Assessment of Bacillus species capacity to protect Nile tilapia from A. hydrophila infection and improve growth performance.

The present study evaluated the capacity of three Bacillus species to improve health status and growth performance of Nile Tilapia fed with high levels of soybean meal and challenged with Aeromonas hydrophila. In vitro experiments showed that ß-hemolysin and metalloprotease enzymes were produced by A. hydrophila throughout the exponential growth phase. In vivo experiments showed that 107 colony-forming units (CFUs)/ml of this pathogen killed 50% of control group fishes in 13 days. To evaluate the influence of Bacillus strains on health status and growth performance in Nile Tilapia, 180 fishes (33.44 + 0.05 g) were distributed in 12 tanks of 200 L each, and animals were fed twice per day until satiety. 1) Control group without Bacillus, 2) Bacillus sp1, 3) Bacillus sp2, and 4) Bacillus sp3 groups were formulated containing 106 CFU/g. After 40 days of feeding, the fishes were intraperitoneally injected with 1 ml of A. hydrophila at 2 × 107 CFU/ml, and mortality was recorded. The results showed that cumulative mortality rate was significantly (p< 0.05) lower in the Bacillus sp1 (25%), sp2 (5%), and sp3 (15%) groups, than the control group (50%). Weight gain was also significantly better (p< 0.05) in the Bacillus sp1 (36%), sp2 (67%), and sp3 (55%) groups with respect to the control group (30%). In conclusion, functional diet formulated with high levels of soybean meal and supplemented with Bacillus sp2 could be an alternative to protect Nile tilapia cultures from A. hydrophila infections and improve fish growth performance.

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.

Protective Effect of Leek Extract (Allium ampeloprasum L.) on Catfish (Clarias gariepinus) Experimentally Challenged with Aeromonas hydrophila.

BACKGROUND AND OBJECTIVE: Leek (Allium ampeloprasum) is one of the most commonly used herbal foods all over the world. This study was conducted to evaluate the protective effect of leek extract on catfish experimentally challenged with Aeromonas hydrophila, a problematic bacterial pathogen that affects various freshwater fish species. MATERIALS AND METHODS: Aeromonas hydrophila was isolated and identified from catfish showing clinical signs of septicemia. The in vitro activity of leek extract to control the growth of Aeromonas hydrophila was investigated. In the in vivo experiment, about 240 adult catfish (Clarias gariepinus) were fed three different leek extract concentrations (10, 25 and 50 mg kg-1 body weight) for 1 month. Later on, a challenge study was conducted using an identified A. hydrophila strain. Morbidity and mortality were recorded throughout one week post-challenge. Furthermore, the effect of leek extract on some immune-related genes was investigated. RESULTS: Under the in vitro testing, a significant increase (10 and 13 mm) in the inhibition zone was recorded in wells treated with 25 and 50 mg L-1 leak extract, respectively. A significant reduction in fish mortalities was reported in all leek extract treated groups compared to the control group which was given water. TLR1 gene expression was upregulated in fish treated with leek extract while TNFα gene expression was down-regulated. CONCLUSION: Overall, results suggested that the leek extract has immunostimulating effects that can help control bacterial infections in catfish and probably other fish species.

Ribonuclease1 contributes to the antibacterial response and immune defense in blunt snout bream (Megalobrama amblycephala).

Ribonuclease 1 (RNase1) is a vertebrate-specific enzyme that mainly performs digestive activity in herbivorous mammals. Here we used bacterial viability assays to explore its antimicrobial activity in blunt snout bream (Megalobrama amblycephala). The results showed that Ma-RNase1 rapidly killed Gram-negative and Gram-positive bacteria at micromolar concentrations. Ma-RNase1 increased the permeability of bacterial outer and inner membranes, thus reducing the integrity of bacterial cell wall and membrane. Moreover, Ma-RNase1 effectively counteracted the tissue damage and apoptosis caused by Aeromonas hydrophila infection. Quantitative real-time PCR and immunoblot analysis indicated that RNase1 mRNA and protein were up-regulated in the kidney and gut during infection. Furthermore, A. hydrophila infection significantly induced Tnf-α and Il-1ß mRNA expression in liver, but not in the RNase1 pre-treatment group. In addition, a significant increase in the expression of immune-related genes (Nf-κb and Tlr4) was found in liver, kidney and gut of A. hydrophila-infected fish, while a decrease in Myd88 and Tlr4 levels was found in liver, spleen, kidney and gut in the group pre-treated with RNase1. Collectively, these data suggest that Ma-RNase1 has antimicrobial function both in vitro and in vivo, and contributes to the protective effect and immune defense of blunt snout bream.

Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture.

BACKGROUND: Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture that results in the loss of over 3 million pounds of marketable channel catfish, Ictalurus punctatus, and channel catfish hybrids (I. punctatus, ♀ x blue catfish, I. furcatus, ♂) each year from freshwater catfish production systems in Alabama, U.S.A. vAh isolates are clonal in nature and are genetically unique from, and significantly more virulent than, traditional A. hydrophila isolates from fish. Even with the increased virulence, natural infections cannot be reproduced in aquaria challenges making it difficult to determine modes of infection and the pathophysiology behind the devastating mortalities that are commonly observed. Despite the intimate connection between environmental adaptation and plastic response, the role of environmental adaption on vAh pathogenicity and virulence has not been previously explored. In this study, secreted proteins of vAh cultured as free-living planktonic cells and within a biofilm were compared to elucidate the role of biofilm growth on virulence. RESULTS: Functional proteolytic assays found significantly increased degradative activity in biofilm secretomes; in contrast, planktonic secretomes had significantly increased hemolytic activity, suggesting higher toxigenic potential. Intramuscular injection challenges in a channel catfish model showed that in vitro degradative activity translated into in vivo tissue destruction. Identification of secreted proteins by HPLC-MS/MS revealed the presence of many putative virulence proteins under both growth conditions. Biofilm grown vAh produced higher levels of proteolytic enzymes and adhesins, whereas planktonically grown cells secreted higher levels of toxins, porins, and fimbrial proteins. CONCLUSIONS: This study is the first comparison of the secreted proteomes of vAh when grown in two distinct ecological niches. These data on the adaptive physiological response of vAh based on growth condition increase our understanding of how environmental niche partitioning could affect vAh pathogenicity and virulence. Increased secretion of colonization factors and degradative enzymes during biofilm growth and residency may increase bacterial attachment and host invasiveness, while increased secretion of hemolysins, porins, and other potential toxins under planktonic growth (or after host invasion) could result in increased host mortality. The results of this research underscore the need to use culture methods that more closely mimic natural ecological habitat growth to improve our understanding of vAh pathogenesis.

Use of Bacteriophage to Control Experimental Aeromonas hydrophila Infection in Tilapia (Oreochromis niloticus).

BACKGROUND AND OBJECTIVE: Antibiotics have been used to treat Aeromonas hydrophila infections in fish farming. However, their extensive uses can cause many negative effects including the development of drug-resistant bacterial strains. The main objective of this study was to find an alternative to antibiotics to inhibit A. hydrophila both in vitro and in vivo. MATERIALS AND METHODS: A bacteriophage infecting A. hydrophila was isolated from a fish a pond water sample. It was classified based on its genome type studied by enzymatic digestion and morphology investigated by transmission electron microscopy. Its ability to control experimental A. hydrophila infection in tilapia (Oreochromis niloticus) was examined by feeding tilapia with fish diets supplemented with different titers of the bacteriophage. RESULTS: A bacteriophage specific to Aeromonas hydrophila UR1 designated PAh4 was isolated and classified as a member of the family Myoviridae. When tilapia experimentally infected with A. hydrophila at the median lethal dose (3.16×105 CFU per fish) were fed the fish diets supplemented with the bacteriophage PAh4 at doses ranging from 105-108 PFU g-1 of diet, the diets could reduce the mortality rate of infected tilapia in a dose-dependent manner. CONCLUSION: The bacteriophage PAh4 can be used as an alternative to antibiotics to control A. hydrophila infection in tilapia.

Synergistic effect of biological and advanced oxidation process treatment in the biodegradation of Remazol yellow RR dye.

The current study investigated the efficiency of synergistic biological and Advanced Oxidation Process (AOPs) treatment (B-AOPs) using Aeromonas hydrophila SK16 and AOPs-H2O2 in the removal of Remazol Yellow RR dye. Singly, A. hydrophila and AOPs showed 90 and 63.07% decolourization of Remazol Yellow RR dye (100 mg L-1) at pH 6 and ambient temperature within 9 h respectively. However, the synergistic B-AOPs treatments showed maximum decolorization of Remazol Yellow RR dye within 4 h. Furthermore, the synergistic treatment significantly reduced BOD and COD of the textile wastewater by 84.88 and 82.76% respectively. Increased levels in laccase, tyrosinase, veratryl alcohol oxidase, lignin peroxidase and azo reductase activities further affirmed the role played by enzymes during degradation of the dye. UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS) confirmed the biotransformation of dye. A metabolic pathway was proposed based on enzyme activities and metabolites obtained after GC-MS analysis. Therefore, this study affirmed the efficiency of combined biological and AOPs in the treatment of dyes and textile wastewaters in comparison with other methods.

Antimicrobial Activities of Rhopalaea-Associated Fungus Aspergillus flavus strain MFABU9.

BACKGROUND AND OBJECTIVE: Rhopalaea is a genus of ascidian belonging to the family Diazonidae. Ascidians provide niches for various microorganisms including fungi. This present study describes the potential new source for natural bioactive compounds from Rhopalaea-associated fungi obtained from Bunaken marine park. MATERIALS AND METHODS: As part of an on-going research program to explore the chemical diversity of marine derived fungi, we performed an antimicrobial bioactivity-guided screening of EtOAc extracts of the fungi isolated from ascidian Rhopalaea sp. RESULTS: The study confirms that the ascidian obtained from Bunaken marine park was Rhopalaea sp. The fungus isolated from the ascidian was Aspergillus flavus which showed antimicrobial activity against bacteria Escherichia coli, Staphylococcus aereus, Aeromonas hydrophila and antifungal against the human pathogenic fungus Candida albicans. CONCLUSION: Aspergillus flavus isolated from ascidian Rhopalaea sp. has the potential as antibacterial and antifungal.

Acetylation of lysine 7 of AhyI affects the biological function in Aeromonas hydrophila.

Acyl-homoserine-lactone synthase (AhyI) of Aeromonas hydrophila can produce quorum sensing (QS) auto-inducer 1 (AI-1) type signal molecule, which plays important roles in various biological phenomenons such as biofilm formation, hemolysin production and motility. Previous research revealed that the AhyI of A. hydrophila has acetylation modification on lysine 7 site, but its intrinsic biological function is still largely unknown. To study the effect of AhyI protein and its acetylation modification on the physiological traits of A. hydrophila, the site-directed mutagenesis strains including ΔahyI::ahyI-K7Q and ΔahyI::ahyI-K7R were made in this study. The mutation at K7 site of lysine acetylation in AhyI protein decreased the protease production, but the lysine acetylations do not affect the biofilm formation and hemolysin production. To further study the effect of lysine acetylation on AI-1 signal molecule production, the acyl-homoserine lactones (AHLs) extraction and bioluminescence quantification were performed. Compared with the rescue strain, the acetylation on K7 of AhyI resulted in a decreased level of AHLs and bioluminescence production. It indicated that the lysine acetylation modification on the AhyI protein can regulate the production of signalling molecules. Overall, the obtained data in this study provide a theoretical basis for further understanding the role of lysine acetylation of AhyI protein and lay a foundation to systematically study the regulatory mechanism of QS.

First data on microflora of loggerhead sea turtle (Caretta caretta) nests from the coastlines of Sicily.

Caretta caretta is threatened by many dangers in the Mediterranean basin, but most are human-related. The purposes of this research were: (i) to investigate microflora in samples from six loggerhead sea turtle nests located on the Sicilian coast and (ii) to understand microbial diversity associated with nests, with particular attention to bacteria and fungi involved in failed hatchings. During the 2016 and 2018 summers, 456 eggs and seven dead hatchling from six nests were collected. We performed bacteriological and mycological analyses on 88 egg samples and seven dead hatchlings, allowing us to isolate: Fusarium spp. (80.6%), Aeromonas hydrophila (55.6%), Aspergillus spp. (27.2%) and Citrobacter freundii (9%). Two Fusarium species were identified by microscopy and were confirmed by PCR and internal transcribed spacer sequencing. Statistical analyses showed significant differences between nests and the presence/absence of microflora, whereas no significant differences were observed between eggs and nests. This is the first report that catalogues microflora from C . caretta nests/eggs in the Mediterranean Sea and provides key information on potential pathogens that may affect hatching success. Moreover, our results suggest the need for wider investigations over extensive areas to identify other microflora, and to better understand hatching failures and mortality related to microbial contamination in this important turtle species.

Effects of dietary myo-inositol on growth, antioxidative capacity, and nonspecific immunity in skin mucus of taimen Hucho taimen fry.

In this study, the effects of dietary myo-inositol on the skin mucosal immunity and growth of taimen (Hucho taimen) fry were determined. Triplicate groups of 500 fish (initial weight 5.58 ± 0.15 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, and 738.15 mg kg-1) until satiation for 56 days. Thereafter, the nonspecific skin mucus immune parameters, antioxidative capacity, and growth performance were measured. The skin mucus protein and the activities of alkaline phosphatase were significantly higher than those in the control group (P < 0.05). However, there were no significant differences in lysozyme activity among the treatments (P > 0.05). The antimicrobial activity and minimum inhibitory concentration of the skin mucus were increased significantly by myo-inositol supplementation (P < 0.05). The superoxide dismutase, catalase, and glutathione peroxidase activities were significantly elevated in the treatment groups (P < 0.05), whereas the malondialdehyde contents were significantly decreased (P < 0.05). Low-level myo-inositol (28.75 mg kg-1) led to a significantly lower weight gain, feed efficiency, condition factor, and survival rate compared with the other treatments (P < 0.05). In conclusion, dietary myo-inositol deficiency (28.75 mg kg-1) adversely affects the skin mucus immune parameters, antioxidative capacity, and growth performance of Hucho taimen fry.

Melamine and curcumin enriched diets modulate the haemato-immune response, growth performance, oxidative stress, disease resistance, and cytokine production in oreochromis niloticus.

Currently, feed adulteration and contamination with melamine (MEL) are considered one of the serious issues in the aquatic industry. With the limited studies of MEL exposure alone in fish, its adverse impacts on fish cannot be evaluated well. Accordingly, this study aimed to investigate the effects of MEL containing diets on the immune response, disease resistance to Aeromonas hydrophila, growth performance, chemical composition, immune-related genes expression, and histopathology of both spleen and head kidneys. Also, the efficacy of curcumin (CUR) dietary supplementation to alleviate MEL negative impacts were evaluated. A total of 180 apparently healthy Oreochromis niloticus (O. niloticus) were divided into four groups with three replicates fed the basal diet only, basal diet fortified with 200 mg/kg CUR, basal diet containing 1 % MEL, or a basal diet containing CUR + MEL. The results displayed that MEL significantly reduced growth performance indices and body crude lipid contents. Anemic, leukopenic, lymphocytopenic, heterocytopenic, esonipenic, hypoproteinemic and hypoalbuminic conditions were apparent. Moreover, depleted immune and antioxidant indicators including lysozyme activity, nitric oxide, immunoglobulin M, complement 3, glutathione peroxidase, and superoxide dismutase enzyme activity were recorded. Also, MEL reduced the disease resistance of O. niloticus to bacterial infection. Furthermore, MEL induced downregulation of mRNA levels of interleukin 1ß and tumor necrosis factor α in the spleen together with obvious pathological perturbations in both spleen and head kidneys. The CUR addition resulted in a significant enhancement in most indices. These results may conclude that MEL could alter both innate and adaptive immune responses via the negative transcriptional effect on immune-related genes together with the oxidative damage of the immune organs. Furthermore, CUR dietary supplements could be advantageous for mitigating MEL negative impacts, thus offering a favorable aquafeed additive for O. niloticus.

Use of bacterial cellulose film modified by polypyrrole/TiO2-Ag nanocomposite for detecting and measuring the growth of pathogenic bacteria.

The aim of this study was to use of bacterial cellulose/polypyrrole/TiO2-Ag (BC/PPy/TiO2-Ag) nanocomposite film to detect and measure the growth of 5 pathogenic bacteria. For this purpose, at first, 13 BC/PPy/TiO2-Ag films were fabricated, then bacterial suspensions were prepared according to McFarland standard. The results showed that by increasing the bacterial concentration, the electrical resistance of sensors was decreased and there was a relation between bacterial concentration and bacterial type with electrical resistance change of sensors. The obtained data showed that the sensitivity of the sensors was increased with increasing the concentration of polypyrrole and TiO2-Ag. FT-IR and SEM tests were performed to investigate the interaction between nanoparticles and determine the size of nanoparticles. The BC/PPy/TiO2-Ag biosensors are portable and the response time of these sensors is very short for target analysis. Therefore, these sensors have the potential to improve biological safety as diagnostic tools.

Caffeine supplementation in diet mitigates Aeromonas hydrophila-induced impairment of the gill phosphotransfer network in grass carp Ctenopharyngodon idella.

Some evidence suggests the involvement of phosphotransfer network in the pathogenesis of fish bacterial diseases, catalyzed by creatine kinase (CK), pyruvate kinase (PK) and adenylate kinase (AK); nevertheless, the effects on fish affected by Aeromonas hydrophila remain unknown. Recent evidence suggested a potent protective effect of caffeine on the branchial phosphotransfer network of fish subjected to challenge conditions. Therefore, the aim of this study was to evaluate whether A. hydrophila infection impaired branchial bioenergetics. We also determined whether dietary supplementation with caffeine protected against A. hydrophila-induced gill bioenergetic imbalance. We found that branchial cytosolic CK and AK activities were significant lower in fish experimentally infected with A. hydrophila than in uninfected fish, while mitochondrial CK activity was significant higher. Branchial lactate dehydrogenase (LDH) activity and lactate levels were significant higher in fish experimentally infected by A. hydrophila than in uninfected fish, while sodium-potassium ion pump (Na+, K+-ATPase) activity and adenosine triphosphate (ATP) levels were significant lower. No significant difference was observed between groups with respect to branchial PK activity. The dietary supplementation with 8% caffeine improved the branchial CK (cytosolic and mitochondrial), AK, and LDH activities, as well as ATP levels, but did not prevent increases in branchial lactate levels or the inhibition of Na+, K+-ATPase activity elicited by aeromonosis. Based on this evidence, we believe that reduction of CK (cytosolic) and AK activities contributes to impairment of bioenergetic homeostasis, while augmentation of mitochondrial CK activity can be considered an attempt to prevent or reduce the energetic imbalance during aeromonosis caused by A. hydrophila. The use of 8% caffeine dietary supplementation improved the energetic metabolism via protective effects on CK and AK activities, avoiding the necessity of using anaerobic metabolism. In summary, 8% dietary caffeine can be used to improve branchial energetic homeostasis during aeromonosis caused by A. hydrophila.

Vermamoeba vermiformis in hospital network: a benefit for Aeromonas hydrophila.

Aeromonas hydrophila, considered as an emerging pathogen, is increasingly involved in opportunistic human infections. This bacterium, mainly present in aquatic environments, can therefore develop relationships with the free-living amoeba Vermamoeba vermiformis in hospital water networks. We showed in this study that the joint presence of V. vermiformis and A. hydrophila led to an increased bacterial growth in the first 48 h of contact and moreover to the protection of the bacteria in adverse conditions even after 28 days. These results highlight the fact that strategies should be implemented to control the development of FLA in hospital water systems.

Antibacterial synergy between rutin and florfenicol enhances therapeutic spectrum against drug resistant Aeromonas hydrophila.

Emergence of antibiotic resistant bacteria has necessitated the drive to explore competent antimicrobial agents or to develop novel formulations to treat infections including Aeromonas hydrophila. The present study investigates the synergistic antibacterial effects of citrus flavonoid rutin and florfenicol (FF) against A. hydrophila in vitro and in vivo. Rutin is extracted and purified from Citrus sinensis peel through preparative HPLC and characterized through TLC, GC-MS and 1H and 13C NMR analyses. Though rutin did not display significant antibacterial activity, it modulated FF activity resulting in four-fold reduction in the MIC value for FF. The anti-biofilm potential of synergistic association of rutin and FF was validated by protein analysis, quantification of exopolysaccharide (EPS) and microscopy studies using sub-MIC doses. Besides antibacterial action, in vivo studies showed that Rutin/FF combination enhanced host immunity by improving blood cell count, anti-protease, and lysozyme activities as well as decreased the oxidative stress and the pathological changes of tilapia Oreochromis niloticus against A. hydrophila infection. No significant DNA damages or clastogenic effects were detected in tilapia challenged with A. hydrophila under Rutin/FF treatment. It is shown that an acute-phase Lipopolysaccharide binding protein (LBP) enhances the innate host defence against bacterial challenge. Semi quantitative RT-PCR and western blot results revealed the significant increase of LBP in the supernatant of tilapia monocytes/macrophages challenged with A. hydrophila upon treatment. The study findings substantiate that the combination of natural molecules with antibiotics may open up possibilities to treat MDR strains.

Selection of Phages to Control Aeromonas hydrophila - An Infectious Agent in Striped Catfish.

Striped catfish (Pangasianodon hypophthalmus) farming in the Mekong Delta Vietnam (MKDVN) importantly contributes to national aquaculture export. Currently, however, diseases occur more frequently across the entire MKDVN region. One of the most common types is hemorrhagic septicemia caused by Aeromonas hydrophila. In this study, isolation and selection of the phages for control in vitro Aeromonas hydrophila were conducted. 24 phages were isolated from 100 striped catfish pond water samples. Next, lytic activity of these phages was clarified. Four phages with short latent period (about 25 to 40 min) and/or high burst size (about 67 to 94 PFU/ cell) were selected to evaluate their infection activity to different phage-resistant A. hydrophila strains. Two phages termed as TG25P and CT45P were subjected to the phage cocktail to inactivate A. hydrophila. Re-growth of the host bacteria appeared about eight hours after treatment. Usage of the phage cocktail that attach different host bacterial receptors is not always much effective than usage of single phage. This is the first report about phage therapy to control A. hydrophila isolated from striped catfish. Some challenges in the phage cocktail were shown to achieve strategies in prospective studies in the context of high antibiotic resistance of A. hydrophila.

Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.

The survival and hepatic and muscle glucose and lactate levels of Rhamdia quelen inoculated with Aeromonas hydrophila and treated with terpinen-4-ol, carvacrol or thymol.

The aim of this study was to evaluate the resistance of Rhamdia quelen (silver catfish) to Aeromonas hydrophila infection after treatment with pure and nanoencapsulated forms either terpinen-4-ol, thymol, or carvacrol and the effects of these treatments on fish metabolic responses. After A. hydrophila inoculation, fish were treated with 30 min daily baths for 6 consecutive days with terpinen-4-ol, thymol, or carvacrol in their pure or nanoencapsulated forms at concentrations of 5, 10, 15 or 25 mg L-1. A positive control group, negative control group and saline group were also included. Survival was evaluated at the end of treatment for six consecutive days. Muscle and liver were collected to determine glucose and lactate levels. The fish treated with the nanoencapsulated form of the compounds had a high survival rate, similar to saline group and negative control groups. The carvacrol, thymol and terpinen-4-ol nanoencapsulated forms improved survival of silver catfish infected with A. hydrophila. Muscle and liver glucose and lactate levels are not indicated as biomarkers because they did not present any correlation between the metabolic state of the fish and the bacterial infection.

The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence-related genes expression and virulence factors activity after NE addition in serum-SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE-associated stimulation of A. hydrophila strain growth was not linear-dose-dependent, and only 100 µM, or higher concentrations, could stimulate growth. Real-time PCR analyses revealed that NE notably changed 13 out of the 16 virulence-associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.