Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

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

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

Validity of cold storage and heat treatment on the deactivation of Vibrio parahaemolyticus isolated from fish meat markets.

Vibrio parahaemolyticus is a zoonotic disease transmitted to humans when handling or consuming improperly cooked fish meat. This study aimed to evaluate the effect of thermal treatment on V. parahaemolyticus isolates. Different heat treatment methods are used to determine the best methods for controlling V. parahaemolyticus, isolated from fish meat, which include microwave, low-temperature long-time, and high-temperature short-time methods. The V. parahaemolyticus isolates significantly declined in bacteria count when they were kept at 4°C, and 25°C for a long time, and the V. parahaemolyticus isolates significantly declined in bacteria count manner when they were kept at -20°C for a long time. The high temperature and long-time exposure at 75°C/25 minutes by moist heat, 87°C/5 minutes by dry heat, and 70°C/20 minutes by frying heat were enough to kill V. parahaemolyticus isolates. This work can be useful to decrease the hazards of infections related to V. parahaemolyticus and reduce the causes of fishborne pathogens.

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

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

Dietary administration of yeast (Saccharomyces cerevisiae) hydrolysate from sugar byproducts promotes the growth, survival, immunity, microbial community and disease resistance to VP (AHPND) in Pacific white shrimp (Litopenaeus vannamei).

This study investigated the effects of yeast hydrolysate (YH) from sugar byproducts on various parameters in Pacific white shrimp (Litopenaeus vannamei). The study found no significant differences in water quality parameters across all treatment tanks, ensuring that the observed effects were not due to environmental variations. There were no significant differences in growth parameters between the control group and groups receiving YH at different dosages. However, the group given YH at 10.0 g/kg feed exhibited a notably higher survival rate and higher expression of growth-related genes (IGF-2 and RAP-2A) in various shrimp tissues. YH was associated with enhanced immune responses, including lysozyme activity, NBT dye reduction, bactericidal activity, and phagocytic activity. Notably, the 10.0 g/kg feed group displayed the highest phagocytic index, indicating a dose-dependent immune response. Expression of immune-related genes (ALF, LYZ, ProPO, and SOD) was upregulated in various shrimp tissues. This upregulation was particularly significant in the gills, hepatopancreas, intestine, and hemocytes. While total Vibrio counts remained consistent, a reduction in green Vibrio colonies was observed in the intestine of shrimp treated with YH. YH, especially at 5.0 and 10.0 g/kg feed dosages, significantly increased survival rates and RPS values in response to AHPND infection. The findings of this study suggest that incorporating additives derived from yeast byproducts with possible prebiotic properties obtained from sugar byproducts can lead to positive results in terms of enhancing growth performance, immunity, histological improvements, and resistance to V. parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND).

Whole-genome sequencing and transcriptome-characterized mechanism of streptomycin resistance in Vibrio parahaemolyticus O10: K4.

Streptomycin resistance in V. parahaemolyticus has been widespread in both clinical and environmental isolates. Therefore, it is of great significance to characterize the mechanism of streptomycin resistance in V. parahaemolyticus. O10:K4 has emerged and becoming the new dominant serotype since 2020. In this study, we isolated a total of 36 strains of V. parahaemolyticus O10:K4 from 2020 to 2022 and found that more than half of them were resistant to streptomycin. We obtained streptomycin resistant and sensitive strains by detecting the resistance profiles. Whole-genome sequencing showed that VP_RS10735 and VP_RS05605 were the predominant mutations in streptomycin resistant O10:K4 clinical isolates. In addition, this study provided global insight into the characteristics of the transcriptome signature of streptomycin resistance, revealing that efflux transporters play a key role in streptomycin resistance. Finally, we found that streptomycin resistant strain was more virulent than sensitive strain. The results of this study should advance our understanding of the mechanisms of aminoglycoside resistance.

Anti-Biofilm Activity of Laurel Essential Oil against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a primary seafood-associated pathogen that could cause gastroenteritis. It can attach to various surfaces and form a biofilm, which poses serious threats to food safety. Hence, an effective strategy is urgently needed to control the biofilm formation of V. parahaemolyticus. Laurel essential oil (LEO) is used in food, pharmaceutical and other industries, and is commonly used as a flavoring agent and valuable spice in food industries. The potential antibiofilm effects of LEO against V. parahaemolyticus were examined in this study. LEO obviously reduced biofilm biomass at subinhibitory concentrations (SICs). It decreased the metabolic activity and viability of biofilm cells. Microscopic images and Raman spectrum indicted that LEO interfered with the structure and biochemical compositions of biofilms. Moreover, it also impaired swimming motility, decreased hydrophobicity, inhibited auto-aggregation and reduced attachment to different food-contact surfaces. RT-qPCR revealed that LEO significantly downregulated transcription levels of biofilm-associated genes of V. parahaemolyticus. These findings demonstrate that LEO could be potentially developed as an antibiofilm strategy to control V. parahaemolyticus biofilms in food industries.

Enhancement of loop mediated isothermal amplification's sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus.

The modified loop-mediated isothermal amplification (LAMP), called multiple hybrid, inner primers (MHP)-LAMP, was developed to enhance the efficiency of the existing LAMP-based assay for Vibrio parahaemolyticus detection. The method was built on a conventional LAMP assay by employing 2 newly designed extra sets of primers to increase the initial binding sites of core primers on the V. parahaemolyticus's rpoD gene from 8 to 12. With this strategy, the assay detection sensitivity was increased by 10 folds, with the detection limit (DL) approaching 100 copies of purified target genomic DNA (gDNA) as analyzed by real-time turbidity measurement and gel electrophoresis. The MHP also accelerated the rate of DNA amplification by 30%, rendering the assay faster. The MHP-LAMP assay did not cross- react with other pathogens, indicating that it was highly specific for V. parahaemolyticus detection. Whilst V. parahaemolyticus was used as a study model herein, our idea of using MHP to maximize assay sensitivity and speed is considered as a universal strategy that can be applied to enhance efficiency of LAMP-based assays for detecting any DNA and RNA of interest. •The strategy of using multiple hybrid, inner primers (MHP) to enhance LAMP assay's efficiency was demonstrated with success.•The MHP enhanced the sensitivity and speed of the existing LAMP assay, designed to detect V. parahaemolyticus, by 10 times and 30%, respectively.•The proposed strategy can be applied to boost up any other LAMP-based assay's diagnostic performance.

LysM-containing proteins function in the resistance of Litopenaeus vannamei against Vibrio parahaemolyticus infection.

Lysin motif (LysM) is a functional domain that can bind to peptidoglycans, chitin and their derivatives. The LysM-containing proteins participate in multiple biological processes, such as the hydrolysis of bacterial cell walls and the perception of PAMPs in plants and high animals. In the present study, two genes encoding LysM-containing proteins, designated as LvLysM1 and LvLysM2, were identified in the Pacific white shrimp, Litopenaeus vannamei, and their functions during Vibrio infection were analyzed. The open-reading frame (ORF) of LvLysM1 was 795 bp, only encoding a LysM domain at the N-terminal region. The ORF of LvLysM2 was 834 bp, encoding a LysM domain at the central region and a transmembrane region at the C-terminal region. Both LvLysM1 and LvLysM2 were widely transcribed in all tested shrimp tissues. Enzyme-linked immunosorbent assay (ELISA) showed that the recombinant protein of LvLysM2 could bind to different bacterial polysaccharides, while LvLysM1 showed no direct binding activity. The transcripts of LvLysMs in gills increased significantly after infection with Vibrio parahaemolyticus. When LvLysM1 or LvLysM2 was knocked down by dsRNA, the mortality of shrimp was significantly increased after infection with Vibrio parahaemolyticus. Interestingly, some SNPs existed in these two genes were apparently correlated with the VpAHPND resistance of shrimp. These results suggested that LvLysM1 and LvLysM2 might contribute to the disease resistance of shrimp. The data provide new knowledge about the function of LysM-containing proteins in shrimp and potential genetic markers for disease resistance breeding.

Isolation of Bacillus subtilis and Bacillus pumilus with Anti-Vibrio parahaemolyticus Activity and Identification of the Anti-Vibrio parahaemolyticus Substance.

The adoption of intensive farming has exacerbated disease outbreaks in aquaculture, particularly vibriosis caused by Vibrio parahaemolyticus. The use of probiotics to control V. parahaemolyticus is recognized as a good alternative to antibiotics for avoiding the development of antibiotic-resistant bacteria. In this study, two strains of B. HLJ1 and B. C1 with strong inhibitory activity on V. parahaemolyticus were isolated from aquaculture water and identified as Bacillus subtilis and Bacillus pumilus, respectively. Both B. HLJ1 and B. C1 lacked antibiotic resistance and virulence genes, suggesting that they are safe for use in aquaculture. In addition, these two strains can tolerate acid environments, produce spores, secrete extracellular enzymes, and co-aggregate as well as auto-aggregate with V. parahaemolyticus. B. HLJ1 and B. C1 produced the same anti-V. parahaemolyticus substance, which was identified as AI-77-F and belongs to amicoumacins. Both B. C1 and B. HLJ1 showed inhibitory activity against 11 different V. parahaemolyticus and could effectively control the growth of V. parahaemolyticus in simulated aquaculture wastewater when the concentration of B. C1 and B. HLJ1 reached 1 × 107 CFU/mL. This study shows that B. HLJ1 and B. C1 have great potential as aquaculture probiotics.

Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus.

Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39-100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 101, 102, and 103 CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.

Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Background: Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. Methods: V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. Results: All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Conclusions: Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

Screening of marine lactic acid bacteria for Vibrio parahaemolyticus inhibition and application to depuration in Pacific oysters (Crassostrea gigas).

AIMS: This study aims to assess the use of marine lactic acid bacteria (LAB) to reduce Vibrio parahaemolyticus levels during oyster depuration process. METHODS AND RESULTS: The inhibitory effect of 30 marine LAB strains against V. parahaemolyticus strains was evaluated by in vitro assays. A total of three positive strains (Latilactobacillus sakei SF1583, Lactococcus lactis SF1945, and Vagococcus fluvialis CD264) were selected for V. parahaemolyticus levels reduction during oyster depuration. Pacific oysters Crassostrea gigas were artificially and independently contaminated by four GFP-labelled V. parahaemolyticus strains (IFVp201, IFVp69, IFVp195, and LMG2850T) at 105 CFU ml-1 and then exposed by balneation to 106 CFU ml-1 of each LAB strains during 24 h, at 19°C. Quantification of V. parahaemolyticus in haemolymph by flow cytometry revealed variations in natural depuration of the different V. parahaemolyticus strains alone. Furthermore, the addition of LABs improved up to 1-log bacteria ml-1 the reduction of IFVp201 concentration in comparison to the control condition. CONCLUSIONS: Although further optimizations of procedure are needed, addition of marine LABs during oyster depuration may be an interesting strategy to reduce V. parahaemolyticus levels in Crassostrea gigas.

Emergence of Vibrio parahaemolyticus serotype O10:K4 in Thailand.

An emerging serotype O10:K4 of Vibrio parahaemolyticus has been predominantly isolated from outbreaks and sporadic cases in China. Herein, we report the first case of infection due to V. parahaemolyticus O10:K4 isolated from a hospitalized patient with acute diarrhea in Thailand. We sequenced the whole genome of the O10:K4 strain and compared it with those of the pandemic O3:K6 strain, O10:K4 strains in China, and other clinical and environmental strains. The results suggested that the O10:K4 strains are not a mere serotype variant diverged from the pandemic O3:K6 strain, confirming that the O10:K4 strain emergence has spread to Southeast Asia.

Molecular Epidemiology of Antimicrobial Resistance and Virulence Profiles of Escherichia coli, Salmonella spp., and Vibrio spp. Isolated from Coastal Seawater for Aquaculture.

The occurrence of waterborne antimicrobial-resistant (AMR) bacteria in areas of high-density oyster cultivation is an ongoing environmental and public health threat given the popularity of shellfish consumption, water-related human recreation throughout coastal Thailand, and the geographical expansion of Thailand's shellfish industry. This study characterized the association of phenotypic and genotypic AMR, including extended-spectrum ß-lactamase (ESBL) production, and virulence genes isolated from waterborne Escherichia coli (E. coli) (n = 84), Salmonella enterica (S. enterica) subsp. enterica (n = 12), Vibrio parahaemolyticus (V. parahaemolyticus) (n = 249), and Vibrio cholerae (V. cholerae) (n = 39) from Thailand's coastal aquaculture regions. All Salmonella (100.0%) and half of V. cholerae (51.3%) isolates harbored their unique virulence gene, invA and ompW, respectively. The majority of isolates of V. parahaemolyticus and E. coli, ~25% of S. enterica subsp. enterica, and ~12% of V. cholerae, exhibited phenotypic AMR to multiple antimicrobials, with 8.9% of all coastal water isolates exhibiting multidrug resistance (MDR). Taken together, we recommend that coastal water quality surveillance programs include monitoring for bacterial AMR for food safety and recreational water exposure to water for Thailand's coastal water resources.

Effect of feeding different types of ß-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) on growth performance and immunity of banana shrimp (Penaeus merguiensis).

ß-glucans are produced by many organisms and could be used as supplementary feed to enhance immunity and growth in some aquatic animals. This study aimed to compare the effectiveness of ß-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) as growth promoters and immunity enhancers in banana shrimp (Penaeus merguiensis). Shrimp were divided into 3 groups: the control group was fed without ß-glucan; the second and the third group were fed with 2 g kg-1 of ß-glucan derived from C. muelleri and T. weissflogii, respectively. Shrimp were fed over a 30-day period to determine growth performance (final weight, weight gain, average daily gain (ADG), and feed conversion ratio (FCR)) at day 15 and day 30, respectively. The immune parameters determined were total hemocyte count (THC), phenoloxidase activity (PO) and immune gene expression. Survival rates were measured after 14 days of the feeding trial and Vibrio parahaemolyticus infection (6, 24, 48 h post infection). There was no significant difference (P > 0.05) for growth stimulation of shrimps between the two types of ß-glucans (C. muelleri or T. weissflogii). Notably, shrimps fed with ß-glucans had a higher final weight, weight gain, and ADG (P < 0.05) than shrimps fed with the control diet, while FCR of shrimps fed with both ß-glucans was lower when compared to the control diet. Immune parameters, THC, PO, and gene expression of anti-lipopolysaccharide factor (ALF) and crustin were significantly higher (P < 0.05) in shrimps fed with ß-glucans, especially with ß-glucans from C. muelleri than the control group both before and after V. parahaemolyticus infection. Expression of penaeidin 3 and peroxiredoxin genes was significantly higher in shrimps fed with ß-glucans after bacterial infection. Histopathology of hepatopancreas revealed an increase in blasenzellen hepatopancreatic epithelial cells (B cells) after 14 days of feeding which remained higher following infection with V. parahaemolyticus. The survival rate of shrimps fed with the diet containing ß-glucan derived from either C. muelleri (82.2%) or T. weissflogii (77.8%) after V. parahaemolyticus infection was significantly higher than for the control group (51.1%) (P < 0.05). In conclusion, we propose that feeding banana shrimps with ß-glucans derived from marine diatoms either C. muelleri or T. weissflogii at a 2 g kg-1 diet can significantly improve their growth performance and immunity.

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.

On-Farm Practices Associated with Multi-Drug-Resistant Escherichia coli and Vibrio parahaemolyticus Derived from Cultured Fish.

Aquaculture activities have been implicated as responsible for the emergence of antimicrobial resistance (AMR), leading to broad dissemination and transference of antibiotic resistance to pathogens that affect humans and animals. The current study investigates the on-farm practices and environmental risk factors that can potentially drive the development and emergence of multi-drug-resistant (MDR) Escherichia coli and Vibrio parahaemolyticus in the aquaculture system. A cross-sectional study was conducted on 19 red hybrid tilapia (Oreochromis spp.) and 13 Asian seabass (Lates calcarifer, Bloch 1970) farms on the west coast of peninsular Malaysia. Data were collected using a structured questionnaire pertaining to farm demography, on-farm management practices and environmental characteristics. Multi-drug-resistant E. coli (n = 249) and V. parahaemolyticus (n = 162) isolates were analyzed using multi-level binary logistic regression to identify important drivers for the occurrence and proliferation of the MDR bacteria. On-farm practices such as manuring the pond (OR = 4.5; 95% CI = 1.21-16.57) were significantly associated with the occurrence of MDR E. coli, while earthen ponds (OR = 8.2; 95% CI = 1.47-45.2) and human activity adjacent to the farm (OR = 4.6; 95% CI = 0.75-27.98) were associated with an increased likelihood of MDR V. parahaemolyticus. Considering the paucity of information on the drivers of AMR in the aquaculture production in this region, these findings indicate the targeted interventions implementable at aquaculture farms to efficiently abate the risk of MDR amongst bacteria that affect fish that are of public health importance.

Integrated RNA-seq and RNAi Analysis of the Roles of the Hsp70 and SP Genes in Red-Shell Meretrix meretrix Tolerance to the Pathogen Vibrio parahaemolyticus.

The "Wanlihong" Meretrix meretrix (WLH-M) clam is a new variety of this species that has a red shell and stronger Vibrio tolerance than ordinary M. meretrix (ORI-M). To investigate the molecular mechanisms responsible for the WLH-M strain's tolerance to Vibrio, we challenged clams with Vibrio parahaemolyticus and then assessed physiological indexes and conducted transcriptome analysis and RNA interference experiments. The mortality, tissue bacterial load, and hemocyte reactive oxygen species level of ORI-M were significantly higher than those of WLH-M, whereas the content and activity of lysozyme were significantly lower. Gene Ontology functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that immune and metabolic pathways were enriched in Vibrio-challenged clams. The expressions of the heat shock protein 70 (Hsp70) and serine protease (SP) genes, which are involved in antibacterial immunity, were significantly upregulated in WLH-M but not in ORI-M, while the expression of the kynurenine 3-monooxygenase gene, a proinflammatory factor, was significantly downregulated in WLH-M. RNA interference experiments confirmed that Hsp70 and SP downregulation could result in increased mortality of WLH-M. Therefore, we speculate that Hsp70 and SP may be involved in the antibacterial immunity of WLH-M in vivo. Our data provided a valuable resource for further studies of the antibacterial mechanism of WLH-M and provided a foundation for the breeding of pathogen-resistant strains.

Depolymerized Fractions of Sulfated Galactans Extracted from Gracilaria fisheri and Their Antibacterial Activity against Vibrio parahaemolyticus and Vibrio harveyi.

Various seaweed sulfated polysaccharides have been explored for antimicrobial application. This study aimed to evaluate the antibacterial activity of the native Gracilaria fisheri sulfated galactans (NSG) and depolymerized fractions against the marine pathogenic bacteria Vibrio parahaemolyticus and Vibrio harveyi. NSG was hydrolyzed in different concentrations of H2O2 to generate sulfated galactans degraded fractions (SGF). The molecular weight, structural characteristics, and physicochemical parameters of both NSG and SGF were determined. The results revealed that the high molecular weight NSG (228.33 kDa) was significantly degraded to SGFs of 115.76, 3.79, and 3.19 kDa by hydrolysis with 0.4, 2, and 10% H2O2, respectively. The Fourier transformed spectroscopy (FTIR) and 1H- and 13C-Nuclear magnetic resonance (NMR) analyses demonstrated that the polysaccharide chain structure of SGFs was not affected by H2O2 degradation, but alterations were detected at the peak positions of some functional groups. In vitro study showed that SGFs significantly exerted a stronger antibacterial activity against V. parahaemolyticus and V. harveyi than NSG, which might be due to the low molecular weight and higher sulfation properties of SGF. SGF disrupted the bacterial cell membrane, resulting in leakage of intracellular biological components, and subsequently, cell death. Taken together, this study provides a basis for the exploitation and utilization of low-molecular-weight sulfated galactans from G. fisheri to prevent and control the shrimp pathogens.