Quercetin disrupts biofilm formation and attenuates virulence of Aeromonas hydrophila.

Aeromonas hydrophila poses significant health and economic challenges in aquaculture owing to its pathogenicity and prevalence. Overuse of antibiotics has led to multidrug resistance and environmental pollution, necessitating alternative strategies. This study investigated the antibacterial and antibiofilm potentials of quercetin against A. hydrophila. Efficacy was assessed using various assays, including antibacterial activity, biofilm inhibition, specific growth time, hemolysis inhibition, autoaggregation, and microscopic evaluation. Additionally, docking analysis was performed to explore potential interactions between quercetin and virulence proteins of A. hydrophila, including proaerolysin, chaperone needle-subunit complex of the type III secretion system, and alpha-pore forming toxin (PDB ID: 1PRE, 2Q1K, 6GRK). Quercetin exhibited potent antibacterial activity with 21.1 ± 1.1 mm zone of inhibition at 1.5 mg mL-1. It also demonstrated significant antibiofilm activity, reducing biofilm formation by 46.3 ± 1.3% at the MIC and attenuating autoaggregation by 55.9 ± 1.5%. Hemolysis was inhibited by 41 ± 1.8%. Microscopic analysis revealed the disintegration of the A. hydrophila biofilm matrix. Docking studies indicated active hydrogen bond interactions between quercetin and the targeted virulence proteins with the binding energy -3.2, -5.6, and -5.1 kcal mol⁻1, respectively. These results suggest that quercetin is an excellent alternative to antibiotics for combating A. hydrophila infection in aquaculture. The multifaceted efficacy of quercetin in inhibiting bacterial growth, biofilm formation, virulence factors, and autoaggregation highlights the potential for aquaculture health and sustainability. Future research should delve into the precise mechanisms of action and explore synergistic combinations with other compounds for enhanced efficacy and targeted interventions.

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

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

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

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

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

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

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

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

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

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

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

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

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

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

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

Sequestered Alkaloid Defenses in the Dendrobatid Poison Frog Oophaga pumilio Provide Variable Protection from Microbial Pathogens.

Most amphibians produce their own defensive chemicals; however, poison frogs sequester their alkaloid-based defenses from dietary arthropods. Alkaloids function as a defense against predators, and certain types appear to inhibit microbial growth. Alkaloid defenses vary considerably among populations of poison frogs, reflecting geographic differences in availability of dietary arthropods. Consequently, environmentally driven differences in frog defenses may have significant implications regarding their protection against pathogens. While natural alkaloid mixtures in dendrobatid poison frogs have recently been shown to inhibit growth of non-pathogenic microbes, no studies have examined the effectiveness of alkaloids against microbes that infect these frogs. Herein, we examined how alkaloid defenses in the dendrobatid poison frog, Oophaga pumilio, affect growth of the known anuran pathogens Aeromonas hydrophila and Klebsiella pneumoniae. Frogs were collected from five locations throughout Costa Rica that are known to vary in their alkaloid profiles. Alkaloids were isolated from individual skins, and extracts were assayed against both pathogens. Microbe subcultures were inoculated with extracted alkaloids to create dose-response curves. Subsequent spectrophotometry and cell counting assays were used to assess growth inhibition. GC-MS was used to characterize and quantify alkaloids in frog extracts, and our results suggest that variation in alkaloid defenses lead to differences in inhibition of these pathogens. The present study provides the first evidence that alkaloid variation in a dendrobatid poison frog is associated with differences in inhibition of anuran pathogens, and offers further support that alkaloid defenses in poison frogs confer protection against both pathogens and predators.

Aeromonas hydrophila biofilm, exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces.

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.

Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila.

BACKGROUND: Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. RESULTS: We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 µM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 µg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 µM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 µM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 µM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. CONCLUSIONS: Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.

The Influences of Bacillus subtilis on the Virulence of Aeromonas hydrophila and Expression of luxS Gene of Both Bacteria Under Co-cultivation.

The aim of this study was to explore the influence of Bacillus subtilis CH9 on Aeromonas hydrophila SC2005. The transcription level of virulence genes of A. hydrophila SC2005 and its hemolysin activity as well as its cytotoxicity were analyzed when B. subtilis CH9 and A. hydrophila SC2005 were co-cultured. The results indicated that the transcription levels of four virulence genes of A. hydrophila, including aer, ahyB, hcp, and emp, decreased when A. hydrophila was cultured with B. subtilis CH9. Furthermore, the extracellular products of A. hydrophila showed attenuated hemolysin activity as well as cytotoxicity when A. hydrophila was cultured with B. subtilis CH9. Finally, the transcriptional levels of luxS genes of B. subtilis CH9 and A. hydrophila SC2005 were determined when these two species were co-cultured. RT-qPCR results suggested that the transcription level of A. hydrophila was down-regulated significantly. On the contrary, the transcription level of B. subtilis CH9 was up-regulated significantly. These results suggested that the probiotic role of B. subtilis CH9 is related to the inhibition of growth and virulence of A. hydrophila SC2005, and quorum sensing may be involved.

The guanidinobutyrase GbuA is essential for the alkylquinolone-regulated pyocyanin production during parasitic growth of Pseudomonas aeruginosa in co-culture with Aeromonas hydrophila.

The opportunistic pathogen Pseudomonas aeruginosa controls the production of virulence factors by quorum sensing (QS). Besides cell density, QS in P. aeruginosa is co-regulated by metabolic influences, especially nutrient limitation. Previously, a co-culture model system was established consisting of P. aeruginosa and the chitinolytic bacterium Aeromonas hydrophila, in which parasitic growth of P. aeruginosa is strictly dependent on the QS-controlled production of pyocyanin in response to nutrient limitation (Jagmann et al., ). In this study, the co-culture was employed to identify novel genes involved in the regulation of pyocyanin production. Via transposon mutagenesis, the gene gbuA encoding a guanidinobutyrase was identified, deletion of which led to a loss of pyocyanin production in co-cultures and to a reduced pyocyanin production in single cultures. Addition of the natural substrate of GbuA to the mutant strain enhanced the negative effect on pyocyanin production in single cultures. The gbuA mutant showed a reduced transcription of the pqsABCDE operon and could be complemented by PqsE overexpression and addition of alkylquinolone signal molecules. The strong effect of gbuA deletion on the QS-controlled pyocyanin production in co-cultures showed the value of this approach for the discovery of novel gene functions linking metabolism and QS in P. aeruginosa.

Macroalgal activity against multiple drug resistant Aeromonas hydrophila: A novel treatment study towards enhancement of fish growth performance.

OBJECTIVE: The aim of this study was to evaluate the efficiency of macroalgal extracts as antibacterial agent against multidrug-resistant (MDR) bacteria isolated from Nile tilapia (Oreochromis niloticus) as well as to enhance the fish growth performance by macroalgae diet application. METHODS: A total of 50 swabs were collected from the diseased organs of tilapia fish including gills, skin, spleen, intestine, liver, kidney and muscle. The isolated bacteria were identified and then confirmed by using VITEK 2. Eight macroalgal species were collected from Abu-Qir, Alexandria coast, Egypt. After determination of their biomass, three solvents were used to prepare algal extracts. The antibacterial activities of different macroalgal extracts were measured against MDR Aeromonas hydrophila 6 (MDRAH6) using well-diffusion method. The mechanism by which macroalgal extract affects MDR bacteria was conducted by using transmission electron microscope (TEM). To evaluate the safety of the promising algal extract, GC-MS was performed to detect the composition of S. vulgare extract. In addition, growth performance was measured as an application of algal extracts into fish feed. RESULTS: Between eight collected macroalgal species, Sargassum vulgare showed the highest biomass production (53.4 g m-2). In addition, its ethanolic extract showed the highest significant antibacterial activity with MIC value of 250 µg ml-1. TEM examination showed distinctive changes in the treated MDRAH6 cells including rupture of the cell wall, leakage of cytoplasmic contents, alterations in the cytoplasm density in addition to totally cell deformation. In addition, GC-MS analysis revealed eleven identified components in S. vulgare ethanolic extract, in which 9,12-octadecadienoyl chloride and hexadecanoic acid methyl ester were dominant (46.6 and 19.7 %, respectively). Furthermore, dietary replacement of fish meal with S. vulgare ethanolic extract significantly enhanced the growth performance and survival of Nile tilapia with a significant reduction in the total bacterial count. CONCLUSION: Ethanol extract of the brown macroalga S. vulgare could be a promising antibacterial and a new active agent against MDR A. hydrophila, which could be a major causative agent of Nile tilapia fish diseases. In addition, this study recommended S. vulgare as a natural and effective source to enhance the growth performance of Nile tilapia. In fact, isolation and examination of the individual antibacterial active compounds of the S. vulgar ethanolic extract are under investigation.

Characterisation of a novel Type I crustin involved in antibacterial and antifungal responses in the red claw crayfish, Cherax quadricarinatus.

Antimicrobial peptides are important immune effectors involved in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Type I crustin from the red claw crayfish Cherax quadricarinatus. The full-length cDNA encoded by this gene, designated CqCrs, comprised 608 bp, containing a 5'-untranslated region (UTR) of 55 bp, a 3'-UTR of 229 bp with a poly (A) tail, and an open reading frame (ORF) of 324 bp encoding a polypeptide of 107 amino acids. The deduced amino acid sequence of CqCrs exhibited a configuration typical of other crustacean Type I crustin orthologs, including one signal peptide region at the N-terminus between residues 1 and 16 and a long whey acidic protein (WAP) domain at the C-terminus between residues 60 and 107, along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrs was clustered first with other crustacean Type I crustins, then with other crustacean Type II crustins, and finally with other crustacean Type III crustins. Transcription of CqCrs was detected in all tissues, especially in immune tissues and was differentially induced in hemocytes post-stimulation with ß-1, 3-glucan, lipopolysaccharides (LPS) and peptidoglycans (PG) at selected time-points. To clarify the biological activity of CqCrs, the recombinant CqCrs protein (rCqCrs) was constructed and expressed in Escherichia coli BL21 (DE3). Purified rCqCrs bound to diverse bacteria and inhibited the growth of different microbes to varying degrees. These findings suggest that CqCrs is involved in a specific innate immune recognition and defense mechanisms against bacterial and fungal in C. quadricarinatus.

Molecular characterization and upregulation of cytosolic manganese superoxide dismutase by imidazole derivative KK-42 in Macrobrachium nipponense.

Imidazole derivative KK-42 is a well-known regulator of insect growth. KK-42 pretreatment has been shown to promote the survival of Macrobrachium nipponense infected with Aeromonas hydrophila, possibly via activation of superoxide dismutase (SOD). In this study, the cytMnSOD gene was cloned from the hepatopancreas of M. nipponense using the rapid amplification of cDNA ends technique. The full-length cDNA of cytMnSOD was 1233 bp long, and the open reading frame was 858 bp long, encoding a 286-aa protein with a 60-aa leader sequence. The calculated molecular mass of the translated cytMnSOD protein was 31.33 kDa, with an estimated isoelectric point of 5.62. cytMnSOD contained two N-glycosylation sites, four conserved amino acids responsible for binding manganese, and a manganese SOD domain (DVWEHAYY). Real-time RT-PCR analysis showed that cytMnSOD was expressed in all tissues examined with the highest expression observed in the hepatopancreas. Levels of the cytMnSOD transcript in the hepatopancreas were highest in stage C of the molting cycle. Real-time PCR analysis revealed that cytMnSOD expression increased significantly 3, 6, and 12 h after KK-42 treatment, with simultaneous increases in SOD activity from 6 to 12 h. Our results demonstrate that cytMnSOD expression and SOD activity may be induced by KK-42, which may represent one of the molecular mechanisms through which KK-42 promotes increased survival of prawns infected with A. hydrophila.

Plant essential oils against Aeromonas hydrophila: in vitro activity and their use in experimentally infected fish.

AIMS: The aims of this study were to investigate the in vitro antibacterial activity of the essential oils (EOs) of Hesperozygis ringens (HREO), popularly known as 'espanta-pulga' and two different species of basil, Ocimum gratissimum (OGEO) and Ocimum americanum (OAEO), as well as, the potential of these products to be used in silver catfish (Rhamdia quelen) infected with Aeromonas hydrophila. METHODS AND RESULTS: OGEO and HREO showed better antibacterial activity in vitro. Subinhibitory concentrations of all EOs inhibited haemolysis caused by Aer. hydrophila in fish erythrocytes (100% reduction for OAEO at 100 µg ml(-1) and more than 90% for HREO and OGEO at 150 µg ml(-1) ). However, OAEO and HREO showed the best survival results (75 and 70% respectively) after their use as treatment (therapeutic baths-1 h daily/5 days) in silver catfish experimentally infected with Aer. hydrophila. A second in vivo assay using healthy fish was conducted to verify the potential of the EOs (preventive baths-1 h daily/5 days) to promote fish survival. Fish exposed to HREO and OAEO and their diluent (ethanol) showed significant lower haematocrit values and higher complement system activity compared to control. Plasma cortisol level was significantly higher in the groups exposed to both EOs. There was no significant difference in survival of silver catfish challenged with Aer. hydrophila after preventive baths with HREO, OAEO and control group. CONCLUSIONS: All tested EOs showed in vitro antibacterial properties against Aer. hydrophila and HREO and OAEO showed potential to be used in the treatment of infected fish. SIGNIFICANCE AND IMPACT OF THE STUDY: These products can be used in aquaculture as therapeutic and prophylactic agents against fish pathogens, with antimicrobial and/or immunostimulant properties.

Inactivation kinetics of various chemical disinfectants on Aeromonas hydrophila planktonic cells and biofilms.

The present article focuses on the inactivation kinetics of various disinfectants including ethanol, sodium hypochlorite, hydrogen peroxide, peracetic acid, and benzalkonium chloride against Aeromonas hydrophila biofilms and planktonic cells. Efficacy was determined by viable plate count and compared using a modified Weibull model. The removal of the biofilms matrix was determined by the crystal violet assay and was confirmed by field-emission scanning electron microscope. The results revealed that all the experimental data and calculated Weibull α (scale) and ß (shape) parameters had a good fit, as the R(2) values were between 0.88 and 0.99. Biofilms are more resistant to disinfectants than planktonic cells. Ethanol (70%) was the most effective in killing cells in the biofilms and significantly reduced (p<0.05) the biofilms matrix. The Weibull parameter b-value correlated (R(2)=0.6835) with the biofilms matrix removal. The present findings deduce that the Weibull model is suitable to determine biofilms matrix reduction as well as the effectiveness of chemical disinfectants on biofilms. The study showed that the Weibull model could successfully be used on food and food contact surfaces to determine the exact contact time for killing biofilms-forming foodborne pathogens.

Detection of Aeromonas hydrophila in liquid media by volatile production similarity patterns, using a FF-2A electronic nose.

A technique for rapid detection of pathogenic microorganisms is essential for the diagnosis of associated infections and for food safety analysis. Aeromonas hydrophila is one such food contaminant. Several methods for rapid detection of this pathogen have been developed; these include multiplex polymerase chain reaction assays and the colony overlay procedure for peptidases. However, these conventional methods can only be used to detect the microorganisms at high accuracy after symptomatic onset of the disease. Therefore, in the future, simple pre-screening methods may be useful for preventing food poisoning and disease. In this paper, we present a novel system for the rapid detection of the microorganism A. hydrophila in cultured media (in <2 h), with the use of an electronic nose (FF-2A). With this electronic nose, we detected the changes of volatile patterns produced by A. hydrophila after 30 min culture. Our calculations revealed that the increased volatiles were similar to the odours of organic acids and esters. In future, distinctive volatile production patterns of microorganisms identified with the electronic nose may have the potential in microorganism detection.