Quercetin disrupts biofilm formation and attenuates virulence of Aeromonas hydrophila.

Aeromonas hydrophila poses significant health and economic challenges in aquaculture owing to its pathogenicity and prevalence. Overuse of antibiotics has led to multidrug resistance and environmental pollution, necessitating alternative strategies. This study investigated the antibacterial and antibiofilm potentials of quercetin against A. hydrophila. Efficacy was assessed using various assays, including antibacterial activity, biofilm inhibition, specific growth time, hemolysis inhibition, autoaggregation, and microscopic evaluation. Additionally, docking analysis was performed to explore potential interactions between quercetin and virulence proteins of A. hydrophila, including proaerolysin, chaperone needle-subunit complex of the type III secretion system, and alpha-pore forming toxin (PDB ID: 1PRE, 2Q1K, 6GRK). Quercetin exhibited potent antibacterial activity with 21.1 ± 1.1 mm zone of inhibition at 1.5 mg mL-1. It also demonstrated significant antibiofilm activity, reducing biofilm formation by 46.3 ± 1.3% at the MIC and attenuating autoaggregation by 55.9 ± 1.5%. Hemolysis was inhibited by 41 ± 1.8%. Microscopic analysis revealed the disintegration of the A. hydrophila biofilm matrix. Docking studies indicated active hydrogen bond interactions between quercetin and the targeted virulence proteins with the binding energy -3.2, -5.6, and -5.1 kcal mol⁻1, respectively. These results suggest that quercetin is an excellent alternative to antibiotics for combating A. hydrophila infection in aquaculture. The multifaceted efficacy of quercetin in inhibiting bacterial growth, biofilm formation, virulence factors, and autoaggregation highlights the potential for aquaculture health and sustainability. Future research should delve into the precise mechanisms of action and explore synergistic combinations with other compounds for enhanced efficacy and targeted interventions.

Preparation of valine-curcumin conjugate and its in vitro antibacterial and antitumor activity and in vivo biological effects on American eels (Anguilla rostrata).

Curcumin (Cur) exhibits diverse natural pharmacological activities, despite its limited water solubility (hydrophobicity) and low bioavailability. In this investigation, a valine-curcumin conjugate (Val-Cur) was synthesized through amino acid side chain modification, and its solubility increased to 1.78 mg/mL. In vitro experimental findings demonstrated that the antibacterial activity of Val-Cur against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus was significantly superior to that of Cur. The inhibition rate of Val-Cur against HepG2 (human hepatocellular carcinoma) cells was higher than that of Cur at low concentrations (below 25 µmol/L), although the IC50 value of Val-Cur did not differ significantly from that of Cur. In vivo biological effects of Val-Cur were assessed by adding it into the feed (150 mg/kg) of American eels (Anguilla rostrata). Val-Cur significantly improved the growth performance (↑weight gain rate, ↑specific growth rate, and ↓feed conversion rate) and activities of intestinal digestive enzymes (amylase and lipase) and antioxidant enzymes (superoxide dismutase) in American eels. Additionally, Val-Cur significantly improved serum biochemical indices (↑high-density lipoprotein cholesterol, ↓low-density lipoprotein cholesterol, ↓aspartate and alanine aminotransferases). Furthermore, Val-Cur increased intestinal microbial diversity, reduced the abundance of potentially pathogenic bacteria (Spiroplasma, Clostridium, and Pseudomonas), and elevated the abundance of beneficial digestion-promoting bacteria (Romboutsia, Phyllobacterium, Romboutsia sedimentorum, and Clostridium butyricum) conducive to glucose metabolism (P < 0.05). To the best of our knowledge, this study is the first to explore water-soluble curcumin in aquaculture, and the findings will lay the groundwork for the potential application of water-soluble curcumin in the field of aquaculture.

Structure-activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus.

BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host's immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure-activity relationships of its intramolecular disulphide bonds. RESULTS: The predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa. CONCLUSIONS: Our study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Rapid discrimination methods for clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using the N-terminal sequence of the flaA gene and investigation of antimicrobial resistance.

Although the genus Aeromonas inhabits the natural environment, it has also been isolated from hospital patient specimens as a causative agent of Aeromonas infections. However, it is not known whether clinical strains live in the natural environment, and if these strains have acquired antimicrobial resistance. In this study, we performed the typing of flagellin A gene (flaA) of clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using Polymerase Chain Reaction (PCR) assay with newly designed primers. Detection rates of the clinical and environmental flaA types of A. hydrophila were 66.7% and 88.2%, and the corresponding rates for A. veronii biovar sobria were 66.7% and 90.9%. The PCR assays could significantly discriminate between clinical and environmental strains of both species in approximately 4 h. Also, among the 63 clinical Aeromonas strains used, only one extended-spectrum ß-lactamase-producing bacteria, no plasmid-mediated quinolone resistance bacteria, and only four multidrug-resistant bacteria were detected. Therefore, the PCR assays could be useful for the rapid diagnosis of these Aeromonas infections and the monitoring of clinical strain invasion into water-related facilities and environments. Also, the frequency of drug-resistant Aeromonas in clinical isolates from Okinawa Prefecture, Japan, appeared to be low.

Synergistic effect of green synthesis magnesium oxide nanoparticles and seaweed extract on improving water quality, health benefits, and disease resistance in Nile tilapia.

This study aimed to evaluate the effect of adding liquid extract of algae (Hypnea musciformis, Grateloupia acuminata, and Sargassum muticum) (HGS) and Magnesium oxide nanoparticles (MgO NPs) using this extract to rear water of Oreochromis niloticus, on improving culture water indices, growth performance, digestive enzyme, hemato-biochemical characters, immune, antioxidative responses, and resistance after challenged by Aeromonas hydrophila with specific refer to the potential role of the mixture in vitro as resistance against three strains bacteria (Aeromonas sobria, Pseudomonas fluorescens, P. aeruginosa) and one parasite (Cichlidogyrus tilapia). The first group represented control, HGS0, whereas the other group, HGS5, HGS10, and HGS15 mL-1 of liquid extract, as well as all groups with 7.5 µg mL-1 MgO-NPs added to culture water of O. niloticus, for 60 days. Data showed that increasing levels at HGS 10 and HGS15 mL-1 in to-culture water significantly enhanced growth-stimulating digestive enzyme activity and a significantly improved survival rate of O. niloticus after being challenged with A. hydrophila than in the control group. The total viability, coliform, fecal coliform count, and heavy metal in muscle partially decreased at HGS 10 and HGS15 mL-1 than in the control group. Correspondingly, the highest positive effect on hemato-biochemical indices was noticed at levels HGS 10 and HGS15 mL-1. Fish noticed an improvement in immune and antioxidant indices compared to control groups partially at HGS 10 and HGS15 mL-1. Interestingly, fish cultured in rearing water with the mixture provided downregulated the related inflammatory genes (HSP70, TNF, IL-1ß, and IL-8) partially at HGS15 mL-1. In vitro, the mixture showed positive efficiency as an antibacterial and partially antiparasitic at HGS 10 and HGS15 mL-1. This study proposes utilizing a mixture of (HGS) and (MgO-NPs) with optimum levels of 10-15 mL-1 in cultured water to improve water indices, growth, health status, and increased resistance of O. niloticus against bacterial and parasitic infection.

Natural Sunlight-Mediated Emodin Photoinactivation of Aeromonas hydrophila.

Aeromonas hydrophila can be a substantial concern, as it causes various diseases in aquaculture. An effective and green method for inhibiting A. hydrophila is urgently required. Emodin, a naturally occurring anthraquinone compound, was exploited as a photo-antimicrobial agent against A. hydrophila. At the minimum inhibitory concentration of emodin (256 mg/L) to inactivate A. hydrophilia in 30 min, an 11.32% survival rate was observed under 45 W white compact fluorescent light irradiation. In addition, the antibacterial activity under natural sunlight (0.78%) indicated its potential for practical application. Morphological observations demonstrated that the cell walls and membranes of A. hydrophila were susceptible to damage by emodin when exposed to light irradiation. More importantly, the photoinactivation of A. hydrophila was predominantly attributed to the hydroxyl radicals and superoxide radicals produced by emodin, according to the trapping experiment and electron spin resonance spectroscopy. Finally, a light-dependent reactive oxygen species punching mechanism of emodin to photoinactivate A. hydrophila was proposed. This study highlights the potential use of emodin in sunlight-mediated applications for bacterial control, thereby providing new possibilities for the use of Chinese herbal medicine in aquatic diseases prevention.

The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance.

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

Dual effect of selenium loaded chitosan nanoparticles on growth, antioxidant, immune related genes expression, transcriptomics modulation of caspase 1, cytochrome P450 and heat shock protein and Aeromonas hydrophila resistance of Nile Tilapia (Oreochromis niloticus).

Nowadays there is a great attention for nanotechnology in aquaculture production. It has an efficient role in nutrients and drugs delivery, ponds sterilization, water treatment and aquatic diseases reduction. Till now, there is no available data on impact of selenite-loaded chitosan nanoparticles (SeChNPs) on Nile tilapia. Hence, the current study investigated the effects of selenite-loaded chitosan nanoparticles supplementation on the growth, immune, antioxidant and apoptotic related genes as well as resistance to Aeromonas hydrophila of Nile tilapia, Oreochromis niloticus. A total of 400 fish were randomly divided into four groups, and each group retained five replicates. The control group was fed a basal diet (with inorganic se), other groups fed diets supplemented with SeChNPs 0.5, 1 and 2 g/kg diet. The loading concentration of Se to ChNPs was 0.3, 0.6 and 1.2 mg/0.5, 1 and 2 gm respectively. Fish groups fed SeChNPs (0.5 and 1 g/kg) exhibited the highest final body gain, better feed utilization. Additionally, the expression of myostatin gene was down-regulated by 0.2 and 0.3 fold in group fed 0.5 and 1 g/kg SeChNPs when compared with control group. Dietary inclusion of SeChNPs increased serum lysozyme, alternative complement and myeloperoxidase activities and immunoglobulin type M level. Supplementation of SeChNPs at the level of 2 g/kg up-regulated glutathione peroxidase, superoxide dismutase and catalase expression by 1.12, 4.9 and 2.31 folds respectively, in comparison with control group. In contrast, the levels of C- reactive protein and malondialdehyde were reduced. The expression of IL-10, IL-8, TNF-α and IL-1ß genes was up-regulated after dietary inclusion of different levels of SeChNPs in a dose dependent manner. Post-challenge, the highest survival rate was detected in group fed 2 g/kg SeChNPs (93%) in contrast, the control group was displayed the lowest survival rate (45%). After challenge with A. hydrophila, the expression of caspase 1 was up-regulated in groups fed 1 and 2 g/kg of SeChNPs. Moreover, the maximum down-regulation of cytochromes P450 and heat shock protein were found in 2 g/kg SeChNPs supplemented group (reduced by 0.4 and 0.6-fold, respectively, when compared with control group). In conclusion, the ameliorative effects of SeChNPs on Nile tilapia growth resulted from immune stimulatory and free radicals scavenging effects of selenium loaded chitosan nano composite.

Antibacterial potential and mechanism of action of dithiocyano-methane as a bactericidal agent for farm disinfection.

AIMS: This study aimed to investigate the antibacterial ability and action mechanism of dithiocyano-methane against Aeromonas hydrophila, so as to provide a reference for its application in farm disinfection. METHODS AND RESULTS: After exposing the bacteria to dithiocyano-methane, the minimum inhibitory concentration (MIC), minimum bactericide concentration (MBC), activities of alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase and electric conductivity in bacterial suspensions were determined, transmission electron microscope images on cellular structure and SDS-PAGE profile of bacterial proteins were analysed and the expression of genes related to the above experimental observations was confirmed by real-time quantitative PCR. The MIC and MBC of dithiocyano-methane against three tested strains was 1·46 and 2·93 mg l-1 respectively. The results showed that dithiocyano-methane significantly damaged bacterial cell structure, inhibited the biosynthesis of bacterial proteins and changed the integrity and permeability of bacterial cell wall and cell membrane. CONCLUSIONS: Dithiocyano-methane showed remarkable antibacterial ability against three tested strains, indicating it is a potential effective bactericidal agent for preventing animal diseases resulted from Aer. hydrophila. SIGNIFICANCE AND IMPACT OF THE STUDY: To our best knowledge, this is the first report to examine the antibacterial ability and action mechanism of dithiocyano-methane against bacteria. The results demonstrate the great potential of dithiocyano-methane as a disinfectant against Aer. hydrophila in settings such as aquaculture ponds and livestock farms.

Virulence and antimicrobial resistance potential of Aeromonas spp. associated with shellfish.

Aeromonas spp. are associated with seafood-related outbreaks worldwide. In seafood industry, shellfish play a major role in global seafood production. With this emerging trend of shellfish consumption, shellfish-related bacterial infections are being reported frequently. Aeromonas spp. are natural contaminants found in shellfish. Although 36 species have been identified, some species including Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria have dragged major attention as foodborne pathogenic bacteria. The ability to elaborate a variety of virulence factors of Aeromonas spp. contributes to the pathogenic activities. Also, emerging antimicrobial resistance in Aeromonas spp. has become a huge challenge in seafood industry. Furthermore, multidrug resistance increases the risk of consumer health. Studies have supplied pieces of evidence about the emerging health risk of Aeromonas spp. isolated from seafood. Therefore, the present review was intended to highlight the prevalence, virulence and antimicrobial resistance of Aeromonas spp. isolated from various types of shellfish.

Characterization and application of rhamnolipid from Pseudomonas plecoglossicida BP03.

The production of rhamnolipid (glycolipid) biosurfactant was achieved under optimized conditions from newly isolated bacteria (Pseudomonas plecoglossicida BP03) from rice mill effluent. The isolated biosurfactant was structurally characterized using FTIR and NMR spectroscopic studies. The obtained biosurfactant (1·39 g l-1 ) showed a variety of applications including larvicidal and pupicidal activity against malarial vector (Anopheles sunadicus). It also exhibited antimicrobial activity against human pathogens, and possessed potent anti-biofilm activity against Staphylococcus aureus, Bacillus subtilis and Aeromonas hydrophila. The obtained biosurfactant showed a dose-dependent inhibition of exopolymeric substance (EPS) and growth curve in S. aureus. Furthermore, the cytotoxicity assays revealed that the biosurfactant exhibit a cytotoxic potency against the human fibroblastic sarcoma cells Ht-1080. An in silco analysis was also performed using Schrodinger maestro 9.3 against surface protein (SasG) of S. aureus, and the resultant analysis revealed an interactive docking score of -3·4 kcal mol-1 . The obtained result indicates that the synthesized economically viable biosurfactant ensures excellent applications towards various fields.

Synergistic Effect of Biosynthesized Silver Nanoparticles and Natural Phenolic Compounds against Drug-Resistant Fish Pathogens and Their Cytotoxicity: An In Vitro Study.

Fish pathogens causing disease outbreaks represent a major threat to aquaculture industry and food security. The aim of the presented study is to develop safe and effective bioactive agents against two bacterial isolates: Aeromonas hydrophila and Pseudomonas fluorescens. We employed a broth microdilution method to investigate the antibacterial effect of biosynthesized silver nanoparticles (AgNPs); rutin, a natural flavonoid extracted from Ruta graveneoles; and heliomycin, a secondary metabolite produced by marine actinomycetes AB5, as monotherapeutic agents. Moreover, AgNPs in combination with rutin (AgNP + R) and heliomycin (AgNPs + H) were examined for their synergistic effect. The cytotoxic effect of individual bioactive compounds and in combination with AgNPs was investigated on epithelioma papulosum cyprini (EPC) fish cell lines. Individual treatment of AgNPs, rutin, and heliomycin exhibited a dose-dependent antimicrobial activity against A. hydrophila and P. fluorescens. Rutin minimum inhibitory concentration (MIC) showed the lowest cytotoxicity when tested on EPC cell lines, while heliomycin MIC was highly cytotoxic. Combined subtherapeutic doses of AgNPs + R and AgNPs + H displayed additive and synergistic effects against A. hydrophila and P. fluorescens, respectively, with improved results and relative safety profile. The study findings demonstrate that a combination of AgNPs and natural bioactive compounds may represent novel therapeutics fighting fish pathogens potentially affecting the fish farming industry.

Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multi-drug-resistant Aeromonas hydrophila.

A rapid increase in multi-drug-resistant (MDR) bacteria in aquaculture highlights the risk of production losses due to diseases and potential public health concerns. Previously, we reported that ozone nanobubbles (NB-O3 ) were effective at reducing concentrations of pathogenic bacteria in water and modulating fish immunity against pathogens; however, multiple treatments with direct NB-O3 exposures caused alterations to the gills of exposed fish. Here, we set up a modified recirculation system (MRS) assembled with an NB-O3 device (MRS-NB-O3 ) to investigate whether MRS-NB-O3 (a) were safe for tilapia (Oreochromis niloticus), (b) were effective at reducing bacterial load in rearing water and (c) improved survivability of Nile tilapia following an immersion challenge with a lethal dose of MDR Aeromonas hydrophila. The results showed no behavioural abnormalities or mortality of Nile tilapia during the 14-day study using the MRS-NB-O3 system. In the immersion challenge, although high bacterial concentration (~2 × 107  CFU/ml) was used, multiple NB-O3 treatments in the first two days reduced the bacteria between 15.9% and 35.6% of bacterial load in water, while bacterial concentration increased from 13.1% to 27.9% in the untreated control. There was slight up-regulation of non-specific immune-related genes in the gills of the fish receiving NB-O3 treatments. Most importantly, this treatment significantly improved survivability of Nile tilapia with relative percentage survival (RPS) of 64.7% - 66.7% in treated fish and surviving fish developed specific antibody against MDR A. hydrophila. In summary, the result suggests that NB-O3 is a promising non-antibiotic approach to control bacterial diseases, including MDR bacteria, and has high potential for application in recirculation aquaculture system (RAS).

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A. hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20 ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24 hr. Without the existence of the phage, the highest mortality rate was 68.3 ± 2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33 ± 2.9% or 16.67 ± 2.9% at the phage dose of log 6.2 ± 0.09 or log 4.2 ± 0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Linalool Nanoemulsion Preparation, Characterization and Antimicrobial Activity against Aeromonas hydrophila.

Aeromonas hydrophila is one of the most important aquatic pathogens causing huge economic losses to aquaculture. Linalool, a vital ingredient of a variety of essential oils, was proved as a good antimicrobial agent in our previous studies. However, the low solubility and volatility of Linalool obstruct its application in the field of aquatic drugs. Thus, in this study, Linalool nano-emulsion (LN) was prepared to solve these obstructions. We investigated the physicochemical properties, antibacterial activity, and mode of action of LN against A. hydrophila. LN with different medium chain triglycerides (MCT) concentrations were prepared by ultrasonic method. The results showed that the emulsion droplet size of LN was the smallest when MCT was not added to the formulation. Nano-emulsions are usually less than 500 nm in diameter. In our study, LN in this formulation were spherical droplet with a diameter of 126.57 ± 0.85 nm and showed good stability. LN showed strong antibacterial activity, the MIC and MBC values were 0.3125% v/v and 0.625% v/v, respectively. The bacterial population decreased substantially at 1 × MIC of LN. LN exhibited disruptive effect on cell membranes by scanning electron microscope (SEM) and transmission electron microscope (TEM). The present study provided a formulation of Linalool nano-emulsion preparation. Moreover, the good antibacterial activity of LN showed in our study will promote the application of Linalool for the control and prevention of A. hydrophila in aquaculture.

Comparative proteomic analysis of sensitive and multi-drug resistant Aeromonas hydrophila isolated from diseased fish.

Bacterial hemorrhagic septicemia caused by multi-drug resistant (MDR) Aeromonas hydrophila has exponentially increased in the past decade, and reached an alarming rate making it a major concern in the aquaculture industry in China. The aim of this study was to investigate the difference in the regulation of proteins expression in multi-drug resistance and susceptible A. hydrophila strains isolated from diseased fish using two-dimensional electrophoresis (2-DE) combined with mass spectrometry. 28 isolates of A. hydrophila were successfully identified by biochemical tests. Antibiotic susceptibility test results showed that all the isolates have different drug resistant patterns. A total of 61 and 17 differently expressed proteins were identified in MDR and susceptible A. hydrophila, respectively, evidencing that biological processes related to carbon metabolism, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, cationic antimicrobial peptide (CAMP) resistance and propanoate metabolism were down-regulated in MDR strain, while proteins involved in biosynthesis of antibiotics, glycolysis/gluconeogenesis were highly expressed in the sensitive strain. The analysis of differentially expressed proteins from multi-drug resistance and susceptible strains suggests that a number of proteins are involved in several metabolic metabolism pathways plays an important role in A. hydrophila drug resistance. Our findings provide new insights about mechanisms involved in drug resistance and propose possible novel targets for developing alternative antibacterial drugs.

Antibiofilm and immunological properties of lectin purified from shrimp Penaeus semisulcatus.

Penaeid prawns are considered as most demanding fishery resources. The current study aims to purify and characterize lectin from the haemolymph of Penaeus semisulcatus. The semisulcatus-lectin was purified by affinity chromatography using mannose coupled Sepharose CL-4B column and purified lectin exhibited a single band of 66 kDa in SDS-PAGE. The purity and crystalline structure of purified lectin was confirmed by HPLC and X-ray diffraction analysis. Semisulcatus-lectin exhibited yeast agglutination activity against Saccharomyces cerevisiae and agglutinated human erythrocytes. Semisulcatus-lectin was evaluated for phenol oxidase activation and phagocytic activities. It was observed that semisulcatus-lectin had antibacterial activity against Gram-negative Vibrio parahaemolyticus and Aeromonas hydrophila, suggesting a potential therapeutic strategy in aquaculture industry for disease management.

Rheum officinale extract promotes the innate immunity of orange-spotted grouper (Epinephelus coioides) and exerts strong bactericidal activity against six aquatic pathogens.

We investigated the antimicrobial properties and the effects of Rheum officinale extract (ROE) on nonspecific immune parameters of orange-spotted grouper (Epinephelus coioides) in vitro and in vivo. The in vitro analysis was conducted by treating grouper primary head kidney leukocytes with various concentrations of ROE. The phagocytic rate of the leukocytes was elevated in a dose-dependent manner from 0.01 to 0.1 mg/ml, but decreased with higher concentrations of ROE (0.5 and 1.0 mg/ml). The production of reactive oxygen species (ROS) was strongly enhanced in a dose-dependent manner by treatment with ROE doses of 0.1-10.0 mg/ml. However, morphological changes (e.g., rounding and shrinkage of cells, chromatin condensation, fragmentation, and appearance of apoptotic bodies) were observed in the leukocytes after incubation with higher concentrations of ROE (1.0 and 10.0 mg/ml). A 28-day feeding trial was performed to assess the impact of dietary administration of ROE on grouper innate immunity parameters. Fish were fed with feed supplemented with 0, 0.1, 1.0, or 5.0 g ROE per kg of feed. The phagocytic activity of the animals' leukocytes was significantly elevated in all ROE-fed groups on day 1 and in groups fed with ROE at 0.1 or 1.0 g/kg on day 14. Production of ROS was substantially increased on day 1 in fish fed with ROE at 1.0 and 5.0 g/kg, but decreased steadily later on. The ability to generate ROS increased steadily until day 7 in fish fed the lowest concentration of ROE (0.1 mg/ml), but decreased thereafter. ROE showed excellent antibacterial activity against six pathogens of aquatic animals: Vibrio parahaemolyticus, V. vulnificus, V. alginolyticus, V. carchariae, Aeromonas hydrophila, and Edwardsiella tarda. The minimum inhibitory and bactericidal concentrations of measured ROE-derived anthraquinones were 10.57-84.53 µg/ml and 10.57-169.05 µg/ml, respectively.

Molecular characterization and immunohistochemical localization of tilapia piscidin 3 in response to Aeromonas hydrophila infection in Nile tilapia.

The antimicrobial activity of tilapia piscidin 3 (TP3) was determined in vitro against a locally isolated Aeromonas hydrophila. A 388 bp fragment was amplified from the TP3 cDNA and sequenced. The coding sequence (CDS) of TP3 was estimated to be 231 bp codes for 76 amino acids long and stop codon. In silico analysis was performed to detect both the signal peptide and the prodomain cleavage sites to follow the amino acids number 22 and 70, respectively. Based on this, a peptide 23 amino acids long with a remarkably high computed antimicrobial probability was synthesized and used in the subsequent experiments. The antimicrobial activity of TP3 was determined with minimum inhibitory concentration (MIC) and minim um bactericidal concentration (MBC) methods. TP3 exhibited relatively weak antimicrobial activities against the tested bacteria. A challenge experiment was then performed in Nile tilapia with low and high doses of A. hydrophila, followed by timely recognition; after 3, 6, 24 h, and 7 days of the specific TP3 gene expression, immunohistochemical localization was also performed. Histopathological examination revealed provoked inflammatory responses and congestion in the same organs of TP3 expression. Immunohistochemical localization showed that A. hydrophila induced tilapia fish to express TP3 after 24 h within the gills, intestine, hepatopancreas, spleen, and posterior kidney. In quantitative real time (RT)-polymerase chain reaction analysis, the high dose showed higher mRNA expression levels than the low dose, and its expression levels increased in the A. hydrophila-infected fish. It was therefore concluded that TP3 plays an essential role in fish immunity.

A C-type lectin with antibacterial activity in weather loach, Misgurnus anguillicaudatus.

C-type lectins are carbohydrate-binding proteins that play important roles in immunity by serving as pattern recognition receptors. In the present study, a novel nattectin-like C-type lectin was obtained from the weather loach, Misgurnus anguillicaudatus, designated as MaCTL. MaCTL encodes a peptide with 165 amino acids, with a signal peptide and a single C-type lectin domain (CTLD), containing a galactose-specific QPD motif and a conserved Ca2+ -binding site. Transcripts of MaCTL were significantly upregulated after immune challenge with its pathogen A. hydrophila. In vitro assays with recombinant MaCTL protein revealed that it exhibited hemagglutinating and bacterial agglutinating activities, in a Ca2+ -dependent manner. MaCTL was found to bind to a wide range of bacteria, as well as bind to bacterial polysaccharides LPS and PGN. Moreover, MaCTL displayed antimicrobial activity by inhibiting the growth of bacteria. These results collectively suggest that MaCTL is involved in the antibacterial defence of weather loach.