Effects of berberine hydrochloride on antioxidant response and gut microflora in the Charybdis japonica infected with Aeromonas hydrophila.

This study used berberine hydrochloride to treat the Asian paddle crab, Charybdis japonica infected with the Gram-negative bacterium Aeromonas hydrophila at concentrations of 0, 100, 200 and 300 mg/L. The effect of berberine hydrochloride on the survival rate and gut microbiota of C. japonica was investigated. Berberine hydrochloride improved the stability of the intestinal flora, with an increase in the abundance of probiotic species and a decrease in the abundance of both pathogenic bacteria after treatment with high concentrations of berberine hydrochloride. Berberine hydrochloride altered peroxidase activity (POD), malondialdehyde (MDA), and lipid peroxidation (LPO) in the intestinal tract compared to the control. Berberine hydrochloride could modulate the energy released from the enzyme activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the intestinal tract of C. japonica infected with A. hydrophila. Zona occludens 1 (ZO-1), Zinc finger E-box binding homeobox 1 (ZEB1), occludin and signal transducer, and activator of transcription5b (STAT5b) expression were also increased, which improved intestinal barrier function. The results of this study provide new insights into the role of berberine hydrochloride in intestinal immune mechanisms and oxidative stress in crustaceans.

Hypoxia-inducible factor-1α promotes macrophage functional activities in protecting hypoxia-tolerant large yellow croaker (Larimichthys crocea) against Aeromonas hydrophila infection.

The immune system requires a high energy expenditure to resist pathogen invasion. Macrophages undergo metabolic reprogramming to meet these energy requirements and immunologic activity and polarize to M1-type macrophages. Understanding the metabolic pathway switching in large yellow croaker (Larimichthys crocea) macrophages in response to lipopolysaccharide (LPS) stimulation and whether this switching affects immunity is helpful in explaining the stronger immunity of hypoxia-tolerant L. crocea. In this study, transcript levels of glycolytic pathway genes (Glut1 and Pdk1), mRNA levels or enzyme activities of glycolytic enzymes [hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase A (LDHA)], aerobic respiratory enzymes [pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (IDH), and succinate dehydrogenase (SDH)], metabolites [lactic acid (LA) and adenosine triphosphate (ATP)], levels of bactericidal products [reactive oxygen species (ROS) and nitric oxide (NO)], and transcripts and level changes of inflammatory factors [IL1ß, TNFα, and interferon (IFN) γ] were detected in LPS-stimulated L. crocea head kidney macrophages. We showed that glycolysis was significantly induced, the tricarboxylic acid (TCA) cycle was inhibited, and metabolic reprogramming occurred, showing the Warburg effect when immune cells were activated. To determine the potential regulatory mechanism behind these changes, LcHIF-1α was detected and found to be significantly induced and transferred to the nucleus after LPS stimulation. LcHif-1α interference led to a significant reduction in glycolytic pathway gene transcript expression, enzyme activity, metabolites, bactericidal substances, and inflammatory factor levels; a significant increase in the aerobic respiration enzymes; and decreased migration, invasion, and phagocytosis. Further ultrastructural observation by electron microscopy showed that fewer microspheres contained phagocytes and that more cells were damaged after LcHif-1α interference. LcHif-1α overexpression L. crocea head kidney macrophages showed the opposite trend, and promoter activities of Ldha and Il1ß were significantly enhanced after LcHif-1α overexpression in HEK293T cells. Our data showed that LcHIF-1α acted as a metabolic switch in L. crocea macrophages and was important in polarization. Hypoxia-tolerant L. crocea head kidney showed a stronger Warburg effect and inhibited the TCA cycle, higher metabolites, and bactericidal substance levels. These results collectively revealed that LcHif-1α may promote the functional activities of head kidney macrophages in protecting hypoxia-tolerant L. crocea from Aeromonas hydrophila infection.

Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila.

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.

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.

Differential polyvalent passive immune protection of egg yolk antibodies (IgY) against live and inactivated Vibrio fluvialis in fish.

Egg yolk antibodies (IgY) can be prepared in large quantities and economically, and have potential value as polyvalent passive vaccines (against multiple bacteria) in aquaculture. This study prepared live and inactivated Vibrio fluvialis IgY and immunized Carassius auratus prior to infection with V. fluvialis and Aeromonas hydrophila. The results showed that the two IgY antibodies hold effective passive protective rates against V. fluvialis and A. hydrophila in C. auratus. Further, the serum of C. auratus recognized the two bacteria in vitro, with a decrease in the bacteria content of the kidney. The phagocytic activity of C. auratus plasma was enhanced, with a decrease in the expression of inflammatory and antioxidant factors. Pathological sections showed that the kidney, spleen, and intestinal tissue structures were intact, and apoptosis and DNA damage decreased in kidney cells. Moreover, the immunoprotection conferred by the live V. fluvialis IgY was higher than that of the inactivated IgY. Addition, live V. fluvialis immunity induced IgY antibodies against outer membrane proteins of V. fluvialis were more than inactivated V. fluvialis immunity. Furthermore, heterologous immune bacteria will not cause infection, so V. fluvialis can be used to immunize chickens to obtain a large amount of IgY antibody. These findings suggest that the passive immunization effect of live bacterial IgY antibody on fish is significantly better than that of inactivated bacterial antibody, and the live V. fluvialis IgY hold potential value as polyvalent passive vaccines in aquaculture.

Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila.

Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics.

Response signatures of intestinal microbiota and gene transcription of yellow catfish (Pelteobagrus fulvidraco) to Aeromonas hydrophila infection.

Bacterial intestinal inflammation is a common disease of yellow catfish (Pelteobagrus fulvidraco) in high-density aquaculture. Understanding the interactions between host and intestinal bacteria is helpful to intestinal inflammatory disease control. Here, we constructed a model of intestinal inflammation after Aeromonas hydrophila infection in yellow catfish, and characterized variations in gene expression and microbiome in the gut through high-throughput sequencing. Furthermore, host gene-microbiome interactions were identified. Histology observation showed disordered distribution of columnar epithelial cells and decrease of goblet cells in intestine. A total of 4741 genes showed differentially expression, mostly in comparisons between 12 hpi group with each other groups respectively, including control, 24 hpi and 48 hpi groups. These genes were enriched in immune-related pathways including the IL-17 signaling pathway, triggering strong inflammatory response at the invading stage within 12 h. Subsequently, the host strengthened energy consumption by activating carbohydrate and lipid metabolism pathways to repair the intestinal mucosal immune defense line. In addition, fish with A. hydrophila infection show decreased richness of gut microbial, reduced relative abundance of probiotics including Akkermansia, and elevated pathogenic bacteria such as Plesimonas. An integrative analysis identified A. hydrophila-related genes, such as il22 and stat3, for which expression level is close associated with the shift of A. hydrophila-related bacteria relative abundance, such as Akkermansia and Cetobacterium. Aside from picturing the variations of intestine gene expression and mucosal microbiome of yellow catfish coping with A. hydrophila infection, our study probed the underlying host-microbe interactions in A. hydrophila infection induced intestinal inflammatory, providing new insights for disease control in aquaculture.

Fish decay-accelerating factor (DAF) regulates intestinal complement pathway and immune response to bacterial challenge.

Decay-accelerating factor (DAF) is an essential member of the complement regulatory protein family that plays an important role in immune response and host homeostasis in mammals. However, the immune function of DAF has not been well characterized in bony fish. In this study, a complement regulatory protein named CiDAF was firstly characterized from Ctenopharyngodon idella and its potential roles were investigated in intestine following bacterial infection. Similar to mammalian DAFs, CiDAF has multiple complement control protein (CCP) functional domains, suggesting the evolutionary conservation of DAFs. CiDAF was broadly expressed in all tested tissues, with a relatively high expression level detected in the spleen and kidney. In vivo immune challenge experiments revealed that CiDAF strongly responded to bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and PAMPs (lipopolysaccharide (LPS) or muramyl dipeptide (MDP)) challenges. In vitro RNAi experiments indicated that knockdown of CiDAF could upregulate the expression of complement genes (C4b, C5 and C7) and inflammatory cytokines (TNF-α, IL-1ß and IL-8). Moreover, 2000 ng/mL of CiDAF agonist progesterone effectively alleviated LPS- or MDP-induced intestinal inflammation by regulating expression of complement factors, TLR/PepT1 pathway genes and inflammatory cytokines. Overall, these findings revealed that CiDAF may act as a negative regulator of intestinal complement pathway and immune response to bacterial challenge in grass carp.

Effects of dietary ginger (Zingiber officinale) polysaccharide on the growth, antioxidant, immunity response, intestinal microbiota, and disease resistance to Aeromonas hydrophila in crucian carp (Carassius auratus).

Ginger polysaccharides (GP) promote growth and development in fish. However, the effects of GP on crucian carp remain unclear. The present study investigated the effects of GP on the growth performance, immunity, intestinal microbiota, and disease resistance in crucian carp. Four treatment groups were established with different concentrations of GP (0.1 %, 0.2 %, 0.4 %, and 0.8 %). GP was not added as the control group, and the feeding period lasted for 56 d, followed by a 96-h anti-infection treatment using Aeromonas hydrophila. The results showed that dietary GP significantly improved growth performance, especially in the 0.4 % GP group. Furthermore, GP administration notably increased serum lysozyme (LMZ) activity, digestive enzyme performance, and antioxidant capacity of crucian carp. Moreover, dietary inclusion of GP up-regulated the expression of tumour necrosis factor-α (TNF-α), interleukin-8 (IL-8), interferon-γ (IFN-γ), and nuclear factor kappa-B (NF-κB) genes while down-regulating IL-10 and transforming growth factor-ß (TGF-ß) gene expressions, thus promoting liver health in crucian carp. Additionally, incorporating GP into the diet regulated both the diversity and composition of the intestinal microbiota in crucian carp, explicitly enhancing the relative abundance of beneficial bacteria, such as Fusobacteriota and Firmicutes. Therefore, GP reduces the mortality of crucian carp infected with A. hydrophila. In conclusion, this study provides novel insights into the application of dietary GP in cultured fish and evaluates the value of traditional Chinese medicinal polysaccharides against pathogenic bacteria.

Changes of intestinal barrier in the process of intestinal inflammation induced by Aeromonas hydrophila in snakehead (Channa argus).

Bacterial intestinal inflammation frequently occurs in cultured fish. Nevertheless, research on intestinal barrier dysfunction in the process of intestinal inflammation is deficient. In this study, we explored the changes of intestinal inflammation induced by Aeromonas hydrophila (A. hydrophila) in snakehead and the relationship between intestinal barrier and inflammation. Snakehead [(13.05 ± 2.39) g] were infected via anus with A. hydrophila. Specimens were collected for analysis at 0, 1, 3, 7 and 21 d post-injection. The results showed that with the increase of exposure time, the hindgut underwent stages of normal function, damage, damage deterioration, repair and recovery. Relative to 0 d, the levels of IL-1ß and TNF-α in serum, and the expression of nod1, tlr1, tlr5, nf-κb, tnf-α and il-1ß in intestine were significantly increased, and showed an upward then downward pattern over time. However, the expression of tlr2 and il-10 were markedly decreased, and showed the opposite trend. In addition, with the development of intestinal inflammation, the diversity and richness of species, and the levels of phylum and genus in intestine were obviously altered. The levels of trypsin, LPS, AMS, T-SOD, CAT, GPx, AKP, LZM and C3 in intestine were markedly reduced, and displayed a trend of first decreasing and then rebounding. The ultrastructure observation showed that the microvilli and tight junction structure of intestinal epithelial cells experienced normal function initially, then damage, and finally recovery over time. The expression of claudin-3 and zo-1 in intestine were significantly decreased, and showed a trend of first decreasing and then rebounding. Conversely, the expression of mhc-i, igm, igt and pigr in intestine were markedly increased, and displayed a trend of increasing first and then decreasing. The above results revealed the changes in intestinal barrier during the occurrence and development of intestinal inflammation, which provided a theoretical basis for explaining the relationship between the two.

CcPTGS2a-like gene-mediated NF-κB/ERK signaling regulation in the common carp (Cyprinus carpio).

Prostaglandin-endoperoxide synthase 2 (PTGS2), a common biological macromolecule, is pivotal for innate immunity and pathogen recognition. In this study, we identified and characterized a CcPTGS2a-like gene in the common carp (Cyprinus carpio) with an open reading frame (ORF) of 1821 bp and epidermal growth factor and peroxidase domains. Our multiple sequence analysis revealed high homology between the amino acid sequence of CcPTGS2a-like and those of its homologs in other fish. CcPTGS2a-like mRNA and protein expressions were significantly upregulated in the spleen, head kidney, liver, and gill tissues upon exposure to Aeromonas hydrophila stimulation. CcPTGS2a-like protein recognized the conserved bacterial surface components and exhibited detectable bacterial binding activity. CcPTGS2a-like overexpression before exposure to A. hydrophila notably enhanced the survival rate of common carp, concomitant with decreased bacterial burden. The NF-κB/ERK signaling pathway initiated the immune response in common carp upon infection with A. hydrophila. CcPTGS2a-like overexpression or interference in the head kidney and Epithelioma papulosum cyprinid cells could modulate the p-NF-κB (p-p-65), p-IκBα, and p-ERK1/2 levels as well as the IL-1ß and IL-6 mRNA expression. These results indicated potential CcPTGS2a-like involvement in the immune response of the common carp to bacterial infections through the NF-κB/ERK signaling pathway.

Isolation, characterization, and immunomodulatory effects of extracellular vesicles isolated from fish pathogenic Aeromonas hydrophila.

Bacterial extracellular vesicles (BEVs) are natural nanocarriers that have shown great potential for biomedical applications such as biomarkers, cancer therapy, immunomodulators, vaccines, wound healing, tissue engineering, and drug carriers. In the present study, BEVs were isolated from the gram-negative bacterium, Aeromonas hydrophila using the ultracentrifugation method and denoted as AhEVs. Using transmission electron microscopy imaging, we confirmed the ultrastructure and spherical shape morphology of AhEVs. Nanoparticle-tracking analysis results showed a mean particle size of 105.5 ± 2.0 nm for AhEVs. Moreover, the particle concentration of AhEVs was 2.34 ± 0.12 × 1011 particles/mL of bacterial supernatant. AhEV-treated fathead minnow (FHM) cells did not show cytotoxicity effects up to 50 µg/mL with no significant decrease in cells. Moreover, no mortality was observed in larval zebrafish up to 50 µg/mL which indicates that the AhEVs are biocompatible at this concentration. Furthermore, fluorescent-labeled AhEVs were internalized into FHM cells. Results of qRT-PCR analysis in FHM cells revealed that cellular pro-inflammatory cytokines such as nuclear factor (NF)-κB, interferon (Ifn), Irf7, interleukin (Il) 8, and Il11 were upregulated while downregulating the expression of anti-inflammatory Il10 in a concentration-dependent manner. AhEV-treated adult zebrafish (5 µg/fish) induced toll-like receptor (tlr) 2 and tlr4; tumor necrosis factor-alpha (tnfα); heat shock protein (hsp) 70; and il10, il6, and il1ß in kidney. Protein expression of NF-κB p65 and Tnfα presented amplified levels in the spleen of AhEVs-treated zebrafish. Based on the collective findings, we conclude that AhEVs exhibited morphological and physicochemical characteristics to known EVs of gram (-)ve bacteria. At biocompatible concentrations, the immunomodulatory activity of AhEVs was demonstrated by inducing different immune response genes in FHM cells and zebrafish. Hence, we suggest that AhEVs could be a novel vaccine candidate in fish medicine due to their ability to elicit strong immune responses.

Exploring the protective role of Bacillus velezensis BV1704-Y in zebrafish health and disease resistance against Aeromonas hydrophila infection.

Bacillus genus, particularly Bacillus velezensis, is increasingly considered as viable alternatives to antibiotics in aquaculture due to their safety and probiotic potential. However, the specific mechanisms through which probiotic B. velezensis confers protection against Aeromonas hydrophila infection in fish remain poorly understood. This study delved into the multifaceted impacts of B. velezensis BV1704-Y on diverse facets of zebrafish health, including gut barrier function, immune response, oxidative stress, gut environment, microbiome composition, and disease resistance. Our findings demonstrate that supplementation with B. velezensis BV1704-Y significantly alleviated symptoms and reduced mortality in zebrafish infected with A. hydrophila. Furthermore, a notable reduction in the expression of pivotal immune-related genes, such as IL-1ß, IL6, and TNF-α, was evident in the gut and head kidney of zebrafish upon infection. Moreover, B. velezensis BV1704-Y supplementation resulted in elevated activity levels of essential antioxidant enzymes, including SOD, CAT, and GSH, in gut tissue. Notably, B. velezensis BV1704-Y positively modulated the structure and function of the intestinal microbiome, potentially enhancing immune response and resilience in zebrafish. Specifically, supplementation with B. velezensis BV1704-Y promoted the relative abundance of beneficial bacteria, such as Cetobacterium, which showed a noteworthy negative correlation with the expression of pro-inflammatory genes and a positive correlation with gut barrier-related genes. Altogether, our study suggests that B. velezensis BV1704-Y holds promise as an effective probiotic for protecting zebrafish against A. hydrophila infection, offering potential benefits for the aquaculture industry.

Erythrocytes of the common carp are immune sentinels that sense pathogen molecular patterns, engulf particles and secrete pro-inflammatory cytokines against bacterial infection.

Introduction: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers. Methods: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria. Results: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo. Discussion: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

Aquatic whispers: Decoding skin manifestation of Aeromonashydrophila.

The Aeromonadaceae family, comprised of gram-negative bacilli, is ubiquitously distributed across the globe. Infections by Aeromonas species encompass gastroenteritis, septicaemia, skin and soft tissue infections (SSTIs), pneumonia, and peritonitis. This report delineates a case of Aeromonas hydrophila infection, manifesting as an array of pustules on the patient's lower extremities subsequent to the ingestion of marine crustaceans, specifically prawns. Prompt diagnosis and the initiation of an appropriate antibiotic regimen are imperative to mitigate the risk of further complications.

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.

Antimicrobial efficiency against fish pathogens on the green synthesized silver nanoparticles.

Fish-borne pathogens such as A. hydrophila and F. aquidurense are the most resistant strains in pisciculture farming. Removing the aforementioned pathogens without antibiotics presents a formidable challenge. To overcome this problem, silver nanoparticles (AgNPs) are synthesized using silver nitrate, water medium, and as an AzadirachtaIndica leaf extract via the green synthesis route. X-ray diffraction (XRD) pattern results authenticate the synthesized material is the face-centered cubic structure of silver. The optical absorption edge of the synthesized product was found at the wavelength of 440 nm from the UV-visible spectra, which is confirmed to relate to the Surface Plasmon Resonance peaks of silver particles. In addition, the optical band gap value of the synthesized Ag sample is measured to be 2.81 eV from the obtained optical absorption spectra. EDX spectrum of the synthesized product also supports confirming the silver particle formation. The FT-IR spectra of the neem extract and silver nanoparticles showed their characteristic functional groups, respectively. The presence of bands between 1000 cm-1 to 500 cm-1 indicates to the formation of silver particles. Spherical particles appeared in the synthesized Ag using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The particle size of Ag NPs was measured as 40 nm and 62 ± 10 nm by TEM and Dynamic Light Scattering (DLS). The zeta potential was also measured as -12 mV showing the synthesized sample's stable nature. Using the DPPH assay, synthesized AgNPs were taken along with the various concentrations of ascorbic acid (20, 40, 60, 80, and 100 µg/mL) to examine the free radical scavenging activity (RSA). RSA value is higher (84 ± 2 %) for synthesized AgNPs at higher concentration (100 µg/mL) than 21 ± 2 % at low concentration (100 µg/mL). The antimicrobial efficacy of the AgNPs against A. hydrophila and F. aquidurense was performed through the agar diffusion method and its results showed the inhibitory zones of the F.aquidurense and A. hydrophila were measured as 25 ± 3 mm, and 28 ± 4 mm respectively. The synthesized Ag particles showed excellent antimicrobial and antioxidant properties confirmed by antimicrobial and DPPH experiments. It implies that the green synthesized silver nanoparticles could be a good alternative for antibiotics in aquaculture farms. The exposure of low concentrations of silver nanoparticles to zebrafish and brine shrimp does not affect the viability and morphology. The exposure of silver nanoparticles in the fisheries in optimized concentration and time could control the fish-borne pathogens without antibiotics.

Meningitis caused by Aeromonas hydrophila in Oreochromis niloticus: Proteomics and druggability of virulence factors.

Meningitis caused by Gram-negative bacteria is a serious public health problem, causing morbidity and mortality in both children and adults. Here, we propose a novel experimental model using Nile tilapia (Oreochromis niloticus) to study neuroinflammation. The fish were infected with Aeromonas hydrophila, and the course of infection was monitored in the peripheral blood. Septicemia was obvious in the blood, while in the brain tissue, infection of the meninges was present. The histopathological examination showed suppurative meningitis, and the cellular immune response in the brain tissue during infection was mediated by microglia. These cells were morphologically characterized and phenotyped by MHC class II markers and CD68. The increased production of TNF-α, IL-1ß and iNOS supported the infiltration of these cells during the neuroinflammatory process. In the proteomic analysis of A. hydrophila isolated from brain tissue, we found chemotactic and transport proteins, proteolytic enzymes and enzymes associated with the dismutation of nitric oxide (NO), as well as motor proteins and those responsible for cell division. After characterizing the most abundant proteins during the course of infection, we investigated the druggability index of these proteins and identified promising peptide sequences as molecular targets that are similar among bacteria. Thus, these findings deepened the understanding of the pathophysiology of meningitis caused by A. hydrophila. Moreover, through the proteomics analysis, important mechanisms and pathways used by the pathogen to subvert the host response were revealed, providing insights for the development of novel antibiotics and vaccines.

Immune protection of grass carp by oral vaccination with recombinant Bacillus methylotrophicus expressing the heterologous tolC gene.

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1ß, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.

Immunostimulatory effects of dietary verjuice (Vitis vinifera) on immune response and transcription of immune-related genes in juvenile rainbow trout (Oncorhynchusmykiss).

The development of green aquaculture practices has led to the supplementation of fish diets with natural immunostimulants such as organic acids. This study aimed to assess the dietary effects of verjuice (VJ; unfermented unripe grapes; Vitis vinifera) on hematological parameters, skin mucosal immunity, transcriptional immune responses, and antibacterial serum activity against Aeromonas hydrophila in rainbow trout. The fish (51.0 ± 2.4 g) were randomly distributed into 15 tanks and fed ad-libitum thrice daily with diets containing different levels of VJ including 0 (control; VJ-0), 3 (VJ-3), 6 (VJ-6), 9 (VJ-9), and 12 (VJ-12) mL/kg VJ for 56 d. Results showed that immuno-hematological parameters (total white blood cells, neutrophils, and monocytes) were improved in VJ-added groups (P < 0.05). In addition, dietary VJ (9 mL/kg) modulated serum immunological parameters. Skin mucus immunology exhibited a notable increase in alkaline phosphatase, lysozyme activity, alkaline protease, total protein, total immunoglobulin, and esterase levels in VJ-9 group compared with those in the control group (P < 0.05). The mRNA expression of interleukin-1ß, interleukin-6, interleukin-8, and immunoglobulin M were significantly higher in VJ-9 group than in the control (P < 0.05). Furthermore, the results of the antibacterial evaluation showed that A. hydrophila growth was significantly inhibited in the serum samples from VJ-3 to VJ-9 groups after the 56th day and in all VJ-treated groups after the 70th (P < 0.05). In conclusion, dietary VJ is a novel immunostimulant and the optimal dietary supplementation level of 6.65-7.46 mL/kg can effectively improve immune responses in rainbow trout.