Functional and mechanistic studies of a phytogenic formulation, Shrimp Best, in growth performance and vibriosis in whiteleg shrimp.

Climate change and disease threaten shrimp farming. Here, we studied the beneficial properties of a phytogenic formulation, Shrimp Best (SB), in whiteleg shrimp. Functional studies showed that SB dose-dependently increased shrimp body weight and decreased feed conversion ratio. We found that SB protected against Vibrio parahaemolyticus as evidenced by survival rate, bacterial load, and hepatopancreatic pathology in shrimp. Finally, we explored the likely mechanism by which SB affects growth performance and vibriosis in shrimp. The 16S rRNA sequencing data showed that SB increased 6 probiotic genera and decreased 6 genera of pathogenic bacteria in shrimp. Among these, SB increased the proportion of Lactobacillus johnsonii and decreased that of V. parahaemolyticus in shrimp guts. To dissect the relationship among SB, Lactobacillus and Vibrio, we investigated the in vitro regulation of Lactobacillus and Vibrio by SB. SB at ≥ 0.25 µg/mL promoted L. johnsonii growth. Additionally, L. johnsonii and its supernatant could inhibit V. parahaemolyticus. Furthermore, SB could up-regulate five anti-Vibrio metabolites of L. johnsonii, which caused bacterial membrane destruction. In parallel, we identified 3 fatty acids as active compounds from SB. Overall, this work demonstrated that SB improved growth performance and vibriosis protection in shrimp via the regulation of gut microbiota.

Multidrug resistant Vibrio spp. identified from mussels farmed for human consumption in Central Italy.

AIMS: This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy. METHODS AND RESULTS: A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aacC2 (62.0%) and aadA (58.0%) for aminoglycosides, blaTEM (54.0%) for beta-lactams, qnrS (24.0%) for quinolones, tetD (66.0%) for tetracyclines, and vanB (60.0%) for glycopeptides were mainly amplified by PCR assays. CONCLUSIONS: Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic "dark world".

Oral Administration of Antimicrobial Peptide NZ2114 Through the Microalgal Bait Tetraselmis subcordiformis (Wille) Butcher for Improving the Immunity and Gut Health in Turbot (Scophthalmus maximus L.).

Antibiotics are widely used in aquaculture to treat the bacterial diseases. However, the improper use of antibiotics could lead to environmental pollution and development of resistance. As a safe and eco-friendly alternative, antimicrobial peptides (AMPs) are commonly explored as therapeutic agents. In this study, a mutant strain of Tetraselmis subcordiformis containing AMP NZ2114 was developed and used as an oral drug delivery system to reduce the use of antibiotics in turbot (Scophthalmus maximus) aquaculture. The gut, kidney, and liver immune-related genes and their effects on gut digestion and bacterial communities in turbot fed with NZ2114 were evaluated in an 11-day feeding experiment. The results showed that compared with the group fed with wild-type T. subcordiformis, the group fed with T. subcordiformis transformants containing NZ2114 was revealed with decreased levels of both pro-inflammatory factors (TNF-α and IL-1ß), inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio splendidus demonstrated by the in vitro simulation experiments, and increased richness and diversity of the gut microbiota of turbot. In conclusion, our study provided a novel, beneficial, and low-cost method for controlling bacteria in turbot culture through the oral drug delivery systems.

Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.

Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.

AMR Threat Perception Assessment of Heterotrophic Bacteria From Shrimp Aquaculture Through Epidemiological Cut off Values.

BACKGROUND: Emergence and dissemination of antibiotic resistance is one of the major risks associated with the rampant usage of antibiotics in food-producing animals including aquaculture. OBJECTIVE: To determine Epidemiological Cut-OFF (ECOFF) values of heterotrophic bacterial populations from shrimp culture environments against five different antibiotics. METHODS: In this present study, bacterial samples were isolated from Penaeus vannamei culture environment in different locations of Andhra Pradesh, which is the aquaculture hub of India. The bacterial isolates were assessed for antibiotic resistance towards five antibiotics belonging to different classes (oxytetracycline, chloramphenicol, erythromycin, ciprofloxacin, and co-trimoxazole) by the disc diffusion method. Determination of Epidemiological Cut-OFF (ECOFF) values and analysis by employing normalized resistance interpretation (NRI) was carried out. RESULTS: The most dominant bacterial populations from shrimp culture were Vibrio spp. (pathogenic bacteria) followed by Bacillus spp. (probiotic bacteria). The bacterial isolates showed highest resistance towards oxytetracycline (overall 23.38%) and in location L6 (59.4%) followed by co-trimoxazole (31.1%). ECOFF values calculated by employing NRI showed that the disc diffusion data were distributed in a normalized manner. The maximum ECOFF value was obtained for ciprofloxacin (23.32 mm), while the minimum value was observed for oxytetracycline (9.05 mm). The antibiotic resistant phenotypes showed that the majority of the heterotrophic bacterial isolates (>60%) belonged to the non-wild type phenotype and primarily towards oxytetracycline (90%). CONCLUSION: The presence of non-wild antibiotic-resistant phenotypes of heterotrophic bacterial populations (which include not only pathogenic bacteria but also probiotic bacteria) indicates that shrimp culture ponds may be a reservoir for drug-resistant bacteria and there is a greater risk associated with transmission of resistant genes across bacterial flora. HIGHLIGHTS: NRI analysis of antibiotic disc diffusion data of heterotrophic bacterial populations in shrimp aquaculture environments revealed that majority of them belonged to non-wild type (90%) paticularly to oxytetracycline in comparison to other studied antibiotics (chloramphenicol, erythromycin, ciprofloxacin and co-trimoxazole).

Phytochemical analysis and antibacterial activity of palm waste extract against Vibrio harveyi and Vibrio parahaemolyticus.

Vibrio harveyi and Vibrio parahaemolyticus are species of the Vibrio genus that often cause disease and mass mortality in crustaceans. If not handled quickly and appropriately, these diseases can cause considerable losses to farmers. Therefore, it is necessary to find a solution with safe and environmentally friendly disease prevention technology using natural ingredients, among others from plants, namely oil palm. Some parts of oil palm, namely leaves, fronds, fibres and oil palm pulp, which are palm waste, contain antibacterial compounds. This study aimed to assess the antibacterial activity of palm waste extracts, namely pulp, leaves, fronds and fibres using n-hexane, ethyl acetate, chloroform, ethanol and water maceration solvents against pathogenic bacteria V. harveyi and V. parahaemolyticus, and identify active compounds contained in palm waste. The results of the research are expected to produce innovative and sustainable solutions to control diseases in shrimp farming, contribute to the development of a sustainable fishing industry and open up the potential for utilizing palm waste as a value-added resource in the field of aquatic health. The results of observations on antibacterial activity tests and identifying the content of palm waste extract compounds were analysed descriptively displayed in the form of figures, tables and graphs. The results showed that palm waste extracts (pulp, leaves, fronds and fibres) with ethyl acetate and ethanol maceration solvents had very strong antibacterial potential, namely 20.14 ± 0.31 mm-25.52 ± 1.42 mm on V. harveyi bacteria and 20.41 ± 0.55 mm-25.00 ± 0.51 mm on V. parahaemolyticus bacteria. Palm extracts with n-hexane (>20 mm) and chloroform solvents generally have strong category antibacterial potential (10-20 mm), and palm extracts in water solvents have medium category potential (5-10 mm) against V. harveyi and V. parahemolyticus bacteria. The results of phytochemical tests on palm waste extracts with ethyl acetate and ethanol maceration solvents contain bioactive compounds of flavonoids, saponins, polyphenols and alkaloid tannins, steroids and triterpenoids. Palm extracts with n-hexane and chloroform solvents generally contain saponins, alkaloids, steroids and triterpenoids, while palm waste extracts with water solvents contain saponins.

Oxepine-containing pyrazinopyrimidine alkaloids and quinolinone derivatives produced by Aspergillus versicolor AS-212, a deep-sea-derived endozoic fungus.

Four new oxepine-containing pyrazinopyrimidine alkaloids, versicoxepines A - D (1-4), two quinolinone alkaloid analogs including 3-hydroxy-6-methoxy-4-phenylquinolin-2(1H)-one (5) and 3-methoxy-6-hydroxy-4-phenylquinolin-2(1H)-one (6) which were new naturally occurring compounds, together with two known compounds (7 and 8) were isolated from Aspergillus versicolor AS-212, an endozoic fungus isolated from the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts in the Western Pacific Ocean. Their structures were determined by extensive analysis of the spectroscopic and X-ray crystallographic data as well as by chiral HPLC analysis, ECD calculation, and DP4+ probability prediction. Structurally, versicoxepines B and C (2 and 3) represent the first example of a new oxepine-containing pyrazinopyrimidine alkaloid whose cyclic dipeptide moiety is composed of the same type of amino acid (Val or Ile). Compound 5 displayed antibacterial activity against aquatic pathogens, Vibrio harveyi and V. alginolyticus, with MICs of 8 µg/mL.

Occurrence of Virulence and Antimicrobial Resistance Determinants in Vibrio harveyi Isolated from Marine Food Fish Cultured in Korea.

Vibrio harveyi is a significant cause of infection in both marine animals and humans. It has been reported frequently in seafood-borne infections worldwide. This study was conducted to determine the potential health impact of the V. harveyi isolated from marine food fish cultured in Korea concerning their virulence and antimicrobial resistance. A total of 49 V. harveyi samples were isolated by biochemical tests and multiplex PCR. Phenotypic detection of virulence factors resulted DNase activity (81.63%), hemolysis (α = 75.51% and ß = 12.25), gelatinase activity (71.43%), protease production (71.43%), phospholipase activity (65.31%), and lipase production (34.69%). Virulence genes, including VPI, tlh, tdh, toxR, VAC, and ctxAB, were detected in 57.14%, 44.90%, 36.73%, 22.45%, 12.24%, and 8.16% of the isolates, respectively. Resistance to ampicillin (77.55%), oxacillin (69.39%), nalidixic acid (53.06%), amoxicillin (46.94%), oxytetracycline (46.94%), colistin sulfate (34.69%), fosfomycin (34.69%), chloramphenicol (32.65%), streptomycin (32.65%), cephalothin (28.57%), oxytetracycline (26.53%), ceftriaxone (20.41%), erythromycin (14.29%), and cefoxitin (12.24%) was detected in disc diffusion assay. Most of the isolates were classified as multidrug resistant as they scored multiple antimicrobial resistance index ≥0.2. Furthermore, antimicrobial resistance genes tetB, qnrA, intI1 (Class 1 integron integrase), aac(6')-Ib, blaSHV, blaCTX-M, strA-strB, tetA, aphAI-IAB, qnrC, qnrS, and blaTEM were found in 81.63%, 67.35%, 61.22%, 46.94%, 44.90%, 44.90%, 36.73%, 18.37%, 10.20%, 10.20%, 8.16% and 6.12% of the isolates, respectively. In conclusion, the development of antimicrobial resistance among V. harveyi will ultimately reduce the efficacy of antimicrobials used for treating and can favor the development of more virulent V. harveyi strains.

Calreticulin functions in antimicrobial immunity of obscure puffer Takifugu obscurus.

Calreticulin (Crt) is a highly conserved and multi-functional protein with lectin-like properties and important immunological activities. In this study, a Crt homolog, namely, ToCrt, was cloned and characterized from the obscure puffer Takifugu obscurus with an open reading frame of 1278 bp encoding a putative protein of 425 amino acids. The deduced amino acid sequence of ToCrt consisted of three conserved structural domains: N-domain, P-domain, and C-terminal domain. In the phylogenetic tree, ToCrt formed a separate cluster with three Crts from other pufferfish species (Takifugu rubripes, Takifugu flavidus, and Takifugu bimaculatus). The mRNA transcript of ToCrt was ubiquitously expressed in all the examined tissues in a decreasing order: liver, spleen, kidney, gills, intestine, and heart. After Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila stimulations, the levels of ToCrt mRNA in the kidney and spleen were significantly upregulated compared with that in the control group. The recombinant calreticulin domain of ToCrt (rToCrt) could bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and polysaccharides from bacterial cell walls such as lipopolysaccharide and peptidoglycan. Meanwhile, rToCrt could agglutinate different kinds of microorganisms and exhibit antimicrobial activity. These results suggested that T. obscurus ToCrt could serve as an antimicrobial effector in the host immune response against invading microorganisms.

Analysis of the antimicrobial resistance gene frequency in whole-genome sequenced Vibrio from Latin American countries.

Vibrio species are important environmental-related bacteria responsible for diverse infections in humans due to consumption of contaminated water and seafood in underdeveloped areas of the world. This study aimed to investigate the frequency of antimicrobial resistance genes in 577 sequenced Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus strains isolated in Latin American countries available at the NCBI Pathogen Detection database and to determine the sequence type (ST) of the strains. Almost all strains studied (99.8%) carried at least one antimicrobial resistance gene, while 54.2 % presented a multidrug-resistance profile. The Vibrio strains exhibited genotypic resistance to 11 antimicrobial classes and almG, varG, and catB9, which confer resistance to antibiotic peptides, ß-lactams and amphenicols, respectively, were the most detected genes. Vibrio parahaemolyticus and V. vulnificus showed a broad diversity of STs. Vibrio cholerae strains isolated in Haiti after 2010's earthquake presented the highest diversity and amount of resistance genes in the set of strains analysed and mostly belonged to ST69. In conclusion, the detection of resistance genes from 11 antimicrobial classes and the high number of multidrug-resistant Vibrio species strains emphasize that Latin American public health authorities should employ more efficient control measures and that special attention should be given for the rational use of antimicrobials in human therapy and aquaculture, since the consumption of contaminated water and seafood with resistant Vibrio may result in human infections difficult to be treated.

Antibacterial activity of four recombinant carbohydrate recognition domain proteins identified from the kuruma shrimp Marsupenaeus japonicus.

The carbohydrate recognition domain (CRD) is the key component of C-type lectins (CTLs) with the capacity to recognize and eliminate invading pathogens. Herein, the recombinant proteins of four CRDs identified from the kuruma shrimp, Marsupenaeus japonicus, were produced and purified by an Escherichia coli expression system and affinity chromatography. Bacterial binding and antibacterial assays showed that the four CRDs displayed various bacterial binding and antibacterial activities against different bacteria. Among the four recombinant CRDs, His-CRD2-3 exhibited the broadest spectrum of bacterial binding and antibacterial activities against gram-negative bacteria (Vibrio parahaemolyticus, V. alginolyticus and V. harveyi) and gram-positive bacteria (Staphylococcus aureus and Micrococcus lysodeikticus). Moreover, the four recombinant CRDs showed different capacities to regulate the expression of several immune effector genes (MjCTL3, MjCTL4, MjCTL, Mjily and Mjsty), among which His-CRD2-3 displayed broader and stronger inductive effects on these immune effector genes. This study indicated that the four CRDs participated in immune defense by binding and killing bacteria and regulating the transcription of other immune effector genes. In addition, our results suggested that His-CRD2-3 might be a promising agent for the prevention and treatment of bacteriosis.

Anti-vibrio and immune-enhancing activity of medicinal plants in shrimp: A comprehensive review.

Disease epidemics in shrimp aquaculture increase apace with the development of aquaculture systems throughout the world. The disease caused by Vibrio spp. (vibriosis) is considered the most devastating, which has made it the most feared bacterial disease in the shrimp sector. In aquaculture, several strategies have already been applied to control Vibrio strains, including chemicals, probiotics, antibiotics, natural products from plants, including plant oils; hence, there has been considerable attention for using plants in shrimp aquaculture to provide sustainable, eco-friendly and safe compounds, such as alkaloids, saponins, terpenoids and flavonoids for replacing chemical compounds and antibiotics in current aquaculture. Medicinal plants may also have immunostimulating activity, increase growth and resistance in shrimps. The present paper aims to review the inhibition of Vibrio spp. in shrimp by medicinal plants, using both in vitro or/and in vivo techniques. Several medicinal plants appear capable of inhibiting growth of Vibrio pathogens outside living shrimp or in the body of shrimp, through enhancing growth and immune capacity when shrimps are fed or injected with them. In the current review Gracilaria spp. (Gracilariaceae family) and Sargassum spp. (family Sargassaceae) have been used most for in vitro and in vivo experiments. Among the terrestrial plants, Eucalyptus camaldulensis, Psidium guajava, Rhodomyrtus tomentosa, and Syzygium cumini (Myrtaceae family) had significant activity against Vibrio.

Multidirectional anti-melanoma effect of galactolipids (MGDG-1 and DGDG-1) from Impatiens parviflora DC. and their synergy with doxorubicin.

The anti-melanoma potential of galactolipids: MGDG-1 and DGDG-1, isolated from Impatiens parviflora, and their synergistic effect with anticancer drug - doxorubicin (DOX) was investigated. Both compounds demonstrated time- and dose-dependent cytotoxicity against human melanoma cells of different metastatic potential. MGDG-1 was more effective than DGDG-1, with the highest activity against A375 cell line (IC50 = 15.14 µg/mL). Both compounds acted selectively, were devoid of hepatotoxicity or mutagenicity. Additionally, MGDG-1 proved to be a tyrosinase inhibitor. Co-administration of MGDG-1 and DGDG-1 with DOX revealed a synergistic cytotoxic effect on melanoma cells. The cytotoxicity of all tested MGDG-1/DOX and DGDG-1/DOX cocktails was considerably higher than that of each agent administered alone. MGDG-1/DOX (Mix3) reduced the viability of A375 melanoma cells almost totally and this effect was 2-fold more potent as compared to DOX alone. Our study indicates that the overall effect is enhanced with the increasing concentration of MGDG-1 in the cocktail. These results open up a possibility for lowering therapeutic doses of chemotherapeutics such as doxorubicin when co-administrated with galactolipids. Thus, MGDG-1 can be prospectively considered as multidirectional anti-melanoma agent and can be recommended for further in vitro and in vivo studies, especially in search for effective combined therapy.

Two New Phenol Derivatives from the Cold Seep-Derived Fungus Aspergillus insuetus SD-512.

Two new phenol derivatives, namely insphenol A (1) and acetylpeniciphenol (2), along with seven known analogs (3-9), were isolated from the deep-sea cold seep-derived fungus, Aspergillus insuetus SD-512. The structures of 1 and 2 were established by extensive interpretation of NMR and mass spectroscopic data. The absolute configuration of 1 was determined by the combination of coupling constant analysis and acid hydrolysis. Among the isolated compounds, insphenol A (1) represents the first example of isopentenyl phenol derivative with a unique 1-glycosylation from the species Aspergillus insuetus. The isolated new compounds were evaluated for antibacterial activities against six human or aquatic pathogens, while compound 2 exhibited inhibitory effect against Edwardsiella tarda, Vibrio alginolyticus, and V. vulnificus, with MIC values of 4, 8, and 8 µg/mL, respectively.

Characterization of a novel shrimp pathogen, Vibrio brasiliensis, isolated from Pacific white shrimp, Penaeus vannamei.

A novel pathogenic strain Vibrio 20190611023 was isolated from the hepatopancreas of moribund cultured Penaeus vannamei suffering from black gill disease. This strain was identified as V. brasiliensis based on the phylogenetic analyses of 16S rDNA gene and five other housekeeping genes (i.e., gapA, ftsZ, mreB, topA and gyrB). Some biochemical features of this strain were determined with an API 20NE system, and its haemolytic activity was determined using a sheep blood agar plate. The pathogenicity of this isolate 20190611023 was confirmed by the experimental challenge tests and histopathological examinations. P. vannamei were challenged via reverse gavage with different doses of bacterial suspensions. The calculated median lethal dose (LD50 ) was (3.16 ± 1.78) × 105  CFU/g (body weight). Moreover, antibiotic susceptibility tests were performed, the results of which showed that the strain 20190611023 was sensitive to chloramphenicol, compound sulphamethoxazole, ciprofloxacin, doxycycline and oxacillin, but resistant to erythromycin, kanamycin, gentamicin, cefoperazone, ceftriaxone, cefamezin and piperacillin. To our knowledge, this is the first report for demonstrating V. brasiliensis as a shrimp pathogen, which expands the host range of V. brasiliensis infection. The present study highlights that more attention should be paid to this novel pathogen in intensive shrimp aquaculture.

Probiotics: their action against pathogens can be turned around.

Probiotics when applied in complex evolving (micro-)ecosystems, might be selectively beneficial or detrimental to pathogens when their prophylactic efficacies are prone to ambient interactions. Here, we document a counter-intuitive phenomenon that probiotic-treated zebrafish (Danio rerio) were respectively healthy at higher but succumbed at lower level of challenge with a pathogenic Vibrio isolate. This was confirmed by prominent dissimilarities in fish survival and histology. Based upon the profiling of the zebrafish microbiome, and the probiotic and the pathogen shared gene orthogroups (genetic niche overlaps in genomes), this consequently might have modified the probiotic metabolome as well as the virulence of the pathogen. Although it did not reshuffle the architecture of the commensal microbiome of the vertebrate host, it might have altered the probiotic-pathogen inter-genus and intra-species communications. Such in-depth analyses are needed to avoid counteractive phenomena of probiotics and to optimise their efficacies to magnify human and animal well-being. Moreover, such studies will be valuable to improve the relevant guidelines published by organisations such as FAO, OIE and WHO.

Dynamic and quantitative characterization of antagonism within disinfectant mixtures by a modified area-concentration ratio method.

The coexistence of various typical disinfectant pollutants has the potential to produce toxicity interaction towards organisms in the environment. A suitable model is necessary to evaluate the interaction quantitatively. Hence, the area-concentration ratio (ACR) method was modified (MACR) by combing confidence intervals to dynamically and quantitatively evaluate the toxicity interactions within disinfectant mixture pollutants. Disinfectant mixtures were designed by the direct equipartition design ray method using three guanidine disinfectants, chlorhexidine diacetate (CD), chlorhexidine (CHL), and polyhexamethylene biguanidine (POL) and one chlorine-containing disinfectant calcium hypochlorite (CAL). The toxicities of the four disinfectants and their mixtures towards Vibrio qinghaiensis sp.-Q67 (Q67) were determined by the time-dependent toxicity microplate analysis method. And the toxicity mechanism was analyzed by determining the effects of four disinfectants and their binary mixtures on the structure of cell, DNA and proteins (Pro) for Q67. The results show that the toxicities of CD and CHL to Q67vary little with time, but POL and CAL show the obvious time-dependent toxicity. The toxicities of CD, CHL and POL to Q67 are significantly stronger than that of CAL at the same exposure time. The toxicities of three binary mixture systems don't have significant difference in different exposure time. MACR can dynamically, quantitatively and accurately characterize toxicity interactions compared with ACR. According to MACR, the antagonism intensity dynamically changes with the prolongation of exposure time for binary mixture rays of three guanidine disinfectants and CAL, and linearly correlates with the components' concentration ratios. Four disinfectants all can destroy cell membrane and cause desaturation DNA of test organism, and CAL even can destroy the structure of DNA and protein. The probably reason for the antagonism within binary mixtures is the reaction between guanidine group and ClO-, which is called chemical antaogism.

Molecular docking study of bio-inhibitors extracted from marine macro-alga Ulva fasciata against hemolysin protein of luminescence disease-causing Vibrio harveyi.

Shrimp grow-out and hatchery systems are being affected by bacterial disease particularly Vibrios. The use of chemotherapeutic agents in aquaculture practices has to lead to the development of resistance among aquatic bacteria. Thus, health management becomes of major importance in aquaculture. Under this situation, progressing bio-inhibitors from marine resources are most appropriate to be considered against pathogenic bacteria. Molecular docking is an appropriate tool in structural biology and computer-assisted drug design to predict and neutralize a target protein of known diseases. In this study, marine macro-alga Ulva fasciata was aimed at developing inhibitors against luminescence disease-causing pathogenic bacteria Vibrio harveyi. U. fasciata was collected from Thoothukudi, Tamil Nadu, India. Extract of U. fasciata was tested against growth and virulence factors of V. harveyi during Penaeus monodon larviculture. Further U. fasciata extract was subjected to GC-MS analysis to identify the biomolecules. The homology modeling of virulent protein, hemolysin of V. harveyi was designed in this study. Hence, it was aimed for molecular docking against the biomolecules identified from U. fasciata extract. During shrimp larviculture, the extract of U. fasciata (200 µg mL-1) exhibited reduction on Cumulative Percentage of Mortality (32.40%) in postlarvae against challenge of V. harveyi infection. Biomolecule Methyl dehydroabietate had showed highest binding affinity among the compounds was evaluated in molecular docking study. Statistical analysis had revealed significant differences (p < 0.05) in trials. Therefore, it was proved that the bio-inhibitors from U. fasciata will be a better option for controlling luminescence disease-causing V. harveyi in shrimp grow-out practices.

Effects of subcutaneous injection of λ/κ-carrageenin on the immune and liver antioxidant status of gilthead seabream (Sparus aurata).

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1ß and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Immuno-stimulatory effect and toxicology studies of salt pan bacteria as probiotics to combat shrimp diseases in aquaculture.

The shrimp aquaculture industry has experienced serious economic losses due to diseases caused by Vibrio species. The application of antibiotics to combat diseases has led to environmental hazards, antibiotic-resistance in pathogens and accumulation of antibiotics in tissues. This study explores the use of probiotics as an alternative to antibiotics. A probiotic consortium SFSK4 (comprising salt pan bacteria Bacillus licheniformis TSK71, Bacillus amyloliquefaciens SK27, Bacillus subtilis SK07, Pseudomonas sp. ABSK55) was used as a water additive during shrimp culture. It significantly increased shrimp (Litopenaeus vannamei) immunity i.e. total hemocyte count, phagocytosis, total plasma protein, respiratory burst and bactericidal activity as compared to the control. It also stimulated the phenoloxidase activity by two-fold. Proteomic analysis revealed the differential expression of 50 immune proteins (39 up-regulated and 11 down-regulated) in SFSK4 treated shrimps. Four major immune modulation proteins viz. Caspase2, GTPase activating protein, Hemocyanin and Glucan pattern-recognition lipoprotein involved in cell mediated immune response were identified in SFSK4 treated shrimp hemolymph. SFSK4 decreased shrimp mortality by more than 50% against pathogens. Toxicology studies revealed that administration of the highest dose of probiotic (1012 CFU/mL) showed no adverse effect on shrimp survival (LC50 analysis) and neither exhibited cytotoxicity. Genotoxicity study confirmed that the probiotic did not cause DNA damage in shrimps. The findings suggest that the probiotic SFSK4 is an eco-friendly water additive to enhance shrimp immunity against diseases in aquaculture, which could help curtail environmental hazards as an effective alternative to antibiotics.