Antimicrobial and anthelmintic effects of copper nanoparticles against Koi carp parasites and their toxicity.

This study investigated the in vitro antimicrobial and anthelmintic effect of copper nanoparticles (CuNPs) against the bacterium Aeromonas hydrophila, the monogeneans Dactylogyrus minutus, Dactylogyrus extensus, Gyrodactylus cyprini, and the cestode Schyzocotyle acheilognathi, as well as their toxicity to Cyprinus carpio Koi. In the antimicrobial in vitro test, the inhibition zone method and minimum inhibitory concentration (MIC) were performed. In order to determine the time and efficacy of monogenean parasite mortality, the parasites were exposed to CuNP concentrations of 20, 50, 100, 150, 200, and 300 mg L-1, and a control group with tank water and one with copper sulphate pentahydrate (CuSO4.5H2O) at a concentration of 0.3 mg L-1, performed in triplicate. The parasites were observed every 10 min for 300 min, and mortality was recorded. For the cestodes, parasites were immersed in CuNP concentrations of 50, 100, 150, and 300 mg L-1. At the end of the in vitro tests, the anthelmintic efficacy of each treatment was calculated. To assess the tolerance and toxicity in fish, they were exposed to CuNP concentrations of 0.6, 1.25, 2.5, 5, 10, 20, and 50 mg L-1 for 12 h. The MIC demonstrated that CuNPs effectively inhibited the growth of A. hydrophila up to a dilution of 12,500 mg L-1 and showed an inhibition zone of 14.0 ± 1.6 mm for CuNPs. The results of anthelmintic activity showed a dose-dependent effect of concentration for both groups of parasites, with the most effective concentration being 300 mg L-1 in 120 min. In the toxicity test, the carps showed tolerance to lower concentrations. The study indicated that CuNPs were effective against the studied pathogens. However, it proved to be toxic to fish at high concentrations. The use of low concentrations is recommended still requires further investigation.

Co-infection of Lactococcus garvieae and Aeromonas hydrophila in cultured Nile Tilapia in Kerala, India.

Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch's postulates, challenge experiments were conducted and median lethal dose (LD50) of L. garvieae and A. hydrophila was calculated as 1 × 105.91 CFU per mL and 1 × 105.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia.

Virulence-Associated Genes and Antimicrobial Resistance of Aeromonas hydrophila Isolates from Animal, Food, and Human Sources in Brazil.

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.

Quorum quenching potential of Enterococcus faecium QQ12 isolated from gastrointestinal tract of Oreochromis niloticus and its application as a probiotic for the control of Aeromonas hydrophila infection in goldfish Carassius auratus (Linnaeus 1758).

Quorum quenching (QQ), the obstruction of quorum sensing, is the most attractive way to break down the N-acyl-homoserine lactones (AHL) molecules. This work was focused at isolating AHL degrading bacteria from gastrointestinal tract of Oreochromis niloticus, with abilities appropriate for use as probiotic in aquaculture. The presence of an autoinducer inactivation (aiiA) homolog gene and AHL inactivation assay showed that Enterococcus faecium QQ12, which was one among the 20 isolates, could rapidly degrade synthetic C6-HSL in vitro and hampered violacein production by Chromobacterium violaceum. It had excellent biodegrading ability of natural N-AHL produced by Aeromonas hydrophila, suggesting that it can be used as a potential quencher bacterium for disrupting the virulence of A. hydrophila. It was susceptible to all the five antibiotics tried out. The isolate grew well at pH 3.0-7.0, was resistant to high level of bile salts (0-0.9%) and 0.5% of phenol. QQ12 also exhibited high degree of auto-aggregation and co-aggregation, confirming that it possessed good probiotic attributes. Goldfish fed diet incorporated with 108 and 1010 CFU g-1 of the QQ12 for 30 days showed 76.66-86.66% survival when challenged with A. hydrophila. The study indicates that Enterococcus faecium QQ12 could be used as a non-antibiotic feed additive in aquaculture to control bacterial diseases.

Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.

The antibacterial and physiological effects of pure and nanoencapsulated Origanum majorana essential oil on fish infected with Aeromonas hydrophila.

This study evaluated the antibacterial activity of Origanum majorana essential oil (EOM) and nanocapsules of this oil (NOM) in silver catfish, Rhamdia quelen, infected with Aeromonas hydrophila, and addressed their effects on silver catfish hematological and metabolic parameters. Fish were inoculated with A. hydrophila (360 µL, at a concentration of 1.5 × 109 CFU mL-1) and submitted to 1 h daily baths with EOM (0 (control), 20 or 30 µL L-1), NOM (0 (control), 5 or 10 µL L-1) or a positive control containing florfenicol (30 µL L-1) called group Maxflor® for five consecutive days. All treatments improved the survival rate of the infected fish, but we suggest the treatment of A. hydrophila infections through daily baths with 20 µL L-1 EOM or 5 µL L-1 NOM for five consecutive days as these were the lowest effective concentrations tested. Silver catfish treated with EOM and NOM had higher lymphocyte levels, indicating stimulation of the immune system in these fish. The lowest liver glucose level was found in the group treated with the lowest concentration of NOM, and the lactate values in the liver and muscle of all groups were within the normal values reported for this species. In addition, nanocapsules required much less EOM to elicit effective antibacterial treatment.

Antibacterial potential of phytochemicals alone or in combination with antimicrobials against fish pathogenic bacteria.

AIMS: This study investigated the antibacterial activity of five phytochemicals (carvacrol, citral, eugenol, linalool and thymol) alone or in combination with florfenicol or oxytetracycline against bacteria isolated from silver catfish (Rhamdia quelen). We also analysed the potential of these compounds to inhibit biofilm formation and haemolysis caused by the bacteria. METHODS AND RESULTS: Bacteria were tested with antimicrobials to calculate the multiple antibiotic resistances. The checkerboard assay was used to evaluate a putative synergy between five phytochemicals and antimicrobials against the strains isolated. The biofilm formation inhibition assay was performed with phytochemicals and antimicrobials, and the haemolysis inhibition assay was performed with the phytochemicals. Carvacrol, eugenol and thymol were the most effective phytochemicals. Three combinations (linalool with florfenicol or oxytetracycline against Aeromonas hydrophila and citral with oxytetracycline against Citrobacter freundii) demonstrated synergy in the checkerboard assay. All phytochemicals inhibited biofilm formation and haemolysis activity. CONCLUSION: The tested phytochemicals showed satisfactory activity against fish pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The phytochemicals did not present antagonistic interactions with the antimicrobials, allowing their combined use, which may contribute to a decrease in the use of conventional drugs and their residues in aquatic environment.

Activity of Brazilian propolis against Aeromonas hydrophila and its effect on Nile tilapia growth, hematological and non-specific immune response under bacterial infection

ABSTRACT The effect of the ethanolic extract of propolis (EEP) on Aeromonas hydrophila was analyzed by determination of minimum inhibitory concentration (MIC). Then, the effects of crude propolis powder (CPP) on growth, hemato-immune parameters of the Nile tilapia, as well as its effects on resistance to A. hydrophila challenge were investigated. The CPP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) was added to the diet of 280 Nile tilapia (50.0 ± 5.7 g fish-1). Hemato-immune parameters were analyzed before and after the bacterial challenge. Red blood cell, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and hydrogen peroxide (H2O2) and nitric oxide (NO) were evaluated. The MIC of the EEP was 13% (v/v) with a bactericidal effect after 24 hours. Growth performance was significantly lower for those fish fed diets containing 2.5 and 3% of CPP compared to the control diet. Differences in CPP levels affected fish hemoglobin, neutrophils number and NO following the bacterial challenge. For others parameters no significant differences were observed. Our results show that although propolis has bactericidal properties in vitro, the addition of crude propolis powder to Nile tilapia extruded diets does not necessarily lead to an improvement of fish health.

Activity of Brazilian propolis against Aeromonas hydrophila and its effect on Nile tilapia growth, hematological and non-specific immune response under bacterial infection.

The effect of the ethanolic extract of propolis (EEP) on Aeromonas hydrophila was analyzed by determination of minimum inhibitory concentration (MIC). Then, the effects of crude propolis powder (CPP) on growth, hemato-immune parameters of the Nile tilapia, as well as its effects on resistance to A. hydrophila challenge were investigated. The CPP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) was added to the diet of 280 Nile tilapia (50.0 ± 5.7 g fish-1). Hemato-immune parameters were analyzed before and after the bacterial challenge. Red blood cell, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and hydrogen peroxide (H2O2) and nitric oxide (NO) were evaluated. The MIC of the EEP was 13% (v/v) with a bactericidal effect after 24 hours. Growth performance was significantly lower for those fish fed diets containing 2.5 and 3% of CPP compared to the control diet. Differences in CPP levels affected fish hemoglobin, neutrophils number and NO following the bacterial challenge. For others parameters no significant differences were observed. Our results show that although propolis has bactericidal properties in vitro, the addition of crude propolis powder to Nile tilapia extruded diets does not necessarily lead to an improvement of fish health.

S-(+)- and R-(-)-linalool: a comparison of the in vitro anti-Aeromonas hydrophila activity and anesthetic properties in fish.

Linalool is the main compound of many essential oils and occurs in two isomeric forms: S-(+)- and R-(-)-linalool. This study aimed to determine if linalool isomers have different antimicrobial and anesthetic properties in fish. For this purpose, these compounds were previously isolated from Lippia alba (Mill.)N. E. Brown and Ocimum americanum L. essential oils. Antimicrobial effects were evaluated through the microdilution test against Aeromonas hydrophila, an important fish disease etiologic agent. Induction time until sedation, anesthesia and recovery time were determined in silver catfish (Rhamdia quelen) through bath exposure (60, 180, 300 or 500 µL L-1). The results showed different biological properties for the isomers being S-(+)-linalool the only active against A. hydrophila at 3.2 mg mL-1. The sedation was induced without differences between the compounds, however R-(-)-linalool promoted faster anesthesia. There were no differences regarding the recovery time of the animals exposed to the linalool isomers. Although both S-(+)- and R-(-)-linalool can be used for sedative purposes, their use in A. hydrophila infection is inadvisable due to the high effective concentration. Considering anesthesia as the main objective, the R-(-)-linalool demonstrated clear advantages at lower concentration.

S-(+)- and R-(-)-linalool: a comparison of the in vitro anti-Aeromonas hydrophila activity and anesthetic properties in fish

ABSTRACT Linalool is the main compound of many essential oils and occurs in two isomeric forms: S-(+)- and R-(-)-linalool. This study aimed to determine if linalool isomers have different antimicrobial and anesthetic properties in fish. For this purpose, these compounds were previously isolated from Lippia alba (Mill.)N. E. Brown and Ocimum americanum L. essential oils. Antimicrobial effects were evaluated through the microdilution test against Aeromonas hydrophila, an important fish disease etiologic agent. Induction time until sedation, anesthesia and recovery time were determined in silver catfish (Rhamdia quelen) through bath exposure (60, 180, 300 or 500 μL L-1). The results showed different biological properties for the isomers being S-(+)-linalool the only active against A. hydrophila at 3.2 mg mL-1. The sedation was induced without differences between the compounds, however R-(-)-linalool promoted faster anesthesia. There were no differences regarding the recovery time of the animals exposed to the linalool isomers. Although both S-(+)- and R-(-)-linalool can be used for sedative purposes, their use in A. hydrophila infection is inadvisable due to the high effective concentration. Considering anesthesia as the main objective, the R-(-)-linalool demonstrated clear advantages at lower concentration.

The use of Ocimum americanum essential oil against the pathogens Aeromonas hydrophila and Gyrodactylus sp. in silver catfish (Rhamdia quelen).

UNLABELLED: The bactericidal activity (minimum inhibitory concentration (MIC)-test) of Ocimum americanum (inflorescences) essential oil (OAEO) against Aeromonas hydrophila was determined in this study. Also investigated was the potential of OAEO and the main compound found in the oil (linalool) at subinhibitory concentrations to be inhibitors of haemolysis caused by Aer. hydrophila in fish erythrocytes. An in vivo experiment was conducted to evaluate the survival of fish (Rhamdia quelen) experimentally infected with Aer. hydrophila and exposed to OAEO. A second experiment was conducted to evaluate the in vitro and in vivo activity of OAEO (mix from inflorescences and leaves) against the parasite Gyrodactylus sp. The OAEO showed weak in vitro activity against Aer. hydrophila (6400 µg ml(-1) ). Subinhibitory concentrations of OAEO (100 µg ml(-1) ) inhibited haemolysis (90%) caused by Aer. hydrophila in fish erythrocytes, however, linalool did not inhibit haemolysis activity. At the low concentrations (10 and 20 mg l(-1) ) added to the water, OAEO promoted the survival of fish experimentally infected with Aer. hydrophila. Lastly, the OAEO mix (50 mg l(-1) ) was effective against Gyrodactylus sp., significantly reducing (60%) the number of parasites in the fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytochemicals, such as essential oils (EOs) are a great source of new molecules and have shown potential to be used in aquaculture systems. However, additional studies focused on the in vivo efficacy, mode of action and identification of the active compounds are needed. This study determined the potential of Ocimum americanum EO for use against two important fish pathogens, Aeromonas hydrophila and Gyrodactylus sp., as well as providing preliminary information about the role of the main EO compound (linalool) against Aer. hydrophila virulence.

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.

Influence of carvacrol and 1,8-cineole on cell viability, membrane integrity, and morphology of Aeromonas hydrophila cultivated in a vegetable-based broth.

This study investigated the effects of carvacrol (CAR) and 1,8-cineole (CIN) alone (at the MIC) or in combination at subinhibitory amounts (both at 1/8 MIC) on the cell viability, membrane permeability, and morphology of Aeromonas hydrophila INCQS 7966 (A. hydrophila) cultivated in a vegetable-based broth. CAR and CIN alone or in combination severely affected the viability of the bacteria and caused dramatic changes in the cell membrane permeability, leading to cell death, as observed by confocal laser microscopy. Scanning and transmission electron microscopy images of bacterial cells exposed to CAR or CIN or the mixture of both compounds revealed severe changes in cell wall structure, rupture of the plasma membrane, shrinking of cells, condensation of cytoplasmic content, leakage of intracellular material, and cell collapse. These findings suggest that CAR and CIN alone or in combination at subinhibitory amounts could be applied to inhibit the growth of A. hydrophila in foods, particularly as sanitizing agents in vegetables.

The use of eugenol against Aeromonas hydrophila and its effect on hematological and immunological parameters in silver catfish (Rhamdia quelen).

The aim of this study was to evaluate the activity of eugenol against the fish pathogen Aeromonas hydrophila and eugenol's effect on hematological and natural immune parameters in silver catfish (Rhamdia quelen). In vitro, eugenol showed weak activity against A. hydrophila, but in vivo, at a subinhibitory concentration (10 mg L(-1)), it promoted survival in infected silver catfish. Eugenol (50 µg mL(-1)) reduced the hemolytic activity of A. hydrophila supernatant in vitro in fish erythrocytes. Subjecting catfish to eugenol baths (5 and 10 mg L(-1)) for five days did not alter the hematological and immunological parameters studied in this work. Based on these results, eugenol can be used to treat or prevent bacterial diseases in fish.

The sensitivity of bacterial foodborne pathogens to Croton blanchetianus Baill essential oil

The aim of this study was to assess the activity of essential oil extracted from the leaves of C. blanchetianus Baill, popularly known as "marmeleiro", in inhibiting the growth and survival of pathogenic microorganisms in food by determining their survival in vitro and by observing the behaviour of Listeria monocytogenes inoculated into a food model (meat cubes) that was stored at refrigeration temperature (7 ± 1 ºC) for 4 days. The results indicated a bactericidal effect against Aeromonas hydrophila and Listeria monocytogenes and bacteriostatic action against Salmonella Enteritidis. A bacteriostatic effect on meat contaminated with L. monocytogenes was found for all concentrations of essential oils tested. These results showed that essential oil from the leaves of C. blanchetianus Baill represents an alternative source of potentially natural antimicrobial agents that may be used as a food preservative.

Resistance and inactivation kinetics of bacterial strains isolated from the non-chlorinated and chlorinated effluents of a WWTP.

The microbiological quality of water from a wastewater treatment plant that uses sodium hypochlorite as a disinfectant was assessed. Mesophilic aerobic bacteria were not removed efficiently. This fact allowed for the isolation of several bacterial strains from the effluents. Molecular identification indicated that the strains were related to Aeromonas hydrophila, Escherichia coli (three strains), Enterobacter cloacae, Kluyvera cryocrescens (three strains), Kluyvera intermedia, Citrobacter freundii (two strains), Bacillus sp. and Enterobacter sp. The first five strains, which were isolated from the non-chlorinated effluent, were used to test resistance to chlorine disinfection using three sets of variables: disinfectant concentration (8, 20 and 30 mg·L(-1)), contact time (0, 15 and 30 min) and water temperature (20, 25 and 30 °C). The results demonstrated that the strains have independent responses to experimental conditions and that the most efficient treatment was an 8 mg·L(-1) dose of disinfectant at a temperature of 20 °C for 30 min. The other eight strains, which were isolated from the chlorinated effluent, were used to analyze inactivation kinetics using the disinfectant at a dose of 15 mg·L(-1) with various retention times (0, 10, 20, 30, 60 and 90 min). The results indicated that during the inactivation process, there was no relationship between removal percentage and retention time and that the strains have no common response to the treatments.

The sensitivity of bacterial foodborne pathogens to Croton blanchetianus Baill essential oil.

The aim of this study was to assess the activity of essential oil extracted from the leaves of C. blanchetianus Baill, popularly known as "marmeleiro", in inhibiting the growth and survival of pathogenic microorganisms in food by determining their survival in vitro and by observing the behaviour of Listeria monocytogenes inoculated into a food model (meat cubes) that was stored at refrigeration temperature (7 ± 1 °C) for 4 days. The results indicated a bactericidal effect against Aeromonas hydrophila and Listeria monocytogenes and bacteriostatic action against Salmonella Enteritidis. A bacteriostatic effect on meat contaminated with L. monocytogenes was found for all concentrations of essential oils tested. These results showed that essential oil from the leaves of C. blanchetianus Baill represents an alternative source of potentially natural antimicrobial agents that may be used as a food preservative.