Bacterium Vibrio Parahaemolyticus Research Articles

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

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

The Use of (O. Basilicum) Extract to Treat Litopenaeus vannamei Disease the Infected Bacteria Vibrio parahaemolyticus

This research aims to treat vaname shrimp disease infected with bacteriaVibrio parahaemolyticus. In this research, we used Completely Randomized Design (CRD) 4 treatments and 3 replications. The treatment being tested was the treatment of vaname shrimp disease by soaking in basil (O. basilicum) leaf extract with different concentrations, namely: Treatment A (0 mg/ml), treatment B (1.56 mg/ml), treatment C (3.12 mg/ml) and treatment D (12.5 mg/ml). The parameter observed was the number of bacterial coloniesV. parahaemolyticus maintenance media and in the hepatopancreas of vaname shrimp. Apart from that, observations were made on the survival rate of vaname shrimp and water quality parameters. The research results found that the best concentration in the treatment of diseaseV. parahaemolyticus in vaname shrimp at a concentration of 12.5 mg/ml with the lowest number of bacterial colonies in the rearing media, namely 1.20 x 102 CFU/ml. In the hepatopancreas of vaname shrimp, the lowest number of colonies was obtained at a treatment concentration of 12.5 mg/ml, namely 1.12 x 102 CFU/ml. The highest survival rate was obtained at a treatment concentration of 12.5 mg/ml of 86,67±5,77%. The range of water quality in research observations was obtained as follows salinity 28.72-29.05 ppt, temperature 26.71- 27.25OC, oxygen ranges from 5.72-7.81, pH 7.18-8.13 and ammonia 0.033-0.041ppm. Water quality during the research was in the optimal range for the life of vaname shrimp.

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

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

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

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

Defence mechanisms of Pinctada fucata martensii to Vibrio parahaemolyticus infection: Insights from proteomics and metabolomics

Survival of pearl oysters is not only challenged by coastal pollution, but also pathogen infection that may eventually incur substantial economic losses in the pearl farming industry. Yet, whether pearl oysters can defend themselves against pathogen infection through molecular mechanisms remains largely unexplored. By using iTRAQ proteomic and metabolomic analyses, we analysed the proteins and metabolites in the serum of pearl oysters (Pinctada fucata martensii) when stimulated by pathogenic bacteria (Vibrio parahaemolyticus). Proteomic results found that a total of 2,242 proteins were identified in the experimental (i.e., Vibrio-stimulated) and control groups, where 166 of them were differentially expressed (120 upregulated and 46 downregulated in the experimental group). Regarding the immune response enrichment results, the pathway of signal transduction was significantly enriched, such as cytoskeleton and calcium signalling pathways. Proteins, including cathepsin L, heat shock protein 20, myosin and astacin-like protein, also contributed to the immune response of oysters. Pathogen stimulation also altered the metabolite profile of oysters, where 49 metabolites associated with metabolism of energy, fatty acids and amino acids were found. Integrated analysis suggests that the oysters could respond to pathogen infection by coordinating multiple cellular processes. Thus, the proteins and metabolites identified herein not only represent valuable genetic resources for developing molecular biomarkers and genetic breeding research, but also open new avenues for studies on the molecular defence mechanisms of pearl oysters to pathogen infection.

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

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

The Effectiveness of Lactic Acid Bacteria on the Immune System of Vannamei Shrimp Infected with Bacteria Vibrio Parahaemolyticus

Diseases are a major obstacle in shrimp farming because they can cause relatively high mortality and decrease the quality of the aquaculture environment. One dangerous shrimp disease is caused by Vibrio bacteria. Today, people are turning to biological control methods by utilizing lactic acid bacteria that live as microflora in the digestive tract of aquaculture animals. This study aimed to analyze the potential of lactic acid bacteria isolates from the gut of vannamei shrimp as probiotic candidates and to analyze selected lactic acid bacteria isolates that were effective in increasing the immune response and survival of vannamei shrimp infected with Vibrio parahaemolyticus bacteria. Four BAL isolates were isolated, designated as 8A, 11B, F1, and G2. BAL isolates showed antibacterial activity against Vibrio parahaemolyticus bacteria with an inhibition zone of up to 11.5 mm, capable of living at acidic to alkaline pH (1.5-7.2), bile salts (3000 ppm) and negative catalase. The characterization of several tests such as gram-positive, inhibition test and sugar fermentation test and comparison of LAB characterization showed that LAB isolates (G2) from the intestine of vannamei shrimp were classified as Pediococcus acidilactici. The application of probiotics, namely LAB (G2) to vannamei shrimp through feed can stimulate the vaname shrimp immune system after being challenged with V. parahaemolyticus, as indicated by an increase in the number of hemocytes (THC), phagocytosis activity, and suppressing the population of V. parahaemolyticus in vannamei shrimp.

Phytochemical screening and antibacterial assessment of two macroalgae Ulva papenfussi and Ulva nematoidea (Chlorophyta) against the bacterium Vibrio parahaemolyticus.

This study was conducted to determine whether the macroalgae Ulva papenfussi and Ulva nematoidea could be alternatives for preventing Litopenaeus vannamei vibriosis caused by the bacterium Vibrio parahaemolyticus. Phytochemical screening was performed on methanolic extracts to qualitatively determine the main groups of bioactive compounds, previous to an in vitro antibacterial test against V. parahaemolitycus. Phenols, polyphenols, flavonoids, and the high presence of carbohydrates were found in both macroalgae. U. papenfussi showed more presence of lipids and alkaloids than U. nematoidea. Macroalgae extracts prepared (v:v) with a 1:1 methanol: dichloromethane solvent was used for the in vitro test using the disc diffusion method (MDD). Filter paper discs impregnated with 1.0, 1.5, 2.0, 3.0, and 4.0 mg of the extracts showed antibacterial activity against V. Parahaemolitycus in a dose-dependent manner in both macroalgae. The inhibition zone varied significantly (p < 0.05) from 8.33 ± 0.12 to 11.41 ± 0.73 mm for 1 to 3 mg of extract levels, respectively. In conclusion, both macroalgae have antibacterial activity in their crude extracts against this bacteria. It is suggested to evaluate it as a feed additive for L. vannamei. This study is the first report on a phytochemical screening and antibacterial activity of these macroalgae against V. parahaemolyticus.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

Vibrio parahaemolyticus T6SS2 effector repertoires

ABSTRACT All strains of the marine bacterium Vibrio parahaemolyticus harbor a type VI secretion system (T6SS) named T6SS2, suggesting that this system plays an important role in the life cycle of this emerging pathogen. Although T6SS2 was recently shown to play a role in interbacterial competition, its effector repertoire remains unknown. Here, we employed proteomics to investigate the T6SS2 secretome of two V. parahaemolyticus strains, and we identified several antibacterial effectors encoded outside of the main T6SS2 gene cluster. We revealed two T6SS2-secreted proteins that are conserved in this species, indicating that they belong to the core secretome of T6SS2; other identified effectors are found only in subsets of strains, suggesting that they comprise an accessory effector arsenal of T6SS2. Remarkably, a conserved Rhs repeat-containing effector serves as a quality control checkpoint and is required for T6SS2 activity. Our results reveal effector repertoires of a conserved T6SS, including effectors that have no known activity and that have not been previously associated with T6SSs.

A novel C-type lectin for Litopenaeus vannamei involved in the innate immune response against Vibrio infection

C-type lectins (CTLs), as a member of pattern recognition receptors, play a vital role in the innate immune response of invertebrates to eliminate micro-invaders. In this study, a novel CTL of Litopenaeus vannamei, namely, LvCTL7, was successfully cloned, with an open reading frame of 501 bp and a capability to encode 166 amino acids. Blast analysis showed that the amino acid sequence similarity between LvCTL7 and MjCTL7 (Marsupenaeus japonicus) was 57.14%. LvCTL7 was mainly expressed in hepatopancreas, muscle, gill and eyestalk. Vibrio harveyi can significantly affect LvCTL7 expression level in hepatopancreases, gills, intestines and muscles (p < 0.05). LvCTL7 recombinant protein can bind to Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Vibrio parahaemolyticus and V. harveyi). It can cause the agglutination of V. alginolyticus and V. harveyi, but it had no effect on Streptococcus agalactiae and B. subtilis. The expression levels of SOD, CAT, HSP 70, Toll 2, IMD and ALF genes in the challenge group added with LvCTL7 protein were more stable than those in the direct challenge group (p < 0.05). Moreover, knockdown of LvCTL7 by double-stranded RNA interference downregulated the expression levels of genes (ALF, IMD and LvCTL5) that protect against bacterial infection (p < 0.05). These results indicated that LvCTL7 had microbial agglutination and immunoregulatory activity, and it was involved in the innate immune response against Vibrio infection in L. vannamei.

Stress and Disease Resistance in Crayfish (Procambarus clarkii) Breed “Huachizhen-1”

Stress and disease are critical factors hindering the industrial development of red swamp crayfish (Procambarus clarkii). Breeding crayfish with stress- and disease-resistant characteristics can overcome these limitations and promote their industrial development. In this study, the crayfish breed F3, which exhibits rapid growth and a favoured haplotype combination of the immune genes R, ALF, and crustin2, encoding the Toll-like receptor, anti-lipopolysaccharide factor, and antimicrobial peptide, respectively, were selected as parents to breed offspring (the selected group (SG)). The genotype, stress resistance, and disease resistance of crayfish in the SG and unselected group (USG) were compared. The results showed that the ratio of the favoured haplotype was higher in the SG crayfish than in the USG crayfish, leading to stronger stress and disease resistance. Compared to that of the USG crayfish, the mortality of the SG crayfish subjected to stress during eight days of transport and challenged with bacteria (Aeromonas hydrophila or Vibrio parahaemolyticus) or white spot syndrome virus were significantly reduced by 60% and 20%, respectively (p &lt; 0.05). Based on these results, the stress- and disease-resistant SG crayfish were named the “Huachizhen-1” breed. Additionally, the ratio of the unfavoured homozygous genotypes of R, ALF, and crustin2 sharply decreased, whereas those of the heterozygous genotypes increased together with stress and disease resistance during crayfish maturation under natural conditions, indicating that the heterozygotes of these genes also exhibit strong stress and disease resistance. All of this taken together, the crayfish breed “Huachizhen-1” may be applicable for improving stress and disease resistance and the production of crayfish.

Growth Rates of Vibrio parahaemolyticus Sequence Type 36 Strains in Live Oysters and in Culture Medium.

The pathogenic marine bacterium Vibrio parahaemolyticus can cause seafood-related gastroenteritis via the consumption of raw or undercooked seafood. Infections originating from relatively cool waters in the northeast United States are typically rare, but recently, this region has shown an increase in infections attributed to the ecological introduction of pathogenic sequence type 36 (ST36) strains, which are endemic to the cool waters of the Pacific Northwest. A 2005 risk assessment performed by the Food and Drug Administration (FDA) modeled the postharvest growth of V. parahaemolyticus in oysters as a function of air temperature and the length of time the oysters remained unrefrigerated. This model, while useful, has raised questions about strain growth differences in oyster tissue and whether invasive pathogenic strains exhibit different growth rates than nonclinical strains, particularly at lower temperatures. To investigate this question, live eastern oysters were injected with ST36 clinical strains and non-ST36 nonclinical strains, and growth rates were measured using the most probable number (MPN) enumeration. The presence of V. parahaemolyticus was confirmed using PCR by targeting the thermolabile hemolysin gene (tlh), thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and a pathogenesis-related protein (prp). The growth rates of the ST36 strains were compared to the FDA model and several other data sets of V. parahaemolyticus growth in naturally inoculated oysters harvested from the Chesapeake Bay. Our data indicate that the growth rates from most studies fall within the mean of the FDA model, but with slightly higher growth at lower temperatures for ST36 strains injected into live oysters. These data suggest that further investigations of ST36 growth capability in oysters at temperatures previously thought unsuitably low for Vibrio growth are warranted. IMPORTANCE Vibrio parahaemolyticus is the leading cause of seafood-related gastroenteritis in the United States, with an estimated 45,000 cases per year. Most individuals who suffer from vibriosis consume raw or undercooked seafood, including oysters. While gastroenteritis vibriosis is usually self-limiting and treatable, V. parahaemolyticus infections are a stressor on the growing aquaculture industry. Much effort has been placed on modeling the growth of Vibrio cells in oysters in order to aid oyster growers in designing harvesting best practices and ultimately, to protect the consumer. However, ecological invasions of nonnative bacterial strains make modeling their growth complicated, as these strains are not accounted for in current models. The National Shellfish Sanitation Program (NSSP) considers 10°C (50°F) a temperature too low to enable Vibrio growth, where 15°C is considered a cutoff temperature for optimal Vibrio growth, with temperatures approaching 20°C supporting higher growth rates. However, invasive strains may be native to cooler waters. This research aimed to understand strain growth in live oysters by measuring growth rates when oysters containing ST36 strains, which may be endemic to the U.S. Pacific Northwest, were exposed to multiple temperatures postharvest. Our results will be used to aid future model development and harvesting best practices for the aquaculture industry.

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM.

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.

Comprehensive analysis of the lncRNAs, mRNAs, and miRNAs implicated in the immune response of Pinctada fucata martensii to Vibrio parahaemolyticus

Non-coding RNAs (ncRNAs) have been implicated in a variety of biological processes. However, most ncRNAs are of unknown function and are as-yet unannotated. The immune-related functions of ncRNAs in the pearl oyster Pinctada fucata martensii were explored based on transcriptomic differences in the expression levels of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in the hemocytes of P.f. martensii after challenge by the pathogenic bacterium Vibrio parahaemolyticus. Across the challenged and control pearl oysters, 144 miRNAs and 14,571 lncRNAs were identified. In total, 13,375 ncRNAs were differentially expressed between the challenged and control pearl oysters; in the challenged pearl oysters as compared to the controls, 15 miRNAs and 5147 lncRNAs were upregulated, while 51 miRNAs and 8162 lncRNAs were downregulated. The sequencing results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. GO and KEGG pathway analysis showed that genes targeted by the differentially expressed ncRNAs were associated with the vascular endothelial growth factor (VEGF) signaling pathway and the nuclear factor kappa-B (NF-κB) signaling pathway. An lncRNA-mRNA-miRNA network that was developed based on the transcriptomic results of this study suggested that lncRNAs may compete with miRNAs for mRNA binding sites. This study may provide a useful framework for the detection of additional novel ncRNAs, as well as new insights into the pathogenic mechanisms underlying the response of P.f. martensii to V. parahaemolyticus.

Investigating the Relationship between Nitrate, Total Dissolved Nitrogen, and Phosphate with Abundance of Pathogenic Vibrios and Harmful Algal Blooms in Rehoboth Bay, Delaware.

Vibrio spp. and phytoplankton are naturally abundant in marine environments. Recent studies have suggested that the co-occurrence of phytoplankton and the pathogenic bacterium Vibrio parahaemolyticus is due to shared ecological factors, such as nutrient requirements. We compared these communities at two locations in the Delaware Inland Bays, representing a site with high anthropogenic inputs (Torquay Canal) and a less developed area (Sloan Cove). In 2017 to 2018, using light microscopy, we were able to identify the presence of many bloom-forming algal species, such as Karlodinium veneficum, Dinophysis acuminata, Heterosigma akashiwo, and Chattonella subsalsa. Dinoflagellate biomass was higher at Torquay Canal than that at Sloan Cove. D. acuminata and Chloromorum toxicum were found only at Torquay Canal and were not observed in Sloan Cove. Most probable number real-time PCR revealed V. parahaemolyticus and Vibrio vulnificus in environmental samples. The abundance of vibrios and their virulence genes varied between sites, with a significant association between total dissolved nitrogen (TDN), PO4-, total dissolved phosphorus (TDP), and pathogenic markers. A generalized linear model revealed that principal component 1 of environmental factors (temperature, dissolved oxygen, salinity, TDN, PO4-, TDP, NO3:NO2, NO2-, and NH4+) was the best at detecting total (tlh+) V. parahaemolyticus, suggesting that they are the prime drivers for the growth and distribution of pathogenic Vibrio spp. IMPORTANCE Vibrio-associated illnesses have been expanding globally over the past several decades (A. Newton, M. Kendall, D. J. Vugia, O. L. Henao, and B. E. Mahon, Clin Infect Dis 54:S391-S395, 2012, https://doi.org/10.1093/cid/cis243). Many studies have linked this expansion with an increase in global temperature (J. Martinez-Urtaza, B. C. John, J. Trinanes, and A. DePaola, Food Res Int 43:10, 2010, https://doi.org/10.1016/j.foodres.2010.04.001; L. Vezzulli, R. R. Colwell, and C. Pruzzo, Microb Ecol 65:817-825, 2013, https://doi.org/10.1007/s00248-012-0163-2; R. N. Paranjpye, W. B. Nilsson, M. Liermann, and E. D. Hilborn, FEMS Microbiol Ecol 91:fiv121, 2015, https://doi.org/10.1093/femsec/fiv121). Temperature and salinity are the two major factors affecting the distribution of Vibrio spp. (D. Ceccarelli and R. R. Colwell, Front Microbiol 5:256, 2014, https://doi.org/10.3389/fmicb.2014.00256). However, Vibrio sp. abundance can also be affected by nutrient load and marine plankton blooms (V. J. McKenzie and A. R. Townsend, EcoHealth 4:384-396, 2007; L. Vezzulli, C. Pruzzo, A. Huq, and R. R. Colwell, Environ Microbiol Rep 2:27-33, 2010, https://doi.org/10.1111/j.1758-2229.2009.00128.x; S. Liu, Z. Jiang, Y. Deng, Y. Wu, J. Zhang, et al. Microbiologyopen 7:e00600, 2018, https://doi.org/10.1002/mbo3.600). The expansion of Vibrio spp. in marine environments calls for a deeper understanding of the biotic and abiotic factors that play a role in their abundance. We observed that pathogenic Vibrio spp. were most abundant in areas that favor the proliferation of harmful algal bloom (HAB) species. These results can inform managers, researchers, and oyster growers on factors that can influence the growth and distribution of pathogenic Vibrio spp. in the Delaware Inland Bays.

A Single Catalytic Endolysin Domain Plychap001: Characterization and Application to Control Vibrio parahaemolyticus and Its Biofilm Directly.

Endolysins are enzymes used by bacteriophages to cleave the host cell wall in the final stages of the lytic cycle. As such, they are considered promising antibacterial agents for controlling and combating multidrug-resistant (MDR) bacteria. However, the application of endolysins targeting Gram-negative bacteria is greatly hindered by the outer membrane on these bacteria. Lysqdvp001, an endolysin with modular structure, has been reported as one of the most efficient endolysins against the Gram-negative bacterium Vibrio parahaemolyticus. In this study, Plychap001, the truncated recombinant catalytic domain of Lysqdvp001, was demonstrated to exhibit a direct and efficient bactericidal activity against broad spectrum of V. parahaemolyticus strains. Plychap001 was shown to be highly stable and retain high bactericidal activity at high temperatures, over a wide pH range, and at high NaCl concentrations. Plychap001 also exhibited a synergistic lytic effect with EDTA. Additionally, Plychap001 was found to efficiently degrade and eliminate V. parahaemolyticus biofilms on polystyrene surfaces. Our study establishes Plychap001 as a promising method for controlling V. parahaemolyticus in the food industry.

Health Status of Mytilus chilensis from Intensive Culture Areas in Chile Assessed by Molecular, Microbiological, and Histological Analyses.

Shellfish farming is a relevant economic activity in Chile, where the inner sea in Chiloé island concentrates 99% of the production of the mussel Mytilus chilensis. This area is characterized by the presence of numerous human activities, which could harm the quality of seawater. Additionally, the presence of potentially pathogenic microorganisms can influence the health status of mussels, which must be constantly monitored. To have a clear viewpoint of the health status of M. chilensis and to study its potential as a host species for exotic diseases, microbiological, molecular, and histological analyses were performed. This study was carried out in October 2018, where M. chilensis gut were studied for: presence of food-borne bacteria (Vibrio parahaemolyticus, Escherichia coli, Salmonella spp.), exotic bacteria (“Candidatus Xenohaliotis californiensis”), viruses (abalone and Ostreid herpes virus), and protozoa (Marteilia spp., Perkinsus spp. and Bonamia spp.). Additionally, 18S rDNA metabarcoding and histology analyses were included to have a complete evaluation of the health status of M. chilensis. Overall, despite the presence of risk factors, abnormal mortality rates were not reported during the monitoring period and the histological examination did not reveal significant lesions. Pathogens of mandatory notification to World Organization for Animal Health (OIE) and the Chilean National Fisheries and Aquaculture Service (SERNAPESCA) were not detected, which confirms that M. chilensis have a good health status, highlighting the importance of an integrated vision of different disciplines to ensure the sustainability of this important mussel industry in Chile.

Inactivation of microbial loads and retardation of quality loss in Asian hard clam (Meretrix lusoria) using high-hydrostatic-pressure processing during refrigerated storage

The effects of high pressure processing (HPP) at 200, 300, 400, 500, and 600 MPa for 3 min on physicochemical quality and inactivation of microbial loads of Asian hard clam (Meretrix lusoria) were evaluated. In addition, the retardation effects in microbial growth and quality loss of pressurized hard clams stored at 4 °C were investigated. Results showed that the expanding rate, moisture content, shucking ratio, pH, L* (lightness), and W (whiteness) of clam meat increased significantly with the increase in pressure, but a* (redness) and b* (yellowness) values decreased. With the increase in pressure, the levels of aerobic plate count (APC), psychrotrophic bacteria count (PBC), coliform, and Vibrio parahaemolyticus in clam meat significantly decreased. Furthermore, HPP with a pressure of >400 MPa on clam samples significantly delayed the increase in APC (by 6–9 days within Taiwan's regulation standard limit) and PBC during storage at 4 °C. However, the samples pressurized more than 300 MPa had significantly lower levels of total volatile basic nitrogen (TVBN) than control samples during storage. Overall, the results indicated that pressurization at least 400 MPa for 3 min on Asian hard clam were able to reach the highest expanding rate and moisture content, and 100 % shucking, while it can reduce the coliform, and V. parahaemolyticus to a non-detectable level. In addition, HPP conditions of ≥400 MPa for 3 min significantly extended the shelf-life of hard clam during refrigerated storage. Therefore, HPP is a useful technique to preserve Asian hard clam meats due to its ability to reduce bacterial load and retard TVBN production.

Discovery and Characterization of a New Crustin Antimicrobial Peptide from Amphibalanus amphitrite.

Crustins are an antimicrobial peptide (AMP) family that plays an important role in innate immunity in crustaceans. It is important to discover new AMPs from natural sources to expand the current database. Here, we identified and characterized a new crustin family member, named AaCrus1, from Amphibalanus amphitrite. AaCrus1 shares high identity (48.10%) with PvCrus, a Type I crustin of Penaeus vannamei that possesses a whey acidic protein (WAP) domain. AaCrus1 contains 237 amino acids and eight cysteine residues forming conserved ‘four-disulfide core’ structure. Our recombinant AaCrus1 (rAaCrus 1) could inhibit the growth of two Gram-positive bacteria (Staphylococcus aureus, Bacillus sp. T2) and four Gram-negative bacteria (Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus) with a minimum inhibitory concentration of 3.5–28 μM. It can further induce agglutination of both Gram-positive and Gram-negative bacteria. rAaCrus1 can bind to bacteria and damage bacterial cell membranes. Furthermore, rAaCrus1 disrupted biofilm development of S. aureus and V. parahaemolyticus. Our discovery and characterization of this new crustin can be further optimized as a good alternative to antibiotics.