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

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

Isolation, characterization and functional analysis of a bacteriophage targeting Culex pipiens pallens resistance-associated Aeromonas hydrophila.

BACKGROUND: Culex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance. METHODS: Phages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays. RESULTS: An Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut. CONCLUSIONS: Our findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance.

Metagenomic and transcriptomic analysis revealing the impact of oxytetracycline and ciprofloxacin on gut microbiota and gene expression in the Chinese giant salamander (Andrias davidianus).

Excessive antibiotic use has led to the spread of antibiotic resistance genes (ARGs), impacting gut microbiota and host health. However, the effects of antibiotics on amphibian populations remain unclear. We investigated the impact of oxytetracycline (OTC) and ciprofloxacin (CIP) on Chinese giant salamanders (Andrias davidianus), focusing on gut microbiota, ARGs, and gene expression by performing metagenome and transcriptome sequencing. A. davidianus were given OTC (20 or 40 mg/kg) or CIP (50 or 100 mg/kg) orally for 7 days. The results revealed that oral administration of OTC and CIP led to distinct changes in microbial composition and functional potential, with CIP treatment having a greater impact than OTC. Antibiotic treatment also influenced the abundance of ARGs, with an increase in fluoroquinolone and multi-drug resistance genes observed post-treatment. The construction of metagenome-assembled genomes (MAGs) accurately validated that CIP intervention enriched fish-associated potential pathogens Aeromonas hydrophila carrying an increased number of ARGs. Additionally, mobile genetic elements (MGEs), such as phages and plasmids, were implicated in the dissemination of ARGs. Transcriptomic analysis of the gut revealed significant alterations in gene expression, particularly in immune-related pathways, with differential effects observed between OTC and CIP treatments. Integration of metagenomic and transcriptomic data highlighted potential correlations between gut gene expression and microbial composition, suggesting complex interactions between the host gut and its gut microbiota in response to antibiotic exposure. These findings underscore the importance of understanding the impact of antibiotic intervention on the gut microbiome and host health in amphibians, particularly in the context of antibiotic resistance and immune function.

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

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

Immunomodulatory, antioxidant, and growth-promoting activities of dietary fermented Moringa oleifera in Nile tilapia (Oreochromus niloticus) with in-vivo protection against Aeromonas hydrophila.

BACKGROUND: Moringa oleifera, a well-known medicinal plant, has been used in aquafeed as a dietary supplement. Based on previous studies, insufficient research is available on the dietary supplementation of Nile tilapia with M. oleifera leaf and seed mixtures, specifically the fermented form. Therefore, this study aimed to investigate the efficacy of fermented (FMO) versus non-fermented M. oleifera (MO) leaf and seed mixtures on immunological parameters, antioxidant activity, growth performance, and resistance to A. hydrophila infection after a 30-day feeding trial on Nile tilapia. METHODS: A total of 180 fingerlings were randomly divided into four groups in addition to the control group (36 fish each, in triplicate). Fish in the tested groups were fed on basal diet supplemented with MO5%, MO10%, FMO5%, and FMO10%, while those in control were fed on basal diet only. After the feeding trial, fish were challenged with A. hydrophila. The immunomodulatory activity of M. oleifera was evaluated in terms of phagocytic and lysozyme activities, immune-related cytokines and IgM gene expression. Antioxidants, and growth-promoting activities were also assessed. RESULTS: The results revealed that fish supplemented FMO markedly in FMO10% group followed by FMO5%, exhibited significant (P < 0.05) improvement in the tested immunological, hepatic antioxidants, and growth performance parameters. Furthermore, the highest survival rate post-challenge with mild clinical symptoms, and the lowest A. hydrophila bacterial count were reported in these groups. Meanwhile, MO10%-supplementation exhibited the opposite trend. CONCLUSIONS: The study' conclusion suggests that fermented M. oleifera leaf and seed mixture is a promising growth-promoting and immunostimulatory feed-additive candidate for Nile tilapia and could reduce the losses caused by A. hydrophila infection.

Natural Sunlight-Mediated Emodin Photoinactivation of Aeromonas hydrophila.

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

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

Genomic and Transcriptomic Diversification of Flagellin Genes Provides Insight into Environmental Adaptation and Phylogeographic Characteristics in Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic motile pathogen with a broad host range, infecting both terrestrial and aquatic animals. Environmental and geographical conditions exert selective pressure on both geno- and phenotypes of pathogens. Flagellin, directly exposed to external environments and containing important immunogenic epitopes, may display significant variability in response to external conditions. In this study, we conducted a comparative analysis of ~ 150 A. hydrophila genomes, leading to the identification of six subunits of the flagellin gene (fla-1 to fla-4, flaA, and flaB). Individual strains harbored different composition of flagellin subunits and copies. The composition of subunits showed distinct patterns depending on environmental sources. Strains from aquatic environments were mainly comprised of fla-1 to fla-4 subunits, while terrestrial strains predominated in groups harboring flaA and flaB subunits. Each flagellin showed varying levels of expression, with flaA and flaB demonstrating significantly higher expression compared to others. One of the chemotaxis pathways that control flagellin movement through a two-component system was significantly upregulated in flaA(+ 1)/flaB(+ 1) group, whereas flaA and flaB showed different transcriptomic expressions. The genes positively correlated with flaA expression were relevant to biofilm formation and bacterial chemotaxis, but flaB showed a negative correlation with the genes in ABC transporters and quorum sensing pathway. However, the expression patterns of fla-2 to fla-4 were identical. This suggests various types of flagellin subunits may have different biological functions. The composition and expression levels of flagellin subunits could provide valuable insights into the adaptation of A. hydrophila and the differences among strains in response to various external environments.

Metabolomics and microbiome co-analysis reveals altered innate immune responses in Charybdis japonica following Aeromonas hydrophila infection.

A comprehensive bioinformatics analysis was conducted to elucidate the innate immune response of Charybdis japonica following exposure to Aeromonas hydrophila. This study integrated metabolomics, 16S rRNA sequencing, and enzymatic activity data to dissect the immune mechanisms activated in response to infection. Infection with A. hydrophila resulted in an increased abundance of beneficial intestinal genera such as Photobacterium spp., Rhodobacter spp., Polaribacter spp., Psychrilyobacter spp., and Mesoflavibacter spp. These probiotics appear to suppress A. hydrophila colonization by competitively dominating the intestinal microbiota. Key metabolic pathways affected included fatty acid biosynthesis, galactose metabolism, and nitrogen metabolism, highlighting their role in the crab's intestinal response. Enzymatic analysis revealed a decrease in activities of hexokinase, phosphofructokinase, and pyruvate kinase, which are essential for energy homeostasis and ATP production necessary for stress responses. Additionally, reductions were observed in the activities of acetyl-CoA carboxylase and fatty acid synthase. Gene expression analysis showed downregulation in Peroxiredoxin 1 (PRDX1), Peroxiredoxin 2 (PRDX2), glutathione-S-transferase (GST), catalase (CAT), and glutathione (GSH), with concurrent increases in malondialdehyde (MDA) levels, indicating severe oxidative stress. This study provides insights into the molecular strategies employed by marine crabs to counteract bacterial invasions in their natural habitat.

Integrated time-series biochemical, transcriptomic, and metabolomic analyses reveal key metabolites and signaling pathways in the liver of the Chinese soft-shelled turtle (Pelodiscus sinensis) against Aeromonas hydrophila infection.

Introduction: Aeromonas hydrophila, a bacterium widely distributed in the natural environment, causes multiple diseases in various animals. Exploring the mechanism of the host defense against A. hydrophila can help develop efficient strategies against Aeromonas infection. Methods: Herein, we investigated the temporal influence of A. hydrophila on the Chinese soft-shelled turtle, an economically important species, at the biochemical, transcriptomic, and metabolomic levels. Plasma parameters were detected with the test kits. Transcriptome and metabolome were respectively applied to screen the differentially expressed genes and metabolites. Results: The contents or activities of these plasma parameters were significantly increased at 24 hpi and declined at 96 hpi, indicating that 24 and 96 hpi were two important time points during infection. Totals of 3121 and 274 differentially expressed genes (DEGs) from the transcriptome while 74 and 91 differentially abundant metabolites (DAMs) from the metabolome were detected at 24 and 96 hpi. The top DEGs at 24 hpi included Ccl2, Ccl3, Ccl4, Il1ß, Il6, Il7, Il15, Tnf, and Tnfr1 while Zap70, Cd3g, Cd8a, Itk, Pik3r3, Cd247, Malt1, and Cd4 were the most abundant at 96 hpi. The predominant DAMs included O-phospho-L-serine, γ-Aminobutyric acid, orotate, L-tyrosine, and L-tryptophan at 24 hpi, as well as L-glutamic acid, L-arginine, glutathione, glutathione disulfide, and citric acid at 96 hpi. Discussion: The combined analysis of DEGs and DAMs revealed that tryptophan metabolism, nicotinate and nicotinamide metabolism, as well as starch and sucrose metabolism, were the most important signaling pathways at the early infective stage while tyrosine metabolism, pyrimidine metabolism, as well as alanine, aspartate and glutamate metabolism were the most crucial pathways at the later stage. In general, our results indicated that the Chinese soft-shelled turtle displays stage-specific physiological responses to resist A. hydrophila infection.

Quantitative proteomics reveals the complex regulatory networks of LTTR-type regulators in pleiotropic functions of Aeromonas hydrophila.

LysR-type transcriptional regulators (LTTRs) are ubiquitously distributed and abundant transcriptional regulators in prokaryotes, playing pivotal roles in diverse physiological processes. Nonetheless, despite their prevalence, the intricate functionalities and physiological implications of this protein family remain incompletely elucidated. In this study, we employed a comprehensive approach to deepen our understanding of LTTRs by generating a collection of 20 LTTR gene-deletion strains in Aeromonas hydrophila, accounting for 42.6 % of the predicted total LTTR repertoire, and subjected them to meticulous assessment of their physiological phenotypes. Leveraging quantitative proteomics, we conducted a comparative analysis of protein expression variations between six representative mutants and the wild-type strain. Subsequent bioinformatics analysis unveiled the involvement of these LTTRs in modulating a wide array of biological processes, notably including two-component regulatory systems (TCSs) and intracellular central metabolism. Moreover, employing subsequent microbiological methodologies, we experimentally verified the direct involvement of at least six LTTRs in the regulation of galactose metabolism. Importantly, through ELISA and competitive ELISA assays, we demonstrated the competitive binding capabilities of these LTTRs with the promoter of the α-galactosidase gene AHA_1897 and identified that four LTTRs (XapR, YidZ, YeeY, and AHA_1805) do not engage in competitive binding with other LTTRs. Overall, our comprehensive findings not only provide fundamental insights into the regulatory mechanisms governing crucial physiological functions of bacteria through LTTR family proteins but also uncover an intricate and interactive regulatory network mediated by LTTRs.

Redox profile of silver catfish challenged with Aeromonas hydrophila and treated with hexane extract of Hesperozygis ringens (Benth.) Epling through immersion bath.

The growing increase in the fish farming sector has favored the establishment of bacterial outbreaks caused by Aeromonas hydrophila in several species. The hexane extract of Hesperozygis ringens (HEHR) (Lamiaceae) leaves increased the survival rate of silver catfish (Rhamdia quelen) experimentally infected by A. hydrophila. However, it is noteworthy that no reports have been found on the possible mechanisms of action of this extract in infected fish. This study aimed to evaluate the effect of the HEHR, administered through single immersion bath, on lipid peroxidation and antioxidant defenses in muscle and liver tissue of silver catfish challenged with A. hydrophila. The results showed that the oxidative status of silver catfish was altered, although oxidative stress was not triggered during the experiment. HEHR at 30 mg/L (HEHR30) was not characterized as a pro-oxidant agent in the presence of infection, unlike florfenicol and HEHR at 15 mg/L treatments in some cases. In short, HEHR30 provided an important increase in hepatic catalase activity, characterizing one of the possible mechanisms involved in the greater survival of fish experimentally infected by A. hydrophila. Additionally, HEHR30 did not induce lipid peroxidation, nor reduced antioxidant defenses of silver catfish infected or not by A. hydrophila.

Aeromonas hydrophila induced necrotizing fasciitis following laparoscopic cholecystectomy.

Necrotizi ng fasciit is [NF] i s a m ultifaceted disease of the muscle fascia and body tissues which demands the earliest intervention. Past reviews have documented ver y few cases of Aeromonas Hydroph ila [AH] induced N F fol lo wing abdominal surgery. AH can cause fatal NF as seen in a 72 year old female patient reported at Liaquat National Hospital &Medical College; a ter tiary care center in Karachi, Pakistan on 2nd April, 2022. She had a k nown comorbidity of hypertension and presented with the chief complaint of symptomatic gallstones for which she unde rwent Laparoscopic Cholecystectomy (LAPCHOL). She developed NF of the lower ab domen post- oper atively. Following uneventful Laparoscopic Cholecystectomy our pati ent presented to the ER two days later with severe lower abdo minal pain and overlyi ng celluliti s. Fasc io to my revealed extensive myonecrosis with necrotizing soft tissue in fe ction. Despite u ndergoing extensive surgical debr idement and broad spectr um antibi ot ic administration; the patient died in the ICU on the fifth postoperat ive day followi ng septic complications. Histopathologica l an alysis, confirmed i nflammat ion and necrosis. Culture sensitivity of the debrided tissue revealed AH. Approach should lie towards analyzing the behaviour of such microbes in high risk patients through collective case studies. This is the first clinical case showcasing such parameters e ncountered in the General Surger y Department.

Quantitative proteomics analysis reveals an important role of the transcriptional regulator UidR in the bacterial biofilm formation of Aeromonas hydrophila.

Introduction: Bacterial biofilm is a well-known characteristic that plays important roles in diverse physiological functions, whereas the current intrinsic regulatory mechanism of its formation is still largely unknown. Methods: In the present study, a label-free based quantitative proteomics technology was conducted to compare the differentially expressed proteins (DEPs) between ΔuidR and the wild-type strain in the biofilm state. Results: The results showed that the deletion of gene uidR encoding a TetR transcriptional regulator significantly increased the biofilm formation in Aeromonas hydrophila. And there was a total of 220 DEPs, including 120 up-regulated proteins and 100 down-regulated proteins between ΔuidR and the wild-type strain based on the quantitative proteomics. Bioinformatics analysis suggested that uidR may affect bacterial biofilm formation by regulating some related proteins in glyoxylic acid and dicarboxylic acid pathway. The expressions of selected proteins involved in this pathway were further confirmed by q-PCR assay, and the results was in accordance with the quantitative proteomics data. Moreover, the deletion of four genes (AHA_3063, AHA_3062, AHA_4140 and aceB) related to the glyoxylic acid and dicarboxylic acid pathway lead to a significant decrease in the biofilm formation. Discussion: Thus, the results indicated that uidR involved in the regulatory of bacterial biofilm formation, and it may provide a potential target for the drug development and a new clue for the prevention of pathogenic A. hydrophila in the future.

Impact of Sel-Plex® dietary supplementation on growth performance, physiological response, oxidative status, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings challenged with Aeromonas hydrophila.

Background: Organic selenium (Sel-Plex®) supplementation holds considerable promise for improving the effectiveness of fish production. Aim: This experiment was accomplished to judge the potential benefits of Sel-Plex® nutritional additive on growth outcomes, physiological response, oxidative status, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings exposed to bacterial infection with Aeromonas hydrophila. Methods: Utilizing a basal diet of 30% protein, four experimental diets were prepared, each of which contained Sel-Plex® at concentrations of 0.0, 0.5, 1, and 2 mg/kg, respectively. Three replicates of 20 fish/treatment were used using 240 healthy Nile tilapia fingerlings. Fish were placed in 12 glass aquariums and separated into 4 groups at random. For the entire span of 8 weeks, diets were admitted to fish at a 3% rate of fish biomass/aquarium. After the feeding trial, pathogenic A. hydrophila was intraperitoneally injected into fish of each treatment, and fish were observed for 15 days to track the survival rate (SR) after the challenge. Results: Growth performance, physiological response, immunological parameters (phagocytic activity, phagocytic index, and lysozyme), and antioxidant parameters [catalase, superoxide dismutase (SOD), malondialdehyde, and glutathione peroxidase (GPx)] were noticeably improved in Sel-Plex® treated groups. Moreover, Sel-Plex® increased gene expression linked with the immune system in the liver (tumor necrosis factor-alpha and interleukin 1ß), to growth (insulin-like growth factor 1 and growth hormone receptor), and antioxidants (SOD and GPx). Under pathogen-challenge conditions, the employed dietary Sel-Plex® supplementation could successfully lower fish oxidative stress, offering a potential preventive additive for Nile tilapia instead of antibiotics. On the other hand, Sel-Plex® significantly enhanced each of three intestinal morphological measurements (villus width, villus length, and crypt depth), demonstrating the greatest influence on the improvement of intestinal structure overall. In the Nile tilapia control group, the infection with A. hydrophila caused noticeable degenerative alterations in the gut, hepatopancreas, spleen, and posterior kidney. The severity of the lesion was significantly reduced and significantly improved with higher Sel-Plex® concentrations. Sel-Plex® supplemented groups had 100% SRs among the A. hydrophila-challenged groups. Conclusion: It could be advised to enrich the diets of Nile tilapia fingerlings with 1-2 mg.kg-1 of Sel-Plex® to enhance growth rate, physiological response, immunological reaction, and intestinal absorptive capacity.

Ameliorative effect of BIO-MOS® as a dietary supplementation on growth performance, physiological response, oxidative status, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings challenged with Aeromonas hydrophila.

Background: Mannanoligosaccharides (MOS) usage in fish production has drawn more attention because of their positive benefits on disease resistance and fish performance. Aim: The ongoing research was executed to assess the potential advantages of Bio-Mos® dietary supplementation regarding the growth outcomes, physiological response, oxidative biomarkers, and immunity-linked gene expression in Nile tilapia (Oreochromis niloticus) fingerlings exposed to bacterial infection with Aeromonas hydrophila. Methods: Four experimental diets were developed using a 30% protein baseline diet, with Bio-Mos® added at variable levels; 0.0, 0.5, 1, and 2 g/kg, respectively. 240 healthy Nile tilapia fingerlings were split into 4 groups at random and assigned to 12 glass aquariums (three replicates of 20 fish/treatment). Diets were admitted at a 3% rate of fish biomass/aquarium for 8 weeks. Following the feeding trial, fish from every treatment were intraperitoneally injected with pathogenic A. hydrophila, and then observed for 15 days to record the survival rate percent (SR%) post challenge. Results: Results revealed significant improvement in growth performance, physiological response, immunological parameters (phagocytic index, phagocytic activity, and lysozyme), and antioxidant parameters [catalase, malondialdehyde, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD)] among Bio-Mos® treated groups. Moreover, Bio-Mos® increased the expression of tumor necrosis factor alpha and Interleukin 1ß, genes linked to the liver immune system. Growth-related genes (GHr), antioxidant-related genes (SOD and GSH-Px). In fish subjected to pathogens, dietary MOS supplementation could significantly lower oxidative stress, showing promise as a preventative supplement for Nile tilapia in place of antibiotics. On the other hand, Bio-Mos® considerably improved each of the three intestinal morphological measures (villus width, villus length, and crypt depth), showing the best overall intestinal structure-improving impact. The challenge with A. hydrophila caused marked degenerative alterations in the intestine, hepatopancreas, spleen, and posterior kidney of Nile tilapia, in the control group. However, lesion severity was greatly decreased and showed marked amelioration with an increased concentration of Bio-Mos®. The A. hydrophila-challenged groups revealed a 100% SR% mainly among the Bio-Mos® supplemented groups. Conclusion: It is recommended to enrich the Nile tilapia fingerlings diets with 2 g.kg-1 of MOS for better results on the growth rate, physiological response, immunological response, and intestinal absorptive capacity.

Distribution of bacteria and antimicrobial resistance in retail Nile tilapia (Oreochromis spp.) as potential sources of foodborne illness.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Biocontrol of multidrug resistant pathogens isolated from fish farms using silver nanoparticles combined with hydrogen peroxide insight to its modulatory effect.

This study was divided into two parts. The first part involved the isolation, and detection of the prevalence and antimicrobial resistance profile of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio species from Nile tilapia fish and marine aquatic water. One hundred freshly dead Nile tilapia fish were collected from freshwater aquaculture fish farms located in Al-Abbassah district, Sharkia Governorate, and 100 samples of marine aquatic water were collected from fish farms in Port Said. The second part of the study focused on determining the in vitro inhibitory effect of dual-combination of AgNPs-H2O2 on bacterial growth and its down regulatory effect on crucial virulence factors using RT-PCR. The highest levels of A. hydrophila and P. aeruginosa were detected in 43%, and 34% of Nile tilapia fish samples, respectively. Meanwhile, the highest level of Vibrio species was found in 37% of marine water samples. Additionally, most of the isolated A. hydrophila, P. aeruginosa and Vibrio species exhibited a multi-drug resistance profile. The MIC and MBC results indicated a bactericidal effect of AgNPs-H2O2. Furthermore, a transcriptional modulation effect of AgNPs-H2O2 on the virulence-associated genes resulted in a significant down-regulation of aerA, exoU, and trh genes in A. hydrophila, P. aeruginosa, and Vibrio spp., respectively. The findings of this study suggest the effectiveness of AgNPs-H2O2 against drug resistant pathogens related to aquaculture.

Harnessing black soldier fly (Hermetia illucens) prepupae against Aeromonas hydrophila: Fermentation-based fatty acids production and its bioinformatic assessment.

Background: Aeromonas hydrophila (A. hydrophila) is a bacterium with zoonotic potential and is multidrug-resistant. It utilizes hemolysin and aerolysin to spread infection. Black soldier flies (BSFs) can be antibacterial because of the fatty acids it contains. Aims: This study aimed to investigate and compare the fatty acid profiles of BSF prepupae grown in fermented and nonfermented media using bioinformatics tools and assess their potential as antibacterial agents against A. hydrophila. Methods: The study used BSF prepupae reared on various organic substrates. BSF prepupae grown in fermented or nonfermented substrate were observed against fatty acid. The fatty acid analysis was performed using GC-MS. Fatty acids were analyzed statistically using the one-way ANOVA test with a 95% confidence level. Fatty acid bioactivity was predicted using the online PASS-two-way drug program. Molecular docking on BSF fatty acid compounds was analyzed with PyMol 2.2 and discovery Studio version 21.1.1. Results: The molecular docking test showed the strongest bond was oleic acid with aerolysin and linoleic acid with hemolysin. BSF prepupae grown on fermented media showed higher crude fat and saturated fatty acids (SFAs) but lower unsaturated fatty acids than nonfermented media. Conclusion: Black soldier fly prepupae, particularly those grown on fermented media, possess antibacterial activity against A. hydrophila through potential fatty acid-mediated inhibition of crucial virulence factors.

One Health-based management for sustainably mitigating tetracycline-resistant Aeromonas hydrophila-induced health risk.

Aeromonas hydrophila has ability to spread tetracycline resistance (tetR) under stresses of oxytetracycline (OTC), one of the most important antibiotics in aquaculture industry. Even though environmental reservoir of Aeromonas allows it to be at interfaces across One Health components, a robust modelling framework for rigorously assessing health risks is currently lacking. We proposed a One Health-based approach and leveraged recent advances in quantitative microbial risk assessment appraised by available dataset to interpret interactions at the human-animal-environment interfaces in various exposure scenarios. The dose-response models were constructed considering the effects on mortality for aquaculture species and tetR genes transfer for humans. A scenario-specific risk assessment on pond species-associated A. hydrophila infection and human gut-associated tetR genes transfer was examined. Risk-based control strategies were involved to test their effectiveness. We showed that farmed shrimp exposed to tetracycline-resistant A. hydrophila in OTC-contaminated water experienced higher infection risk (relative risk: 1.25-1.34). The tetR genes transfer risk for farmers in shrimp ponds (∼2 × 10-4) and swimmers in coastal areas (∼4 × 10-6) during autumn exceeded acceptable risk (10-6). This cautionary finding underscores the importance of accounting for monitoring, assessing, and mitigating occupational health hazards among workers in shrimp farming sectors within future One Health-based strategies for managing water infection risks. We recommend that OTC emission rate together with A. hydrophila concentration should be reduced by up to 70-99% to protect human, farmed shrimp, and environmental health. Our predictive framework can be adopted for other systems and be used as a "risk detector" for assessing tetR-related health risks that invoke potential risk management on addressing sustainable mitigation on offsetting residual OTC emission and tetR genes spread in a species-human-environmental health system.