The Antibacterial Efficacy and Mechanism of Tea Polyphenol Against Drug-Resistant Aeromonas veronii TH0426 In Vitro.

The emergence of Motile Aeromonas Septicemia (MAS) caused by Aeromonas veronii in sturgeon farming has become a significant concern due to its high mortality impact on the aquaculture industry. The threat posed by MAS highlights the urgent need for effective control measures to combat bacterial infections in sturgeon populations. Tea polyphenol (TP) has demonstrated promising antibacterial properties against livestock and poultry bacterial infections. However, its antibacterial efficacy and mechanism in bacterial diseases of aquatic animals remain largely unexplored. This study aimed to investigate the in vitro antibacterial effect and mechanism of TP on fish-borne drug-resistant A. veronii TH0426 by assessing the impact of TP on TH0426 cell growth, antibiofilm activity, morphology, as well as measuring electrical conductivity, DNA extravasation, lactate dehydrogenase (LDH) activity, protein, and DNA contents. Results demonstrated that the minimum inhibitory concentration and the minimum bactericidal concentration of TP on TH0426 were 1024 and 2048 µg/mL, respectively. After a 4 h treatment, the growth of TH0426 was completely inhibited at the concentration of 1024 and 2048 µg/mL of TP. Meanwhile, TP exhibited a significant antibiofilm activity. Both scanning electron microscope and transmission electron microscope analyses revealed disrupted cell membrane structure, irregular cell morphology, and loss of intracellular contents following TP treatment. Moreover, increased cell membrane permeability induced by TP led to intracellular ion and DNA leakage, resulting in elevated electrical conductivity and DNA extravasation. Furthermore, TP decreased LDH activity, protein concentration and content, DNA fluorescence intensity, and density in a time-dependent manner, indicating inhibition of protein metabolism and DNA synthesis. In conclusion, TP exhibits potent antibacterial properties by inhibiting biofilm formation, disrupting cell membrane integrity, and interfering with protein metabolism and DNA synthesis in drug-resistant A. veronii TH0426 in vitro.

Ligand modelling of Trachyspermum ammi phytocompounds for Aeromonas hydrophila cell wall synthesis enzyme in Labeo rohita.

Aquaculture faces challenges from Aeromonas hydrophila, causing Motile Aeromonas Septicaemia, particularly affecting Labeo rohita (Rohu) in Pakistan. This study explores potential herbal antibacterials targeting A. hydrophila, molecular docking of Trachyspermum ammi (ajwain) phytocompounds against pathogen. The cell wall synthesis ligase, D-alanine-D-alanine ligase (PDB ID 6ll9) was processed in BIOVIA Discovery Studio and docked with 13 antibacterial phytocompounds found after QSAR analysis of T. ammi. Binding energies were calculated using PyRx to assess complex stability. ADME-TOX assessment for selected phytocompounds and parameterisation in CHARMM-GUI were performed. Docking the two best ligands with highest binding energies and ADME-TOX compliance, we found carvacrol and limonene formed most stable protein-ligand complexes, with raw and processed protein. Our findings suggest these herbal compounds can inhibit D-alanine-D-alanine ligase. These in-silico results support the potential of 'ajwain' in managing A. hydrophila, further in-vivo experiments are necessary to validate these inhibitory properties.

Baicalin enhances antioxidant, inflammatory defense, and microbial diversity of yellow catfish (Pelteobagrus fulvidraco) infected with Aeromonas hydrophila.

Introduction: The aim of this research was to clarify the mechanism through which baicalin exerts its inhibitory effects on Aeromonas hydrophila infection. Methods: The antibacterial efficacy of baicalin was assessed by determining its minimum inhibitory concentration (MIC) against A. hydrophila. Various parameters, including the growth curve, cell wall integrity, biofilm formation, AKP content, and morphological alterations of A. hydrophila, were analyzed. In vivo experiments involved the administration of A. hydrophila 4 h postintraperitoneal injection of varying doses of baicalin to induce infection, with subsequent monitoring of mortality rates. After a 3 d period, liver, spleen, and intestinal tissues were harvested to evaluate organ indices, antioxidant and immune parameters, as well as intestinal microbial composition. Results: The findings indicated that baicalin treatment resulted in the disruption of the cell wall of A. hydrophila, leading to the loss of its normal structural integrity. Furthermore, baicalin significantly inhibited biofilm formation and facilitated the release of intracellular proteins (P < 0.05). In vivo, baicalin enhanced the survival rates of yellow catfish infected with A. hydrophila. Compared to the control group, the liver index of yellow catfish was elevated, while the spleen and intestinal indices were reduced in the baicalin-treated group (P < 0.05). Additionally, baicalin at an appropriate dosage was found to increase levels of SOD, GSH, CAT, ACP, and AKP in yellow catfish (P < 0.05), while simultaneously decreasing MDA accumulation and the mRNA expression of inflammatory markers such as Keap1, IL1, IFN-γ, and TNF-α, (P < 0.05). Moreover, baicalin significantly enhanced the operational taxonomic unit (OTU) count in A. hydrophila-infected yellow catfish (P < 0.05), restoring the abundance of Barnesiellaceae, Enterobacteriaceae, Plesiomonas, and UBA1819 (P < 0.05). Discussion: In summary, baicalin demonstrates the potential to improve the survival rate of yellow catfish subjected to A. hydrophila infection, augment antioxidant and immune responses, mitigate inflammation, and enhance intestinal microbial diversity.

Characterization and Genome Analyses of the Novel Phages P2 and vB_AhydM-H1 Targeting Aeromonas hydrophila.

Background: The emergence of antibiotic-resistant Aeromonas hydrophila strains presents a global health and aquaculture challenge. Bacteriophages offer promise as an alternative to antibiotics for treating drug-resistant Aeromonas infections. Methods: Two new phages, P2 and vB_AhydM-H1, targeting pathogenic A. hydrophila were isolated from sewage water. Their morphology, growth characteristics, lytic activity, stability, and genomes were analyzed. Results: Phage P2, a member of genus Ahphunavirus, and vB_AhydM-H1, a novel member of genus Pahsextavirus, exhibited narrow host ranges, extended latent periods, and typical burst sizes. Both phages remained stable at 40°C for 1 h and within a pH range of 4 to 10 for 3 h. The genomes of P2 and vB_AhydM-H1 spanned 42,660 bp with 49 open reading frames (ORFs) and 52,614 bp with 72 ORFs, respectively. Proteomic (ViPTree) and phylogenetic (VICTOR) analyses confirmed that both phages aligned with their respective families. DeepTMHMM predictions suggested that P2 and vB_AhydM-H1 encode three and four ORFs with transmembrane domains, respectively. Conclusions: Safe for environmental and clinical use because of their lytic nature, and lack of virulence and resistance genes, these newly isolated phages expand the arsenal against antibiotic-resistant Aeromonas infections.

Epidemiology and clinical characteristics of Aeromonas-associated gastroenteritis in Northern Israel: Insights and implications for public health.

The role of Aeromonas spp. in gastroenteritis is controversial due to varied clinical presentations and variable prevalence in asymptomatic. This study, conducted in Northern Israel, aimed to compare positivity rate and demographic characteristics of patients with Aeromonas-associated gastroenteritis (AAG) to asymptomatic, and examine the role of Aeromonas in AAG by comparing clinical and epidemiological characteristics between AAG and Campylobacter-associated gastroenteritis (CAG) patients. Results showed that 4.24%-4.81% of AAG patients had Aeromonas spp. in stools as a sole pathogen in 2020-2022, compared to 4.9% of asymptomatic. Analysis of 243 CAG patients versus 70 AAG patients revealed significantly less diarrhea, fever, abdominal pain, and muscle pain in AAG patients. Multivariate analysis identified higher Ct values, recent restaurant dining, and prolonged diarrhea as predictive factors for AAG versus CAG. In conclusion, similar positivity rates of Aeromonas spp. in symptomatic and asymptomatic making the distinction between true pathogen versus commensal bacteria difficult, unlike CAG.

Two homologous genes encoding interleukin (IL)-34 in the common carp (Cyprinus carpio L.): roles in inflammatory modulation and anti-bacterial defense.

In mammals, interleukin 34 (IL-34) is a ligand for macrophage colony-stimulating factor receptor (M-CSFR), promoting inflammatory responses and inducing the synthesis and secretion of various cytokines. However, studies on its function in lower vertebrates is limited, and its evolutionary relationship with homologous molecules in mammals remains unclear. In this study, two IL-34-encoding genes were cloned and identified in common carp (Cyprinus carpio L.), designated as CcIL-34A and CcIL-34B, with an amino acid sequence similarity of 77.7%. Gene synteny analysis revealed that the IL-34 gene loci are relatively conserved, and both are located downstream of SF3B3. The expression patterns of CcIL-34s were analyzed using qRT-PCR, and this showed that they are expressed across all tested tissues, with higher levels in the liver, spleen, and head kidney and lower levels in the gills and intestines. Following infection with Aeromonas hydrophila, the mRNA expression levels of CcIL-34s in the gills, head kidney, intestines, and spleen were significantly upregulated. Immunofluorescence was also employed to assess changes in CcIL-34 protein expression, showing a significant increase in carp spleens 24 hours after A. hydrophila infection, suggesting that CcIL-34s contribute to host defense against this bacterium. To investigate the immunological function of IL-34 in vivo, pc-CcIL-34A and pc-CcIL-34B eukaryotic expression plasmids were constructed and injected intramuscularly into fish. Five days after injection, the expression levels of inflammation-related cytokines in the head kidney and spleen were significantly altered. Furthermore, 24 h post-A. hydrophila infection, the bacterial loads in the liver, spleen, and kidneys were significantly reduced. Ten days post-infection, the survival rates in the groups with CcIL-34A and CcIL-34B overexpression were 40% and 36.7%, respectively, compared to 16.7% in the control group. These findings suggest that CcIL-34s are involved in modulating inflammatory responses, enhancing the immune response, and improving survival rates in fish following bacterial infection, thus supporting the potential use of IL-34 molecules in aquaculture.

Characterization of Aeromonas salmonicida mesophilic isolates from Alberta (Canada) allows the development of a more sensitive Dictyostelium discoideum predation test.

Aeromonas salmonicida is studied using Dictyostelium discoideum as a model host, with predation resistance measured as a key parameter. A. salmonicida mesophilic isolates exhibit inconclusive results with the amoebic model. This study focuses on new mesophilic isolates (S24-S38, S26-S10, S28-S20) from Alberta, Canada, and introduces an improved predation test method. Phylogenetic analysis reveals two subgroups, with S24-S38 and S26-S10 clustering with the subspecies pectinolytica from Argentina, and S28-S20 with strains from India (Y567) and Spain (AJ83), showcasing surprising mesophilic strain diversity across geographic locations. Predation tests were carried out with various mesophilic and psychrophilic strains of A. salmonicida including Alberta isolates. The amoeba cell lines used were DH1-10 and AX2. Although the mesophilic isolates were very resistant to predation by the amoeba DH1-10, some lost this resistance to the AX2 strain, which appeared more voracious in the conditions tested. In addition, when diluting the culture medium used in a predation test with AX2, a loss of the capacity to predation resistance was observed for all the mesophilic isolates, including the highly resistant S28-S20 isolate. This study provides insights into predation resistance of A. salmonicida isolates and offers avenues for better characterizing mesophilic isolates.

First report of a carbapenem-resistant Aeromonas veronii environmental isolate in the United States co-harboring two carbapenemase genes.

OBJECTIVES: The spread of carbapenem-resistant bacteria (CRB), and especially carbapenemase-producing CRB, is a global public health threat. Among them, Aeromonas species are of increasing concern because these emerging opportunistic pathogens are widespread in the environment and have increasingly been found to be resistant to carbapenems. The aim of this study was to investigate the genome and carbapenem-resistance determinants of Aeromonas veronii SS-M2-3, a highly carbapenem-resistant, carbapenemase-producing, river isolate from California (U.S.). METHODS: We first used disk diffusion assays to characterize the susceptibility profile to carbapenems and other antibiotics of A. veronii SS-M2-3. We next used whole-genome sequencing using the Illumina platform and bioinformatics analysis to characterize the resistome of this isolate and identify its carbapenemase genes. RESULTS: A. veronii SS-M2-3 was resistant to all carbapenems tested and amoxicillin-clavulanic acid, whereas it was sensitive to cefotaxime and all non-ß-lactam antibiotics tested. Whole genome sequencing of this isolate revealed a complex resistome that included multidrug efflux pump genes and three chromosomal ß-lactamase genes. These three genes encoded for highly conserved variants (82% to 97% amino acid identity) of the ChpA3 subclass B2 metallo-carbapenemase, OXA-12 class D carbapenemase and the FOX-2 class C ß-lactamase. This is the first report of an environmental A. veronni isolate from the U.S. co-harbouring two carbapenemase genes. CONCLUSIONS: These findings reveal that natural aquatic environments in the U.S. represent an underappreciated reservoir of carbapenem-resistant Aeromonas veronii isolates that can carry multiple carbapenemase genes.

Aeromonas veronii-associated bacteremia in the course of treatment of acute myeloid leukemia: a case report and review of the literature.

PURPOSE: This study aimed to report a case of acute myeloid leukemia (AML) complicated by Aeromonas veronii infection-induced bacteremia and to review relevant literature on the etiology, prevention, treatment, and prognosis of bacteremia in immunocompromised populations, aiming to reduce mortality in individuals with hematologic and other end-stage diseases and improve patient outcomes. METHODS AND RESULTS: We reported the case of a 23-year-old male patient with relapsed AML characterized by AML1:ETO and ASXL positivity, classified as a high-risk group. The patient presented with fever, abdominal pain, diarrhea, nausea, and vomiting after consuming partially cooked fish. The patient was admitted with high leucocytes, C-reactive protein, procalcitonin, and interleukin-6 levels. Peripheral blood high-throughput sequencing (Next-Generation Sequencing NGS) confirmed infection with Aeromonas veronii, while an abdominal CT scan indicated a liver abscess with gas formation. Culture of the drainage fluid from the ultrasound-guided liver abscess puncture demonstrated growth of Aeromonas veronii. Based on the sensitivity results, the patient was treated with intravenous ciprofloxacin and cefoperazone-sulbactam. After treatment with antibiotics, blood transfusion, liver protection, and azacitidine 100 mg ih, combined with dry white sand(interferon alpha-1B, interleukin-2, and thalidomide), the critical condition of the patient improved, and he was discharged. This study was approved by the Ethics Committee of Medical Research in the Second Affiliated Hospital of Henan University of Science and Technology. Informed consent was obtained from this patient and we have obscured the patient's identifying information. All methods were carried out in accordance with relevant guidelines and regulations. CONCLUSION: When patients with a recurrence of AML have a history of consuming or contacting aquatic products, clinicians should be vigilant about Aeromonas veronii infection. The presence of Aeromonas veronii in peripheral blood must alert clinicians to the possibility of severe sepsis and septic shock. Early diagnosis and prompt treatment are crucial to reducing patient mortality.

Immunomodulation and Humoral Immune Response in Teleost Immunized with Aeromonas-Derived Antigenic Extracellular Bioactive Molecules.

The common use of antimicrobials in food-animal production can lead to drug residues in edible tissues for consumers. However, immunomodulators enhance immune responses and vaccine effectiveness. A new perspective explores bacterial extracellular bioactive molecules (EBMs) in food-animal production to modulate host immune responses, potentially transforming pathogen management and antimicrobial use. This study investigates the immunogenic potential of Aeromonas hydrophila-derived EBMs (Antigens) to enhance the immune system. Four Antigens were administered intraperitoneally to Oreochromis niloticus (Nile Tilapia). Antigens 2 and Antigens 3 boosted fish immune competence within 21 days. Remarkably, Antigens 3 induced robust immunity against A. hydrophila with a single dose, notably enhancing antibody-based immune responses. The increased antibody activity suggests Antigens 3 could be a vaccine candidate, promising further research and potential application in food-animal production to improve disease control. This study highlights immunomodulators' potential in reshaping disease management in the food-animal industry, emphasizing the benefits of focusing on bacterial EBMs to reduce reliance on antimicrobials and achieve sustainable disease prevention.

Proteomic insights into extracellular matrix dynamics in the intestine of Labeo rohita during Aeromonas hydrophila infection.

In the aquaculture sector, one of the challenges includes disease outbreaks such as bacterial infections, particularly from Aeromonas hydrophila (Ah), impacting both wild and farmed fish. In this study, we conducted a proteomic analysis of the intestinal tissue in Labeo rohita following Ah infection to elucidate the protein alterations and its implications for immune response. Our findings indicate significant dysregulation in extracellular matrix (ECM)-associated proteins during Ah infection, with increased abundance of elastin and collagen alpha-3(VI). Pathway and enrichment analysis of differentially expressed proteins highlights the involvement of ECM-related pathways, including focal adhesions, integrin cell surface interactions, and actin cytoskeleton organization. Focal adhesions, crucial for connecting intracellular actin bundles to the ECM, play a pivotal role in immune response during infections. Increased abundance of integrin alpha 1, integrin beta 1, and tetraspanin suggests their involvement in the host's response to Ah infection. Proteins associated with actin cytoskeleton reorganization, such as myosin, tropomyosin, and phosphoglucomutase, exhibit increased abundance, influencing changes in cell behavior. Additionally, upregulated proteins like LTBP1 and fibrillin-2 contribute to TGF-ß signaling and focal adhesion, indicating their potential role in immune regulation. The study also identifies elevated levels of laminin, galectin 3, and tenascin-C, which interact with integrins and other ECM components, potentially influencing immune cell migration and function. These proteins, along with decorin and lumican, may act as immunomodulators, coordinating pro- and anti-inflammatory responses. ECM fragments released during pathogen invasion could serve as "danger signals," initiating pathogen clearance and tissue repair through Toll-like receptor signaling. IMPORTANCE: The study underscores the critical role of the extracellular matrix (ECM) and its associated proteins in the immune response of aquatic organisms during bacterial infections like Aeromonas hydrophila. Understanding the intricate interplay between ECM alterations and immune response pathways provides crucial insights for developing effective disease control strategies in aquaculture. By identifying key proteins and pathways involved in host defense mechanisms, this research lays the groundwork for targeted interventions to mitigate the impact of bacterial infections on fish health and aquaculture production.

Impact of Chaetomorpha aerea-enriched diet on growth, feed utilization, and haemato-immunological responses in Clarias batrachus challenged with Aeromonas hydrophila.

The bacteria Aeromonas hydrophila, which causes motile Aeromonas septicemia (MAS), is dangerous to aquaculture because it affects the fish's well-being and production. As the aquaculture industry seeks sustainable and effective methods to enhance fish immunity and growth, natural supplements such as marine algae have gained attention. This study explored the potential benefits of incorporating the green marine algae Chaetomorpha aerea into the fish diet, focusing on disease resistance, growth, feed utilization, and hematological and immunological responses. Five diets were prepared, varying concentrations of C. aerea (0 control, T1: 1 g/kg; T2: 2 g/kg: T3: 5 g/kg: and T4: 10 g/kg) and administered to fish over 30 days. Following the feeding trial, the fish were exposed to A. hydrophila, and their survival rates were observed for the next 14 days. The findings demonstrated that the final weight, weight gain, relative growth rate, specific growth rate, and daily growth rate were all positively impacted by a diet containing 5 g/kg of C. aerea. Additionally, fish in the 5 g/kg C. aerea group demonstrated improved feed conversion efficiency compared to the control group. While there were no significant changes in red and white blood cell counts on the initial day, serum lysozyme activity and overall resistance to infection were enhanced in fish receiving C. aerea at 2 and 5 g/kg. These results imply that C. aerea supplementation with fish supplements may be a useful immunostimulant, boosting improved health and growth in sustainable aquaculture practices.

Immune response for acute Aeromonas hydrophila infection in two distinct color morphs of northern snakehead, Channa argus.

To compare and analyze the differences in immunological response between the two color morphs of Channa argus, a fish cohort was divided into four groups: black C argus + PBS (B-PBS), black C argus + Aeromonas hydrophila (B-Ah), white C. argus + PBS (W-PBS), and white C. argus + A hydrophila (W-Ah). The B-PBS and W-PBS groups received 100 µL PBS, while the B-Ah and W-Ah groups received 3.6 × 105 CFU/mL A. hydrophila in the same volume. The death rate in each group was noted, changes in plasma biochemical indicators and the expression of liver immune-related genes were examined, and transcriptome techniques were used to compare the differences between the two colors of C. argus following stress. No mortality occurred in the B-PBS and W-PBS groups. Mortality in the W-Ah and B-Ah groups showed an upward and then downward trend after A. hydrophila injection. The highest mortality occurred within 24 h and was higher in the W-Ah group than in the B-Ah group. MDA levels in the B-Ah and W-Ah groups increased and then decreased, while SOD and T-AOC showed the reverse tendency. The W-Ah and W-PBS groups differed significantly in MDA at 3, 12, and 24 h, SOD from 6 to 96 h, and T-AOC between 6 and 48 h. Plasma MDA and T-AOC levels at 12 h and SOD levels at 24 and 48 h differed significantly between the B-PBS and B-Ah groups. In both the W-Ah and B-Ah groups, the expression levels of IL-1ß and IL-8 in the liver showed a temporal pattern with an initial increase followed by a decrease, reaching peak levels after 24 h, while IL-10 showed the reverse pattern. Transcriptome analysis of the liver revealed significant differences between the two C. argus colors. Differential genes in black C. argus were mainly enriched in steroid biosynthesis, glycolysis/gluconeogenesis, and glutathione and propanoate metabolism pathways 24 h after infection. In contrast, differential genes in white C. argus were mainly enriched in pathways such as oxidative phosphorylation, pancreatic secretion, and protein digestion and absorption 24 h after infection. After A. hydrophila infection, white C. argus had higher mortality, more severe oxidative stress and inflammatory responses, and lower antioxidant capacity than black C. argus.

Evaluation of virulence of Aeromonas veronii strain GZ21-2 and development of a highly effective vaccine for grass carp with the potential for industrial application.

Bacterial septicemia represents a significant disease affecting cultured grass carp culture, with the primary etiological agent identified as the Gram-negative bacterium Aeromonas veronii. In response to an outbreak of septicemia in Guangzhou, we developed a formaldehyde-inactivated vaccine against an A. veronii strain designated AV-GZ21-2. This strain exhibited high pathogenicity in experimental infections across at all developmental stages of grass carp. Mortality rates for grass carp weighing 15 ± 5 g ranged from 16 % to 92 % at exposure temperatures of 19 °C-34 °C, respectively. The median lethal dose (LD50) for grass carp groups weighing 15 ± 5 g, 60 ± 10 g, 150 ± 30 g and 500 ± 50 g were determined to be 1.43, 2.52, 4.65 and 7.12 × 107(CFU/mL), respectively. We investigated the inactivated vaccine in conbination with aluminum hydroxide gel (AV-AHG), Montanide ISA201VG (AV-201VG), and white oil (AV-WO) adjuvants. This study aimed to optimize inactivation conditions and identify the adjuvant that elicits the most robust immune response. The AV-GZ21-2 inactivated bacterial solution (AV),when combined with various adjuvants, was capable of inducing a strong specific immune response in grass carp. The relative percent survival (RPS) following a lethal challenge with AV-GZ21-2 were 94 % for AV-AHG, 88 % for AV-201VG, 84 % for AV-WO and 78 % for AV alone. The minimum immunization dose of the AV-AHG vaccine was determined to be 6.0 × 107 CFU per fish, providing immunity for a duration of six months with an immune protection level exceeding 75 %. Furthermore, the AV-AHG vaccine demonstrated significant protective efficacy against various epidemic isolates of A. veronii. Consequently, we developed an inactivated vaccine targeting a highly pathogenic strain of A. veronii, incorporating an aluminum hydroxide gel adjuvant, which resulted in high immune protection and a duration of immunity exceeding six months. These findings suggest that the AV-AHG vaccine holds substantial potential for industrial application.

Mechanistic studies on bioremediation of dye using Aeromonas veronii immobilized peanut shell biochar.

Recalcitrant chemicals in the environment not only present obstacles to living organisms but also contribute to the degradation of natural resources. One contribution to environmental pollution is the discharge of synthetic dyes from the textile sector. This study investigates the combined effect of microbial cells and biochar on eliminating methyl orange (MO) dye. The immobilization of Aeromonas veronii on peanut shell biochar (APSB) was conducted to investigate its efficacy in removing MO dye from water. PSB synthesized by pyrolysis at 300 °C for 120 min showed maximum bacterial immobilization potential. The highest degradation rate of 96.19 % was achieved in APSB within 96 h using MO dye concentration of 100 mg L-1, incubation temperature of 37 °C, pH 7, and biocatalyst dosage of 1g L-1. In comparison, free cells achieved degradation rates of 72.53 % and 61.56 % for PSB. Moreover, the adsorption process was primarily controlled by PSB, with subsequent dye mineralization by A. veronii, as supported by FTIR and LC-MS studies. Moreover, this innovative approach exhibited the reusability of the biocatalyst, giving 76.23 % removal after fifth cycle, suggesting sustainable alternative in dye remediation and potential option for real-time applications.

Isolation and complete genome sequence of Aeromonas bacteriophage Gekk3-15.

Bacteria of the genus Aeromonas, especially A. hydrophila and A. veronii are recognized as important fish pathogens that cause significant economic losses in aquaculture. Environmentally friendly bacteriophage-based solutions for the treatment of fish and for the reduction of colonization by pathogenic bacteria in production facilities are currently in high demand. The bacteriophage Gekk3-15 was isolated during a search for novel phage strains potentially suitable for Aeromonas biocontrol applications. Genome sequencing revealed that this virus is a relatively small myovirus with a 64847 bp long dsDNA genome, which is consistent with virion electron microscopy data. Bacteriophage Gekk3-15 is distinct in its nucleotide and encoded aa sequences from all other sequenced bacteriophage genomes, and may represent a new viral taxon at the genus or subfamily level.

The role of aroA and ppk1 in Aeromonas veronii pathogenicity and the efficacy evaluation of mutant strain AV-ΔaroA/ppk1 as a live attenuated vaccine.

Aeromonas veronii is an opportunistic pathogen that poses great threat to aquaculture and human health, so there is an urgent need for green and efficient methods to deal with its infection. In this study, single and double gene deletion strains (AV-ΔaroA, AV-Δppk1 and AV-ΔaroA/ppk1) that can be stably inherited were constructed. Pathogenicity test showed that the toxicity of AV-ΔaroA and AV-ΔaroA/ppk1 was significantly lower compared to wild-type A. veronii. Biological characterization analysis revealed that the decrease in pathogenicity might be due to the declined growth, motility, biofilm formation abilities and the expression of virulence-related genes in mutants. Subsequently, we evaluated the efficacy of AV-ΔaroA/ppk1 as a live attenuated vaccine (LAV). Safety assessment experiments showed that AV-ΔaroA/ppk1 injected at a concentration of 3 × 107 CFU/mL was safe for C. carassius. The relative percentage survival of AV-ΔaroA/ppk1 was 67.85 %, significantly higher than that of the inactivated A. veronii, which had an RPS of 54.84 %. This improved protective effect was mainly attributed to the increased levels of A. veronii specific IgM antibody, enhanced alkaline phosphatase, lysozyme and superoxide dismutase activities, as well as higher expression levels of several immune related genes. Together, these findings deepen our understanding of the functional roles of aroA and ppk1 in A. veronii pathogenicity, provide a good candidate of LAV for A. veronii.

Isolating pathogenic multidrug-resistant Aeromonas hydrophila from diseased fish and assessing the effectiveness of a novel lytic Aeromonas veronii bacteriophage (AVP3) for biocontrol.

The increasing trend of antimicrobial resistance (AMR) pathogens in aquaculture makes it is imperative to find control measures for AMR pathogens causing high economic losses in aquaculture. In the present study, a multidrug resistance (MDR) Aeromonas hydrophila bacterium was isolated from kidney samples of diseased carp originating from a fish farm in Awankot, Rupnagar, Punjab, India. Moribund-infected fish exhibited large irregular hemorrhages on the external body surfaces, exophthalmia and fin-rot-like lesions. Phenotypic characterization using Rimler-Shotts (RS) media showed characteristic yellow color colonies and beta hemolysis on sheep blood agar. Genotyping using species-specific primers for the rpoB and gyrB genes characterized the isolate as A. hydrophila. The Multiple Antibiotic Resistance (MAR) index analysis showed that the isolated A. hydrophila had an MAR score of 0.29 signifying its resistance to more than three antibiotics, which underscores the need of finding treatment methods for MDR A. hydrophila isolates causing disease in aquaculture. Bacteriophages are considered a better eco-friendly alternative to antibiotics because of their inherent properties of not causing drug residues and resistance. Of the 13 phages tested, the Aeromonas veronii phage designated as AVP3, initially isolated against Aeromonas veronii, showed lytic activity against the MDR A. hydrophila isolated from diseased carp in this study. In addition, it also showed the lytic activity against Aeromonas spp. And A. caviae indicating that it had lytic properties against a wide host range within the Aeromonas species. This finding points to the potential efficacy of bacteriophages in mitigating pathogenic infections in aquaculture.

Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco).

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequences alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. In summary, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.

In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks.

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB, and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.