Antimicrobial Agents In Aquaculture Research Articles

Electrochemical detection of nitrofurazone using laser-engraved three-electrode graphene array

BackgroundNitrofurazone (NFZ) is a widely-used antimicrobial agent in aquaculture. The NFZ residue can be transmitted to humans through the food chain, and cause adverse health effects including carcinogenesis and teratogenesis. Until now, a number of modified electrodes have been developed for NFZ detection, however, there are some issues that need to be improved. For example, the reported detection sensitivity is relatively low, the modification procedure is complicated, and conventional three-electrode system is used. Therefore, it is quite important to develop new NFZ detection method with higher sensitivity, simplicity and practicality. ResultsHerein, a kind of integrated three-electrode array consisted with porous graphene is easily prepared through laser engraving of commercial polyimide tape. Five kinds of graphene arrays were prepared at different laser power percentage (i.e. 30 %, 40 %, 50 %, 60 % and 70 %). It is found that their structure, morphology, fluffiness and porosity show great difference, consequently affecting the electrochemical performance of graphene arrays such as conductivity, active area and electron transfer ability. The engraved graphene array at 50 % laser power percentage (LIG-50 array) is superior owing to uniform 3D structure, abundant pores and high stability. More importantly, LIG-50 array is more active for NFZ oxidation, and significantly enhances the detection sensitivity. The linear range of LIG-50 sensor is from 0.2 to 8 μM, and the detection limit is 0.035 μM, which is successfully used in fish meat samples. SignificanceA sensitive, portable and practical electrochemical sensor has been successfully developed for NFZ using laser-engraved graphene array. The demonstration using fish meat samples manifests this new sensor has good accuracy and great potential in application. This study could provide a new possibility for the design and fabrication of other high-performance electrochemical sensor for various applications in the future.

Identification of TonB-dependent siderophore receptor inhibitors against Flavobacterium columnare using a structure-based high-throughput virtual screening method.

TonB-dependent siderophore receptors play a critical transport role for Flavobacterium columnare virulence formation and growth, and have become valuable targets for the development of novel antimicrobial agents. Traditional Chinese medicine has demonstrated notable efficacy in the treatment of fish diseases and includes potential antibacterial agents. Herein, we performed molecular docking-based virtual screening to discover novel TonB-dependent siderophore receptor inhibitors from traditional Chinese medicine and provide information for developing novel antibacterial agents. Firstly, we efficiently obtained 11 potential inhibitors with desirable drug-like characteristics from thousands of compounds in the TCM library based on virtual screening and property prediction. The antibacterial activity of Enoxolone, along with its interaction characteristics, were determined via an MIC assay and molecular dynamic simulation. Transcriptional profiling, along with validation experiments, subsequently revealed that an insufficient uptake of iron ions by bacteria upon binding to the TonB-dependent siderophore receptors is the antibacterial mechanism of Enoxolone. Finally, Enoxolone's acceptable toxicity was illustrated through immersion experiments. In summary, we have used virtual screening techniques for the first time in the development of antimicrobial agents in aquaculture. Through this process, we have identified Enoxolone as a promising compound targeting the TonB-dependent siderophore receptor of F. columnare. In addition, our findings will provide new ideas for the advancement of innovative antimicrobial medications in aquaculture.

Prokaryotic Expression, Purification, and Antibacterial Activity of the Hepcidin Peptide of Crescent Sweetlips (Plectorhinchus cinctus).

The hepcidin peptide of crescent sweetlips (Plectorhinchus cinctus) is a cysteine-rich, cationic antimicrobial peptide that plays a crucial role in the innate immune system's defense against invading microbes. The aim of this study was to identify the optimal parameters for prokaryotic expression and purification of this hepcidin peptide and characterize its antibacterial activity. The recombinant hepcidin peptides were expressed in Escherichia coli strain Arctic Express (DE3), with culture and induction conditions optimized using response surface methodology (RSM). The obtained hepcidin peptides were then purified before tag cleavage, and their antibacterial activity was determined. The obtained results revealed that induction temperature had the most significant impact on the production of soluble recombinant peptides. The optimum induction conditions were determined to be an isopropylthio-β-galactoside (IPTG) concentration of 0.21 mmol/L, induction temperature of 18.81 °C, and an induction time of 16.01 h. Subsequently, the recombinant hepcidin peptide was successfully purified using Ni-IDA affinity chromatography followed by SUMO protease cleavage. The obtained hepcidin peptide (without His-SUMO tag) demonstrated strong antimicrobial activity in vitro against V. parahaemolyticus, E. coli, and S. aureus. The results showed prokaryotic (E. coli) expression is a feasible way to produce the hepcidin peptide of crescent sweetlips in a cost-effective way, which has great potential to be used as an antimicrobial agent in aquaculture.

Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus)

Antimicrobial peptides (AMPs) are members of humoral immunity and particpate in resisting microbial invasion. In this study, an AMP gene hepcidin was obtained from the oriental loach Misgurnus anguillicaudatus and named Ma-Hep. This Ma-Hep encodes a peptide of 90 amino acids, with a predicted active peptide segment (Ma-sHep) of 25 amino acids at C terminus. Stimulation by a bacterial pathogen Aeromonas hydrophila resulted in significant up-regulation of Ma-Hep transcripts in loach midgut, head kidney, and gill. Ma-Hep and Ma-sHep proteins were expressed in Pichia pastoris and their antibacterial activity was examined. Results showed that Ma-sHep possessed stronger antibacterial activity against various Gram-positive and Gram-negative bacteria, compared to Ma-Hep. Scanning electron microscopy showed that Ma-sHep might kill bacteria by destroying bacterial cell membranes. Moreover, we found that Ma-sHep had an inhibitory effect on blood cell apoptosis induced by A. hydrophila and facilitated the bacterial phagocytosis and clearance in loach. Histopathological analysis indicated Ma-sHep could protect liver and gut of loach from bacterial infection. Ma-sHep has high thermal stability and PH stability, which is conducive to further feed addition. Feed supplemented with Ma-sHep expressing yeast improved the intestinal flora of loach by increasing the dominant bacteria and decreasing the harmful bacteria. Feed supplemented with Ma-sHep expressing yeast also regulated the expression of inflammatory related factors in various tissues of loach and reduced the mortality of loach upon bacterial infection. These findings show that the antibacterial peptide Ma-sHep is involved in the antibacterial defense of loach and can be used as a candidate for new antimicrobial agents in aquaculture.

The Antimicrobial Peptide LJ-hep2 from Lateolabrax japonicus Exerting Activities against Multiple Pathogenic Bacteria and Immune Protection In Vivo.

Hepcidin is widely present in many kinds of fish and is an important innate immune factor. A variety of HAMP2-type hepcidins have strong antimicrobial activity and immunomodulatory functions and are expected to be developed as substitutes for antibiotics. In this study, the antimicrobial activity of Hepc2 from Japanese seabass (Lateolabrax japonicus) (designated as LJ-hep2) was investigated using its recombinant precursor protein (rLJ-hep2) expressed in Pichia pastoris and a chemically synthesized mature peptide (LJ-hep2(66–86)). The results showed that both rLJ-hep2 and synthetic LJ-hep2(66–86) displayed broad antimicrobial spectrum with potent activity against gram-negative and gram-positive bacteria, and fungi. Especially, LJ-hep2(66–86) had stronger antimicrobial activity and exhibited potent activity against several clinically isolated multidrug-resistant bacteria, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecium. Moreover, LJ-hep2(66–86) exerted rapid bactericidal kinetic (killed tested bacteria within 2 h), induced significant morphological changes and promoted agglutination of E. coli, P. aeruginosa and Aeromonas hydrophila. The activity of LJ-hep2(66–86) against E. coli, P. aeruginosa and A. hydrophila was stable and remained active when heated for 30 min. In addition, LJ-hep2(66–86) exhibited no cytotoxicity to the mammalian cell line HEK293T and fish cell lines (EPC and ZF4). In vivo study showed that LJ-hep2(66–86) could improve the survival rate of marine medaka (Oryzias melastigma) by about 40% under the challenge of A. hydrophila, indicating its immunoprotective function. Taken together, both rLJ-hep2 and LJ-hep2(66–86) have good prospects to be used as potential antimicrobial agents in aquaculture and medicine in the future.

Pangasius Hypophthalmus Viscera as a Potential Vector of Bacterial Cross-Contamination and Resistance of Escherichia coli to Antibiotics

The viscera of Pangasius fish was studied to provide baseline information about the presence of antibiotic-resistant E. coli on it. This aimed to assess the possible resistance of bacterial pathogens to antibiotics and cross-contamination into the fish's muscles during processing, as well as to evaluate the effect of starvation on the microbial loads of Pangasius fish viscera. The resistance of E. coli was tested against 15 antimicrobial agents using the disk diffusion method. The findings revealed that starvation reduced microbial loads on the viscera compared to non-starvation Pangasius. LAB, coliforms, and E. coli count on viscera of non-starved Pangasius were 7.0±0.5, 5.5±0.9 and 5.4±1.0 log CFU g-1, whereas those of the starved fish were 2.6±0.8, 3.8±0.4 and 3.1±0.3 log CFU g-1, respectively. A total of 55 E. coli isolated from Pangasius viscera were tested for antimicrobial susceptibility as stated above. Surprisingly, 69.09% of E. coli isolates were multi-antibiotic resistant from three to fifteen antibiotics tested. A high level of resistance to ampicillin (63.64%), ceftazidime (69.09%), nalidixic acid (78.18%) was observed. More importantly, 9.09% of the E. coli isolates were resistant to all kinds of antibiotics tested. As E. coli is a potential vector for transfer of antibiotic resistance gene, causing cross-resistance with human enteric pathogens, there is a need for both the prudent use of these antimicrobial agents in aquaculture and stringent appropriate infection control in the processing chain in Vietnam.

Analysis of the chemical compositions of six essential oils and evaluation of their antioxidant and antibacterial activities against some drug-resistant bacteria in aquaculture

Introduction: The extensive use of chemicals and antimicrobial agents in aquaculture has decreased the immune mechanisms of cultivated species and promoted the emergence of drug-resistant microorganisms leading to diseases among cultivated fish, affecting consumers’ health. Thus, the investigation of natural antibacterial and anti-stress agents is crucial. In the current study, we focused on the evaluation of the potential use of essential oils (EOs) as an antioxidant and antimicrobial agents in aquaculture. Methods: The EOs, obtained by hydrodistillation from clove (Syzygium aromaticum), cinnamon (Cinnamomum verum), rosemary (Rosmarinus officinalis), artemisia (Artemisia herba-alba), cedarwood (Cedrus atlantica) and oregano (Origanum compactum) were analyzed by gas chromatography/mass spectrometry (GC/MS). Their antibacterial activities were carried out against five bacteria, pathogenic to fish in aquaculture, using the well diffusion and microatmosphere methods. The pathogens used were Vibrio anguillarum, Photobacterium damselae subsp damselae, Aeromonas salmonicida, Edwardsiella tarda, and Lactococcus garvieae. Then, the minimum inhibitory and bactericidal concentrations of each EO were determined. Furthermore, the antioxidant activity was performed in vitro. Results: The investigated EOs were effective against the pathogenic strains. They showed variable constituents such as phenols, sesquiterpenes, and monoterpenes. Regarding the antioxidant activity, cinnamon, clove, and oregano EOs showed their abilities to donate hydrogen to 2,2-diphenyl-1-picrylhydrazy (DPPH) radical and scavenge free radicals produced by 2,2-azino-bis-3-ethylbenzothiazoline6-sulfonic acid (ABTS), respectively. Conclusion: These results gave insight into the potential use of phytobiotics in aquaculture as a safe strategy to substitute antibiotics to protect fish from oxidative stress and inhibit the emergence of drug-resistant bacteria for safer consumption of cultivated fish.

Preliminary Phytochemical Screening, GC-MS, FTIR, and Antimicrobial Activity of Commelina forskaolii Vahl Leaves

The Commelina forskaolii Vahl (Commelinaceae) is an edible herb with wide therapeutic value. The present study aimed to explore the preliminary phytochemical screening, GC-MS spectrometry analysis, FTIR, and evaluate the antimicrobial activity of C. forskaolii Vahl leaves using various solvent extracts with different polarities tested for their inhibitory property against most prevalent aquatic pathogenic bacteria (Gram-positive and Gramnegative) and fungi by the agar diffusion method. The results of phytochemical and GC-MS analysis of the different solvent extracts revealed the occurrence of secondary metabolites and totally 35 biologically active compounds. The FTIR results confirmed the presence of alkanes, amines, alcohol, aryl disulfides, ethers, hydroxy group, vinylidene, chloro, bromo, and iodo compounds. The in vitro antimicrobial activity of five solvent extracts of C. forskaolii Vahl was investigated against Streptococcus agalactiae and Streptococcus iniae, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio cholerae, Aspergillus niger and Aspergillus flavus. Among all extracts, aqueous and chloroform extract showed maximum antibacterial activity against all bacterial pathogens based on the different concentrations, which inhibited in the range of 11 mm to 18 mm (aqueous), and 11 mm to 27 mm (chloroform) followed by inhibition range of other extracts 9 mm to 12 mm (ethanol), 11 mm to 23 mm (methanol), and 12 mm to 16 mm (acetone). In addition, the highest antifungal activity was reported in chloroform extract against Aspergillus niger and aqueous extract against Aspergillus flavus with inhibition of 16 mm followed by 17 mm at 75 µl concentration. In the present investigation, C. forskaolii Vahl leaves were examined and reported for the first time and the study suggests that leaves of C. forskaolii Vahl could be potentially used as a natural source of antimicrobial agents in aquaculture and fisheries industries.

Three naturally occurring host defense peptides protect largemouth bass (Micropterus salmoides) against bacterial infections

Infectious diseases caused by drug-resistant bacteria are big challenges for aquaculture industry. Innovative anti-infective drugs and/or therapeutic strategies are urgently needed. Host defense peptides (HDPs) are naturally occurring peptides that are regarded as promising alternatives to conventional antibiotics. However, their application as antimicrobial agents in aquaculture has not been well studied. In the present study, three previously characterized HDPs (CATHPb1, Cm-CATH2, and Hc-CATH) were selected and their protective ability on largemouth bass (Micropterus salmoides) against bacterial infections was determined. The three HDPs exhibited potent and broad-spectrum antimicrobial activity against aquatic pathogenic bacteria, with MIC values ranging 2.34–18.75 μg/ml. They rapidly kill bacteria within 30 min by inducing cytomembrane permeabilization and cell disruption. They also showed strong ability to inhibit the formation of bacterial biofilms and eradicate the preformed biofilms, which enable them with great potential to combat biofilm-related infections. Besides, they could effectively modulate the anti-infective immune responses of largemouth bass by simultaneously inhibiting the expression of pro-inflammatory cytokines (TNF-α and IL-1β) and stimulating the expression of chemokine IL-8. Animal challenge test proved that they could significantly enhance the survival rate of bacteria-challenged largemouth bass from less than 40% to over 60% and remarkably decrease the in vivo bacterial numbers. Moreover, compared to the conventional antibiotics, they showed dramatically lower propensity to induce bacterial resistance. All the results above imply the great potential of the three HDPs as novel anti-infective agents in largemouth bass culture.

The potentiality of endophytes bacterial in red algae as anti-microbial agents in aquaculture: A review

There are several types of macro algae including brown, green and red algae, which have bioactive compound as anti-microbial agents. Besides that, there are endophytes bacteria that can prevent the growth of disease bacteria, especially bacteria that often attack cultivated species such as Vibrio sp. and Streptococcus sp. In red Algae, the bioactive compounds produced by endophytes bacteria has many benefits including anti-cancer, anti-coagulant, anti-viral, anti-fungal, anti-bacterial, and anti-inflammatory. Several studies have shown that the use of endophytes bacteria found in red algae can reduce and/or prevent the attack of bacteria on fish and other aquatic organisms. The species from genus Graciliaria has as majority found as a source of endophytes and its bioactive compounds. It can against pathogen bacteria could inhibited effectively Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli but not enough power against Enterobacter aerogenes. The inhibition zone range 3.1 ± 0.33 mm - 16.33 ± 0.58 mm.

TEMPO-oxidized biodegradable bacterial cellulose (BBC) membrane coated with biologically-synthesized silver nanoparticles (AgNPs) as a potential antimicrobial agent in aquaculture (In vitro)

The emergence of drug-resistance pathogens is one of the major challenges in aquaculture. Finding an alternative remedy for diseases control is now crucial and indispensable. The present study aimed to develop different silver nanocomposite BBC membranes and verified their bactericidal activity either synergistically or independently against seven threatening aquatic pathogens (Vibrio harveyi, V. parahaemolyticus, V. alginolyticus, V. vulnificus, Aeromonas hydrophila, A. veronii and Streptococcus iniae) using membrane disc diffusion and antibacterial log reduction assays. The aqueous extract of Pseudomonas sp. was used for the synthesis of AgNPs and the composite BBC materials were characterized using FTIR, XRD, EDS, and FESEM to confirm their holding capacity of integrated AgNPs. Results evidenced that the TEMPO-oxidized BBC membrane coated with bacterial-based AgNPs exhibited an excellent crystallinity, porous properties, and strongest holding capacity. The membrane also showed potent bactericidal activity represented by wide inhibitory zones (17–20 mm), high killing ratios (95.93–99.86%). and high antibacterial log-reduction values (1.39–2.85). In conclusion, the synergism between TEMPO-oxidized BBC membrane and biologically synthesized AgNPs is an eco-friendly alternative remedy to control aquatic diseases without serious impact.

Use of silver nanoparticles to control Vibrio fluvialis in cultured angelfish Pterophyllum scalare.

Nanoparticles have multiple applications, among which is their use as antimicrobial agents in aquaculture. The objective of this work was to determine the antibacterial effect of silver nanoparticles (AgNPs) against Vibrio fluvialis in cultured angelfish Pterophyllum scalare. AgNPs were synthetized through chemical reduction and characterized by UV-visible and infrared spectroscopy. Particle size ranged from 60 to 170.8 nm, and scanning electron microscopy revealed cubic and spherical forms. A minimal inhibitory concentration of 222.5 ppm was determined, as well as inhibition halos between 8.66 and 14.3 mm. Inhibition of V. fluvialis growth was observed upon contact with AgNPs. An 88% survival of infected fish was obtained when treated with AgNPs, in contrast to 100% mortality of fish that were not treated. No damage to internal or external organs was observed in fish exposed to AgNPs. We conclude that AgNPs exert an antimicrobial effect against V. fluvialis, and thus represent a new alternative to control diseases caused by this microorganism in P. scalare culture.

Exploring small cationic peptides of different origin as potential antimicrobial agents in aquaculture

Antimicrobial peptides (AMPs) form part of the innate immune response, which is of vital importance in fish, especially in eggs and early larval stages. Compared to antibiotics, AMPs show action against a wider spectrum of pathogens, including viruses, fungi and parasites, are more friendly to the environment, and do not seem to generate resistance in bacteria. Thus, we have tested in vitro the potential use of several synthetic peptides as antimicrobial agents in aquaculture: frog Caerin1.1, European sea bass Dicentracin (Dic) and NK-lysin peptides (NKLPs) and sole NKLP27. Our results demonstrate that the highest bactericidal activity against both human and fish pathogens was obtained with Caerin1.1 followed by sea bass Dic and NKLPs, having the sea bass NKLP20.2 none to negligible activity. Interestingly, Aeromonas salmonicida was refractory to all the fish peptides tested. Regarding the antiviral activity, synthetic peptides were able to inhibit the viral infection of nodavirus (NNV), viral septicaemia haemorrhagic virus (VHSV), infectious pancreatic necrosis virus (IPNV) and spring viremia carp virus (SVCV), which are some of the most devastating virus for aquaculture. However, their effectiveness was highly dependent on the type of virus. Strikingly, IPNV resulted the most resistant virus since Caeerin1.1 and sea bass NKLP20.2 were unable to reduce its titre and the other peptides tested only reduced it to values in the 43–78% range. These data demonstrate that synthetic peptides have great antibacterial and antiviral in vitro activity against important fish pathogens and point to their use as potential therapeutic agents in aquaculture.

A rapid and simple protocol for the isolation of bacteriophages from coastal organisms

This work details a protocol for recovering bacteriophages from intertidal sessile mussels and testing their lytic activity against pathogenic bacteria. Although bacteriophages were highly abundant in coastal filter-feeding organisms, they were not detectable in the surrounding water column. This difference reflects the high filtering rate of the mussels, which capture and concentrate high amounts of bacteria, generating an ideal environment for bacteriophages. We validated the protocol providing a mean concentration of 4E + 04 PFU mL-1 lytic bacteriophages for the fish-pathogen bacterium Vibrio ordalii. We suggest that this method has particular utility for the recovery of bacteriophages for use as natural antimicrobial agents in aquaculture.

A teleost TFPI-2 peptide that possesses a broad antibacterial spectrum and immune-stimulatory properties

Tissue factor pathway inhibitor 2 (TFPI-2) is an analogue of TFPI-1 and a potent endogenous inhibitor of tissue factor (TF)-mediated blood coagulation. Previous reports have shown that several peptides derived from human and vertebrates TFPI-2 possess antibacterial activity against diverse bacteria. In this study, a C-terminal peptide, TO24 (with 24 amino acids), derived from red drum (Sciaenops ocellatus) TFPI-2, was synthesized and investigated for its antimicrobial spectrum, action mode, as well as the immune-stimulatory property. Our results indicated that TO24 was active against Gram-positive bacteria Micrococcus luteus and Staphylococcus aureus; Gram-negative bacteria Vibrio litoralis, Vibrio ichthyoenteri, Vibrio vulnificus and Vibrio scophthalmi, as well as fish megalocytivirus, infectious spleen and kidney necrosis virus (ISKNV). During its interaction with V. vulnificus, TO24 exerted its antibacterial activity by destroying cell membrane integrity, penetrating the cytoplasm and inducing degradation of genomic DNA and total RNA. In addition, TO24 had no hemolytic activity against red drum blood cells. In vitro, TO24 enhanced bactericidal activity of red drum macrophages. In vivo, administration of red drum with TO24 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. These results indicate that TO24 is a broad-spectrum antimicrobial peptide with immune-stimulatory properties and it has the potential to be used as an antimicrobial agent in aquaculture.

Photosynthetic purple sulfur bacterium Marichromatium purpuratum RuA2 induces changes in water quality parameters, the occurrence of sulfonamide resistance gene and microbial community structure of marine aquaculture

Traditional methods to combat disease with antimicrobial agents in aquaculture have caused serious concern and alternative approaches using probiotics have been sought. A photosynthetic purple sulfur bacteria Marichromatium purpuratum RuA2 that thrives at salt concentrations of 0.5% to 5.0% and has a more temperate tolerance for oxygen was isolated from a marine fish pond located on the southwestern shore of Qigu, Taiwan. The effects of M. purpuratum RuA2 on the water quality parameters, the occurrence of the sulfonamide resistance gene sul1 and the microbial community were investigated in situ. Supplementation with strain RuA2 resulted in an increased removal rate of total organic carbon and nitrate ions, as well as a lower occurrence of sul1, in the bacterial community. The abundance and diversity of the bacterial community in the control and M. purpuratum RuA2-supplemented water samples were investigated. Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Planctomycetes were identified to be the top five predominant phyla in the control and M. purpuratum RuA2-supplemented water samples. However, supplementation with M. purpuratum RuA2 increased the diversity of the bacterial community composition. Fourteen unique genera were detected solely in strain RuA2-supplemented water samples and the relative abundance of seven shared genera was associated with supplementation with strain RuA2. In conclusion, M. purpuratum RuA2 showed a desirable capacity for improving water quality, reducing the occurrence of the resistance gene sul1 and advantageous effects on the microbial community structure. Tolerant to salinity and oxygen, M. purpuratum RuA2 exhibits an attractive potential for resolving the challenges facing aquaculture today.

Confirmation that pulse and continuous peracetic acid administration does not disrupt the acute stress response in rainbow trout

Peracetic acid (PAA) is considered an eco-friendly alternative to other antimicrobial agents of common use in aquaculture. The literature suggests that fish can habituate to PAA exposure based on a reduction of the fish corticosteroid response to PAA administration after repeated exposures. If that is true, PAA would also be a good option from the point of view of fish physiology. However, stronger evidence is needed to confirm that the use of PAA is welfare-friendly to fish. Besides habituation, other hypothetical factors such as desensitization, physiological exhaustion or PAA-mediated endocrine disruption could potentially explain the reduction in the corticosteroid response after repeated/prolonged PAA exposure. In this study, rainbow trout that had been exposed to PAA for several weeks were challenged with a secondary chasing stressor: fish were pursued with a dipnet for 1 min and their acute response was evaluated by measuring plasma cortisol, plasma glucose, plasma lactate and brain serotonergic activity. All fish were equally able to mount a normal physiological stress response to the secondary stressor independent of previous exposure to PAA. This suggests that the decrease in the cortisol response after repeated exposure to PAA, as seen in previous studies, is a true habituation to PAA administration, which supports the use of PAA as a welfare-friendly antimicrobial agent in aquaculture.

Dietary effects of succinic acid on the growth, digestive enzymes, immune response and resistance to ammonia stress of Litopenaeus vannamei

Organic acids acts as an growth promoter and antimicrobial agent in aquaculture. The present study investigated the effects of a natural organic acid - succinic acid (SA) on the growth, digestive enzymes, immune response and resistance to ammonia stress of Litopenaeus vannamei. The shrimps were firstly fed with diets containing different levels of SA: 0% (Control), 0.25% (SA1), 0.50% (SA2), and 1.0% (SA3) (w/w) for 56 days, followed by an acute ammonia stress for 48 h. The results indicated that dietary of SA improved the growth of shrimp, and increased the survival rate of shrimp after ammonia stress for 48 h. The amylase, lipase and pepsin activity increased in hepatopancreas in three SA group, while trypsin activity was only increased in the SA1 and SA2 groups. At 56 d, T-NOS activity, proPO and HSP70 gene expression level increased in the three SA group, PO activity increased in the SA1 and SA2 groups, T-AOC content and Toll gene expression level increased in the SA2 and SA3 groups, Trx and SOD gene expression level increased in the SA2 group, while Imd, GS and GDH gene expression level was no changes. After exposure to ammonia stress for 48 h, immune biochemical parameters (T-AOC and PO) and genes (proPO, HSP70, Trx and GDH) expression level increased in the three SA group, T-NOS activity, Toll, Imd and GS gene expression level increased in the SA2 and SA3 groups, while SOD gene expression level increased in the SA1 and SA2 groups. These results indicated that SA improved growth, enhanced digestive and immune capacities of L. vannamei against ammonia stress, and may be a potential feed additive for shrimp. The optimal dietary supplementation dosage is 0.50% (w/w) in diet.

TO17: A teleost antimicrobial peptide that induces degradation of bacterial nucleic acids and inhibits bacterial infection in red drum, Sciaenops ocellatus

Tissue factor pathway inhibitor (TFPI)-1 is well known for its role as an inhibitor of blood coagulation. Several studies have demonstrated that the C-terminal peptides of TFPI-1 are active against a broad spectrum of microorganisms. In a previous study, we found that TO17 (with 17 amino acids), a TFPI-1 C-terminal peptide from red drum (Sciaenops ocellatus), was active against Edwardsiella tarda. In the present study, we investigated further the antimicrobial spectrum, action mode, as well as the immunostimulatory property of TO17. Our results showed that TO17 displayed antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Vibrio vulnificus, and infectious spleen and kidney necrosis virus, independent of host serum. Furthermore, the activity of TO17 was influenced by the length or type of amino acids at the N and C termini. During its interaction with V. vulnificus, TO17 exerted its antibacterial activity by destroying cell membrane integrity, penetrating the cytoplasm and inducing degradation of genomic DNA and total RNA. In addition, TO17 had no hemolytic activity against red drum blood cells. In vitro, TO17 enhanced production of nitric oxide and bactericidal activity of red drum macrophages. In vivo, administration of red drum with TO17 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. These results indicate that TO17 is a broad-spectrum antimicrobial peptide with immunostimulatory properties and it has the potential to be used as an antimicrobial agent in aquaculture.

English

Intensive use of antimicrobial agents in aquaculture provides a selective pressure creating reservoirs of drug-resistant bacteria and transferable resistance genes in fish pathogens and other bacteria in the aquatic environment. From these reservoirs, resistance genes may disseminate by horizontal gene transfer and reach human pathogens, or drug-resistant pathogens from the aquatic environment may reach humans directly. This study aims at identifying the antibiotic sensitivity patterns of Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli isolates from fish ponds in two localities (Ashiaman and Dawhenya) in the Greater Accra region of Ghana. A total of 43 isolates were tested using the Kirby-Bauer agar disc diffusion method against Ciprofloxacin (5 µg), Erythromycin (15 µg), Cefuroxime (30 µg), Gentamicin (10 µg), and Tetracycline (30 µg).P. aeruginosa was the most isolated organism with 90% prevalence, followed by E. coli (75%) and S. typhi (50%). All the P. aeruginosa isolates were resistant to Erythromycin and Cefuroxime while 90% of the S. typhi isolates were resistant to Tetracycline and Erythromycin. E. coli isolates showed 100, 93.33 and 66.66% resistance to Erythromycin, Cefuroxime and Tetracycline respectively. Gentamicin, Tetracycline and Ciprofloxacin were sensitive to 66.66, 61.10 and 50% of P. aeruginosa isolates respectively. Ciprofloxacin was sensitive to 90% of the S. typhi isolates whiles Gentamicin was sensitive to 70% of the S. typhi isolates. Ciprofloxacin and Gentamicin were more sensitive to E. coli isolates (80 and 66.66% sensitivity respectively). Multi-drug resistant strains were obtained in 77.78% of P. aeruginosa and 70% of S. typhi isolates whiles 66.67% of E. coli isolates were also resistant to more than two classes of the antibiotics tested. High levels of microbial resistance were observed in the isolates with 72.09% of isolates being multidrug resistant strains. Key words: Resistance patterns, multi-drug resistance, fish ponds, antibiotics.