the microbial richness indexes firstly increased and then decreased (P < 0.05); the bacterial community structure of each treatment group was almost close; the relative abundance of pathogenic bacteria decreased significantly (P < 0.05), such as the Proteobacteria and Cyanobacteria at phylum level, besides the Vibrio and Candidatus Bacilloplasma at genus level. In intestinal tissue, the relative expression levels of TOLL1, TRAF6 and Pehaeidih3 showed up-regulated trends, while the expression of Crustin and LZM firstly up-regulated and then down-regulated (P < 0.05). The challenge experiment suggested that the cumulative mortality of FM group was significantly lower than that of ART0 group; besides the cumulative mortality firstly increased and then decreased between the ARTs groups (P < 0.05). In conclusion, the dietary supplementation of ART can improve the growth, antioxidant capacity, immune response, gut health and disease resistance of the shrimp. To be considered as a dietary immune enhancer, the recommended supplementation level of ART in shrimp's cottonseed protein concentrate meal diets is 0.43%.

Metal Ions and Chemical Modification Reagents Inhibit the Enzymatic Activity of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus.

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: ß-mercaptoethanol (ßME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20−95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146−1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.

Ferrous sulfate efficiently kills Vibrio parahaemolyticus and protects salmon sashimi from its contamination.

The primary seafood-borne pathogen Vibrio parahaemolyticus seriously threats the health of consumers preferring raw-fish products, becoming a global concern in food safety. In the present study, we found ferrous sulfate (FeSO4), a nutritional iron supplement, could efficiently induce the death of V. parahaemolyticus. Further, the bactericidal mechanisms of FeSO4 were explored. With a fluorescent probe of Fe2+, a significant influx of Fe2+ was determined in V. parahaemolyticus exposed to FeSO4, and the addition of an intracellular Fe2+ chelator was able to block the cell death. This suggested that cell death in V. parahaemolyticus induced by FeSO4 was dependent on the influx of Fe2+. It was intriguing that we did not observe the eruption of reactive oxygen species (ROS) and lipid hydroperoxides by Fe2+, but the application of liproxstatin-1 (a ferroptosis inhibitor) significantly modified the occurrence of cell death in V. parahaemolyticus. These results suggested FeSO4-induced cell death in V. parahaemolyticus be a ferroptosis differing from that in mammalian cells. Through transcriptome analysis, it was discovered that the exposure of FeSO4 disturbed considerable amounts of gene expression in V. parahaemolyticus including those involved in protein metabolism, amide biosynthesis, two-component system, amino acid degradation, carbon metabolism, citrate cycle, pyruvate metabolism, oxidative phosphorylation, and so on. These data suggested that FeSO4 was a pleiotropic antimicrobial agent against V. parahaemolyticus. Notably, FeSO4 was able to eliminate V. parahaemolyticus in salmon sashimi as well, without affecting the color, texture, shearing force, and sensory characteristics of salmon sashimi. Taken together, our results deciphered a unique ferroptosis in V. parahaemolyticus by FeSO4, and highlighted its potential in raw-fish products to control V. parahaemolyticus.

Dietary of Lactobacillus paracasei and Bifidobacterium longum improve nonspecific immune responses, growth performance, and resistance against Vibrio parahaemolyticus in Penaeus vannamei.

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 105 and 107 CFU/g; these diets were referred to as P5, P7, B5, and B7. After 8 weeks of the diets, regarding growth performance, the B7 group showed the highest weight gain rate (890.34 ± 103.65%), special growth rate (4.08 ± 0.19%), and feed conversion rate (1.52 ± 0.19%) compared with the other groups. Moreover, the total hemocyte counts were significantly increased (p < 0.05) in the P7 groups on day 14 during the 28-day feeding trial. The phagocytosis rate in all experimental groups was increased on day 14 and was persistently significantly activated to day 21, especially in the P7 and B5 group. The phagocytic index of the P7 group showed a significant increase on day 14 and persistent activation to day 21. In the analysis of respiratory burst activity and phenoloxidase activity, the P7 and B5 groups showed a significant increase on day 7 and persistent activation to day 21. The expression level of the immune-related genes of superoxide dismutase, clotting protein, Penaeidin2, Penaeidin3, Penaeidin4, anti-LPS factor, crustin, and lysozyme was significantly increased in the experimental groups, especially in the P7 group. Furthermore, the optimum conditions of feed additives were determined in challenge trials conducted using P7 and B5. Shrimps fed P7 and B5 showed an increased survival rate (72.73% and 66.67%) after the V. parahaemolyticus challenge. In sum, the results revealed that B. longum, as a feed additive at 107 CFU/g, enhanced growth performance. L. paracasei at 107 CFU/g and B. longum at 105 CFU/g can enhance nonspecific immune responses and immune-related gene expression, and 107 CFU/g L. paracasei has the highest resistance ability for V. parahaemolyticus. Thus, dietary supplementation with L. paracasei and B. longum may be a valuable approach in white shrimp aquaculture.

Identification of antibacterial metabolites produced by a marine bacterium Halobacillus marinus HMALI004.

AIMS: This study examined and characterized the extract for metabolites of Halobacillus marinus HMALI004 to understand their antibacterial activities against opportunistic marine pathogens, that is, Vibrio parahaemolyticus and Vibrio cholerae. METHODS AND RESULTS: The bacterial strain HMALI004 was characterized as H. marinus, and an antibacterial spectral test revealed its inhibition against two opportunistic marine pathogens (V. parahaemolyticus and V. cholera). Fermentation broth of strain HMALI004 was subjected to column chromatography and high-performance liquid chromatography to separate antibacterial substances. Two compounds were successfully isolated and identified as 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid by mass spectrometry (MS) and nuclear magnetic resonance. The minimal inhibition concentration (MIC) values of 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid for V. parahaemolyticus were 25 µg/ml, while their MIC values for V. cholerae were 50 and 100 µg/ml, respectively. The reactive oxygen species (ROS) production of two pathogen strains treated with 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid were detected to investigate the antimicrobial mechanism. The results suggested that 4-chloro-1H-pyrrole-2-carboxylic acid exerted enhanced ROS production in V. parahaemolyticus, whereas 1H-pyrrole-2-carboxylic acid had a weaker effect. Both compounds caused a significant rise in ROS production in V. cholerae, causing severe damage to the cell wall and cytoplasm, leading to cell death. CONCLUSIONS: The bacterium H. marinus HMALI004 was isolated from a shrimp pond and was found to produce antimicrobial compounds, which could inhibit the growth of opportunistic marine pathogens V. parahaemolyticus and V. cholerae by increasing ROS. SIGNIFICANCE AND IMPACT OF THE STUDY: Successfully isolated antibacterial-producing strain, H. marinus HMALI004, and its antimicrobial compounds could be used as biological control agents for marine pathogens.

Salpianthus macrodontus Extracts, a Novel Source of Phenolic Compounds with Antibacterial Activity against Potentially Pathogenic Bacteria Isolated from White Shrimp.

This study aimed to evaluate the antibacterial activity in vitro of Salpianthus macrodontus and Azadirachta indica extracts against potentially pathogenic bacteria for Pacific white shrimp. Furthermore, the extracts with higher inhibitory activity were analyzed to identify compounds responsible for bacterial inhibition and evaluate their effect on motility and biofilm formation. S. macrodontus and A. indica extracts were prepared using methanol, acetone, and hexane by ultrasound. The minimum inhibitory concentration (MIC) of the extracts was determined against Vibrio parahaemolyticus, V. harveyi, Photobacterium damselae and P. leiognathi. The polyphenol profile of those extracts showing the highest bacterial inhibition were determined. Besides, the bacterial swimming and swarming motility and biofilm formation were determined. The highest inhibitory activity against the four pathogens was found with the acetonic extract of S. macrodontus leaf (MIC of 50 mg/mL for Vibrio spp. and 25 mg/mL for Photobacterium spp.) and the methanol extract of S. macrodontus flower (MIC of 50 mg/mL for all pathogens tested). Both extracts affected the swarming and swimming motility and the biofilm formation of the tested bacteria. The main phenolic compounds related to Vibrio bacteria inhibition were naringin, vanillic acid, and rosmarinic acid, whilst hesperidin, kaempferol pentosyl-rutinoside, and rhamnetin were related to Photobacterium bacteria inhibition.

Antibiofilm efficacy of Leuconostoc mesenteroides J.27-derived postbiotic and food-grade essential oils against Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli alone and in combination, and their application as a green preservative in the seafood industry.

Foodborne pathogen-mediated biofilms in food processing environments are severe threats to human lives. In the interest of human and environmental safety, natural substances with antimicrobial properties and generally regarded as safe (GRAS) status are the futuristic disinfectants of the food industry. In this study, the efficacy of bioactive, soluble products (metabolic by-products) from lactic acid bacteria (LAB) and plant-derived essential oils (EO) were investigated as biocidal agents. The postbiotic produced by kimchi-derived Leuconostoc mesenteroides J.27 isolate was analyzed for its metabolic components to reveal its antimicrobial potential against three pathogenic microorganisms (Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli). Additionally, the efficacy of food-grade EO (eugenol and thymol, respectively) was also assessed in our study. Determination of the minimum inhibitory concentration (MIC) of postbiotic and EO against three tested pathogens revealed that the sub-MIC (0.5 MIC) of postbiotic and EO could efficiently inhibit the biofilm formation on both seafood (squid) and seafood-processing surfaces (rubber and low-density polyethylene plastic). Moreover, the polymerase chain reaction (PCR) analysis confirmed that the LAB J.27 isolate possesses bacteriocin- and enzyme-coding genes. The residual antibacterial activity of the produced postbiotic was maintained over a diverse pH range (pH 1-6) but was entirely abolished at neutral or higher pH values. However, the activity was unaffected by exposure to high temperatures (100 and 121 °C) and storage (30 days). Notably, the leakage of intracellular metabolites, damage to DNA, and the down-regulation of biofilm-associated gene expression in the pathogens increased significantly (p > 0.05) following the combination treatment of postbiotic with thymol compared to postbiotic with eugenol. Nonetheless, all in vitro results indicated the prospective use of combining Leu. mesenteroides J.27-derived postbiotic with both EO as a "green preservative" in the seafood industry to inhibit the formation of pathogenic microbial biofilms.

Effects of the Clostridium butyricum on growth performance, antioxidant capacity, immunity and disease resistance of Litopenaeus Vannamei fed with cottonseed protein concentrate (CPC) replacement of fishmeal in diet.

Clostridium butyricum (CB) is a gram-positive bacterium that secretes short-chain fatty acids such as butyric acid and so on. An 8-week feeding trial was conducted to investigate the effects of CB on the growth performance, antioxidant capacity, immunity and resistance to Vibrio parahaemolyticus in Litopenaeus Vannamei fed with cottonseed protein concentrate (CPC) replacement of fishmeal. Six iso-nitrogenous (40%) and iso-lipidic (6%) diets were formulated including a positive control group (PC, 25% fishmeal), a negative control group (NC, CPC replaced 30% of fishmeal protein), and 0.03% (C1, 3 × 108 CFU/kg), 0.12% (C2, 1.2 × 109 CFU/kg), 0.48% (C3, 4.8 × 109 CFU/kg) and 1.92% (C4, 1.92 × 1010 CFU/kg) CB were supplemented on the negative control group (NC). After the feeding trial, the remaining shrimp in each treatment group were subjected to a challenge experiment with Vibrio parahaemolyticus. The results indicated that weight gain rate (WGR), specific growth rate (SGR) in C4 group were significantly lower than those in PC and C2 groups (P < 0.05); the feed conversion ratio (FCR) was significantly higher than that of PC and C2 groups (P < 0.05). There was no significant difference in survival rate (SR) among all groups (P > 0.05). Compared to the PC and NC groups, the total superoxide capacity, superoxide dismutase and lysozyme were significantly higher in the C4 group (P < 0.05); the glutathione peroxidase, acid phosphatase and alkaline phosphatase were significantly higher in the C3 group (P < 0.05); and the malondialdehyde was significantly lower in the C4 group (P < 0.05). The relative mRNA expressions of Toll receptor (TLR), innate immune deficiency gene (IMD), penaiedin3a (Pen3) were significantly down-regulated in the NC group than those in the PC group (P < 0.05). In addition, the relative mRNA expressions of TLR, IMD and Pen3 were significantly up-regulated in all groups supplemented with CB than those in the NC group (P < 0.05). Moreover, the cumulative mortality rate in the NC group was not significantly different from the PC group (P > 0.05) and was significantly higher than those in the C3 and C4 groups (P < 0.05). In conclusion, the CB supplementation on the basis of CPC replacement of 30% fishmeal protein enhanced significantly the antioxidant capacity, immunity and disease resistance of shrimp and improved its growth performance. Therefore, considering the factors of the growth, immunity and disease resistance, the CB supplementation of 0.12%-0.48% (1.2 × 109 CFU/kg-4.8 × 109 CFU/kg) was recommended in the diet of L. vannamei based on the results of this experiment.

Effect of High Hydrostatic Pressure Treatment on Urease Activity and Inhibition of Fishy Smell in Mackerel (Scomber japonicus) during Storage.

In this study, the physicochemical changes related to fishy smell were determined by storing high hydrostatic pressure (HHP)-treated mackerel (Scomber japonicus) meat in a refrigerator for 20 days. The inhibition of crude urease activity from Vibrio parahaemolyticus using HHP treatment was also investigated. The mackerel meat storage experiment demonstrated that production of trimethylamine (TMA) and volatile basic nitrogen (VBN), the main components of fishy smell, was significantly reduced on the 20th day of storage after the HHP treatment compared to the untreated mackerels. The results demonstrated that the increased ammonia nitrogen rates in the 2000, 3000, and 4000 bar, HHP-treated groups decreased by 23.8%, 23.8%, and 31.0%, respectively, compared to the untreated groups. The enzyme activity of crude urease was significantly reduced in the HHP-treated group compared to that in the untreated group. Measurement of the volatile organic compounds (VOCs) in mackerel meat during storage indicated that the content of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to spoilage, were significantly reduced by HHP treatment. Collectively, our results suggested that HHP treatment would be useful for inhibiting the activity of urease, thereby reducing the fishy smells from fish and shellfish.