RESUMEN
Aeromonas hydrophila, the pathogen that is the causative agent of motile Aeromonas septicemia (MAS) disease, commonly attacks freshwater fishes, including yellow catfish (Pelteobagrus fulvidraco). Although the kidney is one of the most important organs involved in immunity in fish, its role in disease progression has not been fully elucidated. Understanding the cellular composition and innate immune regulation mechanisms of the kidney of yellow catfish is important for the treatment of MAS. In this study, single-cell RNA sequencing (scRNA-seq) was performed on the kidney of hybrid yellow catfish (Pelteobagrus fulvidraco â × Pelteobagrus vachelli â) after A. hydrophila infection. Nine types of kidney cells were identified using marker genes, and a transcription module of marker genes in the main immune cells of hybrid yellow catfish kidney tissue was constructed using in-situ hybridization. In addition, the single-cell transcriptome data showed that the differentially expressed genes of macrophages were primarily enriched in the Toll-like receptor and Nod-like receptor signaling pathways. The expression levels of genes involved in these pathways were upregulated in macrophages following A. hydrophila infection. Transmission electron microscopy and TUNEL analysis revealed the cellular characteristics of macrophages before and after A. hydrophila infection. These data provide empirical support for in-depth research on the role of the kidney in the innate immune response of hybrid yellow catfish.
Asunto(s)
Aeromonas hydrophila , Bagres , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Riñón , Transcriptoma , Animales , Bagres/inmunología , Bagres/genética , Aeromonas hydrophila/fisiología , Enfermedades de los Peces/inmunología , Inmunidad Innata/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Transcriptoma/inmunología , Riñón/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Análisis de la Célula IndividualRESUMEN
Polymeric immunoglobulin receptor (pIgR) is an important immune factor in the mucosal immune system of fish, which plays a key role in mediating the secretion and transport of immunoglobulin into mucus. In this study, the full-length cDNA sequence of Megalobrama amblycephala pIgR gene was firstly cloned and the immune response to Aeromonas hydrophila was detected. After being challenged by Aeromonas hydrophila at 3 d, significantly pathological features were observed in intestine, head kidney, spleen, liver and gill of Megalobrama amblycephala. The content of lysozyme (Lys) and the activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) increased significantly at 1 d and reached the peak at 3 d, and the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in serum reached the peak at 5 d and 7 d after infection, respectively. The expression level of IL-1ß gene reached the peak at 3 d in intestine, 5 d in gill and spleen, 7 d in head kidney and liver of Megalobrama amblycephala after infected by Aeromonas hydrophila, respectively. The TNF-α gene expression reached the peak at 3 d in intestine and gill, 5 d in head kidney and spleen, 7 d in liver after infection, respectively. The experimental results showed that the infection of Aeromonas hydrophila caused the pathological changes of immune-related tissues and triggered the inflammation responses. The full-length cDNA sequence of Megalobrama amblycephala pIgR was 1828 bp, and its open reading frame (ORF) was 1023 bp, encoding 340 amino acids. The pIgR of Megalobrama amblycephala has a signal peptide sequence, followed by extracellular region, transmembrane region and intracellular region. The extracellular region includes two Ig-like domains (ILDs), and its tertiary structure is twisted "L". The phylogenetic tree was constructed using the adjacency method, and the pIgR genes of Megalobrama amblycephala and cyprinidae fish were clustered into a single branch. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pIgR gene in different tissues of Megalobrama amblycephala. The expression level of pIgR gene was the highest in liver, followed by intestine, head kidney, skin, middle kidney and spleen, lower in heart, gill and brain, and the lowest in muscle. After being infected by Aeromonas hydrophila, the expression level of Megalobrama amblycephala pIgR gene in intestine, head kidney, spleen, liver and gill showed a trend of increasing first and then decreasing within 28 d. The pIgR gene expression reached the peak in mucosal immune-related tissues (gill and intestine) was earlier than that in systemic immune-related tissues (head kidney and spleen), and the relative expression level of pIgR gene at peak in intestine (12.3 fold) was higher than that in head kidney (3.73 fold) and spleen (7.84 fold). These results suggested that Megalobrama amblycephala pIgR might play an important role in the mucosal immune system to against Aeromonas hydrophila infection.
Asunto(s)
Aeromonas hydrophila , Secuencia de Aminoácidos , Cyprinidae , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Filogenia , Receptores de Inmunoglobulina Polimérica , Animales , Aeromonas hydrophila/fisiología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Cyprinidae/inmunología , Cyprinidae/genética , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Receptores de Inmunoglobulina Polimérica/genética , Receptores de Inmunoglobulina Polimérica/inmunología , Receptores de Inmunoglobulina Polimérica/química , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria , Secuencia de BasesRESUMEN
Molting is a crucial biological process of crustaceans. Crustaceans go through three separate stages throughout their molting process, including pre-molt, post-molt and inter-molt. However, the exact mechanism of immunological modulation during molting remains unclear. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been extensively documented to participate in immune defense. In the present study, a TRAF6 gene with two TRAF-type zinc finger domains was identified from Eriocheir sinensis (designed as EsTRAF6), and its role in regulating immune response during molting process was explored. The mRNA expression level of EsTRAF6 at pre-molt stage was higher than that at post-molt stage and inter-molt stage. After Aeromonas hydrophila stimulation, the expression levels of EsTRAF6, EsRelish and anti-lipopolysaccharide factors (ALFs) genes exhibited a considerable increase at three molting stages. Subsequently, the expression patterns of EsTRAF6 and EsRelish in response to the treatment with 20-hydroxyecdysone (20E) were examined. The mRNA expression of EsTRAF6 and EsRelish were significantly increased at 12 h after 20E injection. Additionally, the protein expression level of TRAF6 was also up-regulated in 20E group compared to control group. Furthermore, the role of EsTRAF6 in regulating the anti- ALFs expression at pre-molt stage post A. hydrophila stimulation was investigated. Following the inhibition of the EsTRAF6 transcript using RNAi or the injection of inhibitor (TMBPS), there was a notable decrease of the EsALF1, EsALF2 and EsALF3 transcripts. Moreover, a significant reduction in the phosphorylation level of NF-κB at pre-molt stage was observed after A. hydrophila stimulation in TRAF6-inhibited crabs. Collectively, our results suggest that EsTRAF6 could be induced by 20E and promoted the EsALFs expression by activating NF-κB at pre-molt stage, which provides a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.
Asunto(s)
Proteínas de Artrópodos , Braquiuros , FN-kappa B , Factor 6 Asociado a Receptor de TNF , Animales , Braquiuros/inmunología , Braquiuros/genética , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/inmunología , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Proteínas de Artrópodos/química , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/inmunología , Muda/inmunología , Muda/genética , Inmunidad Innata/genética , Aeromonas hydrophila/fisiología , Regulación de la Expresión Génica/inmunología , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , Filogenia , Perfilación de la Expresión Génica/veterinariaRESUMEN
Cottonseed meal (CSM) and cottonseed protein concentrate (CPC) serve as protein alternatives to fish meal and soybean meal in the feed industry. However, the presence of gossypol residue in CSM and CPC can potentially trigger severe intestinal inflammation, thereby restricting the widespread utilization of these two protein sources. Probiotics are widely used to prevent or alleviate intestinal inflammation, but their efficacy in protecting fish against gossypol-induced enteritis remains uncertain. Here, the protective effect of Pediococcus pentosaceus, a strain isolated from the gut of Nile tilapia (Oreochromis niloticus), was evaluated. Three diets, control diet (CON), gossypol diet (GOS) and GOS supplemented with P. pentosaceus YC diet (GP), were used to feed Nile tilapia for 10 weeks. After the feeding trial, P. pentosaceus YC reduced the activity of myeloperoxidase (MPO) in the proximal intestine (PI) and distal intestine (DI). Following a 7-day exposure to Aeromonas hydrophila, the addition of P. pentosaceus YC was found to increase the survival rate of the fish. P. pentosaceus YC significantly inhibited the oxidative stress caused by gossypol, which was evidenced by lower reactive oxygen species (ROS) and malondialdehyde (MDA), as well as higher activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in PI and DI. Addition of P. pentosaceus YC significantly inhibited enteritis, with the lower expression of pro-inflammatory cytokines (il-1ß, il-6, il-8) and higher expression of anti-inflammatory cytokines tgf-ß. RNA-seq analysis indicated that P. pentosaceus YC supplementation significantly inhibited nlrc3 and promoted nf-κb expression in PI and DI, and the siRNA interference experiment in vivo demonstrated that intestinal inflammation was mediated by NLRC3/NF-κB/IL-1ß signaling pathway. Fecal bacteria transplantation experiment demonstrated that gut microbiota mediated the protective effect of P. pentosaceus YC. These findings offer valuable insights into the application of P. pentosaceus YC for alleviating gossypol-induced intestinal inflammation in fish.
Asunto(s)
Alimentación Animal , Cíclidos , Enfermedades de los Peces , Gosipol , Pediococcus pentosaceus , Probióticos , Transducción de Señal , Animales , Cíclidos/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/inducido químicamente , Enfermedades de los Peces/prevención & control , Probióticos/farmacología , Probióticos/administración & dosificación , Alimentación Animal/análisis , Transducción de Señal/efectos de los fármacos , Gosipol/administración & dosificación , Gosipol/farmacología , Dieta/veterinaria , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Aeromonas hydrophila/fisiología , FN-kappa B/metabolismo , FN-kappa B/genética , Microbioma Gastrointestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Intestinos/inmunología , Inflamación/veterinaria , Inflamación/inducido químicamente , Inflamación/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Proteínas de Peces/inmunología , Enteritis/veterinaria , Enteritis/prevención & control , Enteritis/inducido químicamente , Enteritis/inmunología , Enteritis/microbiologíaRESUMEN
Selenium (Se), a trace element, is vital for the maintenance of cellular redox balance, thyroid hormone metabolism, inflammation, and immunity. Aeromonas hydrophila (A. hydrophila) is a common Gram-negative conditional pathogenic bacterium in fish culture, posing a serious threat to intensive aquaculture. Our study investigated the influence of dietary Se on the intestinal immune function of grass carp (Ctenopharyngodon idella) and the related regulatory mechanisms. The 2160 healthy juvenile grass carp (9.76 ± 0.005 g) were randomly assigned to 6 test groups of 6 replicates each, and fed graded selenomethionine (0.05, 0.20, 0.40, 0.61, 0.77, 0.98 mg Se/kg diet) for 70 days and then injected with A. hydrophila for a 6-day attack test. The results indicated that appropriate Se levels (0.40 mg/kg diet) alleviated intestinal damage caused by A. hydrophila and increased intestinal immune substances C3 and C4 levels as well as the activity of acid phosphatase (ACP) and lysozyme (LZ) (P > 0.05). Appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) decreased intestinal pro-inflammatory cytokines (IFN-γ2, IL-6, IL-12p35, IL-17 A F and IL-17D) mRNA levels (P > 0.05) and increased intestinal anti-inflammatory factors (TGF-ß1, IL-4/13A, IL-4/13B, IL-10 and IL-22) mRNA levels (P > 0.05) in juvenile grass carp. Further studies revealed that Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal endoplasmic reticulum stress (ERS)-related signaling pathway. Furthermore, we found that appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal autophagy in juvenile grass carp, which may be related to ULK1, Beclin 1, ATG5, ATG12, LC3, and P62. In conclusion, appropriate levels of Se can alleviate intestinal inflammation and inhibit ERS and autophagy in juvenile grass carp. A quadratic regression analysis of intestinal ACP and LZ also indicated that the Se requirements of juvenile grass carp were 0.59 and 0.51 mg/kg, respectively.
Asunto(s)
Aeromonas hydrophila , Alimentación Animal , Autofagia , Carpas , Dieta , Suplementos Dietéticos , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Intestinos , Selenio , Animales , Carpas/inmunología , Autofagia/efectos de los fármacos , Aeromonas hydrophila/fisiología , Dieta/veterinaria , Selenio/farmacología , Selenio/administración & dosificación , Intestinos/efectos de los fármacos , Intestinos/inmunología , Alimentación Animal/análisis , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/inmunología , Enfermedades de los Peces/inmunología , Suplementos Dietéticos/análisis , Distribución Aleatoria , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Relación Dosis-Respuesta a DrogaRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Biopelículas , Fimbrias Bacterianas , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Operón , Aeromonas hydrophila/genética , Aeromonas hydrophila/patogenicidad , Aeromonas hydrophila/fisiología , Biopelículas/crecimiento & desarrollo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Virulencia/genética , Animales , Infecciones por Bacterias Gramnegativas/microbiología , Enfermedades de los Peces/microbiología , Adhesión Bacteriana/genética , Bagres/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Inactivación de GenesRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Subunidad alfa del Factor 1 Inducible por Hipoxia , Macrófagos , Perciformes , Animales , Perciformes/inmunología , Perciformes/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Aeromonas hydrophila/fisiología , Aeromonas hydrophila/inmunología , Lipopolisacáridos/inmunología , Glucólisis , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Activación de Macrófagos/inmunología , Hipoxia/inmunología , Hipoxia/metabolismo , Riñón Cefálico/inmunología , Riñón Cefálico/metabolismoRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Intestinos , Animales , Aeromonas hydrophila/fisiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Peces/inmunología , Peces/microbiología , Microbioma Gastrointestinal , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Inflamación/inmunología , Inflamación/veterinaria , Mucosa Intestinal/inmunología , Intestinos/inmunología , Intestinos/patologíaRESUMEN
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.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Intestinos , Animales , Carpas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Enfermedades de los Peces/inmunología , Inmunidad Innata/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Intestinos/inmunología , Regulación de la Expresión Génica/inmunología , Filogenia , Perfilación de la Expresión Génica/veterinaria , Aeromonas hydrophila/fisiología , Secuencia de Aminoácidos , Alineación de Secuencia/veterinaria , Proteínas del Sistema Complemento/inmunologíaRESUMEN
The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.
Asunto(s)
Aeromonas hydrophila , Proteínas del Sistema Complemento , Corticosterona , Estrés Fisiológico , Animales , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Masculino , Estrés Fisiológico/inmunología , Aeromonas hydrophila/fisiología , Aeromonas hydrophila/inmunología , Corticosterona/sangre , Actividad Bactericida de la Sangre , Bufonidae/inmunología , Bufonidae/microbiología , Bufonidae/sangreRESUMEN
Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1ß and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and ß-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.
Asunto(s)
Aeromonas hydrophila , Aeromonas veronii , Carpas , Enfermedades de los Peces , Microbioma Gastrointestinal , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Intestinos , Animales , Carpas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Aeromonas hydrophila/fisiología , Intestinos/inmunología , Intestinos/microbiología , Aeromonas veronii/fisiología , Aeromonas/fisiología , Aeromonas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunologíaRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Carpas , Enfermedades de los Peces , Proteínas de Peces , Regulación de la Expresión Génica , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , FN-kappa B , Animales , Carpas/inmunología , Carpas/genética , Aeromonas hydrophila/fisiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de los Peces/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/inmunología , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Secuencia de Aminoácidos , Filogenia , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria , Transducción de Señal , Sistema de Señalización de MAP Quinasas/inmunología , Secuencia de BasesRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Vesículas Extracelulares , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Pez Cebra , Animales , Aeromonas hydrophila/fisiología , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/química , Enfermedades de los Peces/inmunología , Pez Cebra/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/inmunología , Cyprinidae/inmunología , Factores Inmunológicos/farmacología , Factores Inmunológicos/química , Línea Celular , Agentes Inmunomoduladores/química , Agentes Inmunomoduladores/farmacologíaRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Bacillus , Resistencia a la Enfermedad , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Probióticos , Pez Cebra , Animales , Aeromonas hydrophila/fisiología , Pez Cebra/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de los Peces/inmunología , Probióticos/farmacología , Probióticos/administración & dosificación , Bacillus/química , Bacillus/fisiología , Resistencia a la Enfermedad/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Alimentación Animal/análisis , Dieta/veterinaria , Inmunidad InnataRESUMEN
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.
Asunto(s)
Aeromonas hydrophila , Bagres , Enfermedades de los Peces , Microbioma Gastrointestinal , Infecciones por Bacterias Gramnegativas , Animales , Aeromonas hydrophila/fisiología , Bagres/inmunología , Bagres/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiologíaRESUMEN
Heme oxygenase-1 (HO-1), an inducible rate-limiting metabolic enzyme, exerts critical immunomodulatory functions by potential anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Although accumulative studies have focused on the immune functions of HO-1 in mammals, the roles in fish are poorly understood, and the reports on involvement in the defensive and immune response are very limited. In this study, On-HO-1 gene from Oreochromis niloticus was successfully cloned and identified, which contained an open reading frame (ORF) of 816 bp and coded for a protein of 271 amino acids. The On-HO-1 protein phylogenetically shared a high homology with HO-1 in other teleost fish (76.10%-98.89 %) and a lowly homology with HO-1 in mammals (38.98%-41.55 %). The expression levels of On-HO-1 were highest in the liver of healthy tilapias and sharply induced by Streptococcus agalactiae or Aeromonas hydrophila. Besides, On-HO-1 overexpression significantly increased non-specific immunological parameters in serum during bacterial infection, including LZM, SOD, CAT, ACP, and AKP. It also exerted anti-inflammatory and anti-apoptotic effects in response to the immune response of the infection with S. agalactiae or A. hydrophila by upregulating anti-inflammatory factors (IL-10, TGF-ß), autophagy factors (ATG6, ATG8) and immune-related pathway factors (P65, P38), and down-regulating pro-inflammatory factors (IL-1ß, IL-6, TNF-α), apoptotic factors (Caspase3, Caspase9), pyroptosis factor (Caspase1), and inflammasome (NLRP3). These results suggested that On-HO-1 involved in immunomodulatory functions and host defense in Nile tilapia.
Asunto(s)
Aeromonas hydrophila , Cíclidos , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Hemo-Oxigenasa 1 , Inmunidad Innata , Filogenia , Animales , Cíclidos/inmunología , Cíclidos/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Aeromonas hydrophila/fisiología , Inmunidad Innata/genética , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiae/fisiología , Regulación de la Expresión Génica/inmunología , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria , Secuencia de AminoácidosRESUMEN
Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.
Asunto(s)
Aeromonas hydrophila , Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Filogenia , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Carpas/inmunología , Carpas/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Aeromonas hydrophila/fisiología , Inmunidad Innata/genética , Rhabdoviridae/fisiología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Regulación de la Expresión Génica/inmunología , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/inmunología , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , Autofagia/inmunologíaRESUMEN
Cytokine-like factor 1 (CYTL1) is a small cytokine and has diverse biological functions in mammals. However, whether CYTL1 exists in lower vertebrates is not clear. In this study, we identified cytl homologs in fish and characterized the immune functions in a teleost species, grass carp (Ctenopharyngodon idella). Fish CYTL1 homologs share conserved molecular features with their mammalian counterparts, including 6 cysteine residues in the mature peptide, genomic organization and synteny. Gene expression analysis revealed that cytl1 was constitutively expressed in tissues of grass carp, with the highest expression detected in the heart. Upon infection with Aeromonas hydrophila (A. hydrophila), cytl1 was downregulated in the hindgut, head kidney, skin, and spleen. In the primary head kidney leukocytes (HKLs), stimulation with inactivated A. hydrophila, LPS, poly(I:C), IL-22, IFN-a or IFN-γrel resulted in downregulation of cytl1 expression. Recombinant grass carp CYTL1 protein produced in the HEK293-F cells was potent to induce il-10 expression, but had little effect on the expression of il-1ß and il-6. In vivo experiments revealed that CYTL1 was effective to recruit macrophages to the muscle injected with cytl expression plasmids. Taken together, our results indicate that CYTL1 is a potent chemokine for recruitment of macrophages in fish.
Asunto(s)
Aeromonas hydrophila , Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Macrófagos , Carpas/inmunología , Carpas/genética , Animales , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Enfermedades de los Peces/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Aeromonas hydrophila/fisiología , Macrófagos/inmunología , Filogenia , Regulación de la Expresión Génica/inmunología , Secuencia de Aminoácidos , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria , Inmunidad Innata/genética , Factores Quimiotácticos/genética , Factores Quimiotácticos/inmunologíaRESUMEN
The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish (Pangasisanodon hypophthalmus) and giant snakehead (Channa micropeltes), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae, Enterobacteriaceae, and Pseudomonadaceae. Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (> 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia.
Asunto(s)
Acuicultura , Farmacorresistencia Bacteriana , Microbiología del Agua , Cambodia , Bagres/microbiología , Especies Centinela , Fenotipo , Genotipo , Aeromonadaceae/clasificación , Aeromonadaceae/aislamiento & purificación , Aeromonadaceae/fisiología , Enterobacteriaceae/clasificación , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/fisiología , Pseudomonadaceae/clasificación , Pseudomonadaceae/aislamiento & purificación , Pseudomonadaceae/fisiología , Aeromonas caviae/aislamiento & purificación , Aeromonas caviae/fisiología , Aeromonas hydrophila/aislamiento & purificación , Aeromonas hydrophila/fisiología , Ciprofloxacina/farmacología , Ciprofloxacina/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/veterinaria , Monitoreo del AmbienteRESUMEN
OBJECTIVES: The aim of this study was to quantify and identify metabolites of Ice Nucleation Active (INA) bacteria as an anti-biofilm agent against biofilms of fish pathogens such as Aeromonas hydrophila and Streptococcus agalactiae. RESULTS: Ice nucleation active bacteria, which have the ability to catalyze ice nucleation, isolated from rainwater in previous studies, were used. All INA isolates were tested in several assays, including the antimicrobial test, which uses streptomycin as the positive control and none of the isolates were found positive in the antimicrobial test. As for the quorum quenching assay, it was found that four out of ten isolates were able to disturb the communication system in Chromobacterium violaceum wild type, which was used as the indicator bacteria. On the next assay, all ten isolates were tested for Biofilm Inhibition and Destruction and showed anti-biofilm activity with the highest percentage inhibition of 33.49% by isolate A40 against A. hydrophila and 77.26% by isolate A19 against S. agalactiae. C1 performed the highest destruction against A. hydrophila and S. agalactiae, with percentages of 32.11% and 51.88%, respectively. As for the GC-MS analysis, supernatants of INA bacteria contain bioactive compounds such as sarcosine and fatty acids, which are known to have antibiofilm activity against several biofilm-forming bacteria. Through 16s rRNA sequencing, identified bacteria are from the Pantoea, Enterobacter, and Acinetobacter genera. As for the conclusion, ice nucleation active bacteria metabolites tested showed positive results against pathogenic bacteria Aeromonas hydrophila and Streptococcus agalactiae in destructing and inhibiting biofilm growth.