Evaluation of the Biological Activities of Peptides from Epidermal Mucus of Marine Fish Species from Chilean Aquaculture.

The skin of fish is a physicochemical barrier that is characterized by being formed by cells that secrete molecules responsible for the first defense against pathogenic organisms. In this study, the biological activity of peptides from mucus of Seriola lalandi and Seriolella violacea were identified and characterized. To this purpose, peptide extraction was carried out from epidermal mucus samples of juveniles of both species, using chromatographic strategies for purification. Then, the peptide extracts were characterized to obtain the amino acid sequence by mass spectrometry. Using bioinformatics tools for predicting antimicrobial and antioxidant activity, 12 peptides were selected that were chemically produced by simultaneous synthesis using the Fmoc-Tbu strategy. The results revealed that the synthetic peptides presented a random coil or extended secondary structure. The analysis of antimicrobial activity allowed it to be discriminated that four peptides, named by their synthesis code 5065, 5069, 5070, and 5076, had the ability to inhibit the growth of Vibrio anguillarum and affected the copepodite stage of C. rogercresseyi. On the other hand, peptides 5066, 5067, 5070, and 5077 had the highest antioxidant capacity. Finally, peptides 5067, 5069, 5070, and 5076 were the most effective for inducing respiratory burst in fish leukocytes. The analysis of association between composition and biological function revealed that the antimicrobial activity depended on the presence of basic and aromatic amino acids, while the presence of cysteine residues increased the antioxidant activity of the peptides. Additionally, it was observed that those peptides that presented the highest antimicrobial capacity were those that also stimulated respiratory burst in leukocytes. This is the first work that demonstrates the presence of functional peptides in the epidermal mucus of Chilean marine fish, which provide different biological properties when the fish face opportunistic pathogens.

New Cyclic Pentapeptides from the Mangrove-Derived Aspergillus fumigatus GXIMD 03099.

Four new cyclic pentapeptides, avellanins D-G (1-4), together with four known compounds (5-8), were isolated from a mangrove-derived Aspergillus fumigatus GXIMD 03099 fungus from Acanthus ilicifolius L. Their structures were elucidated by analysis of HRESIMS, NMR, and ESI-MS/MS data. Their absolute configurations were determined by X-ray diffraction analysis and Marfey's method. Compounds 1-8 were screened for insecticidal and antibacterial activities. Compound 2 showed insecticidal activity against newly hatched larvae of Culex quinquefasciatus with an LC50 value of 86.6 µM; compound 4 had weak activity against Vibrio harveyi with an MIC value of 5.85 µM.

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Cyclic olefin copolymer (COC) as a promising biomaterial for affecting bacterial colonization: investigation on Vibrio campbellii.

Cyclic olefin copolymer (COC) has emerged as an interesting biocompatible material for Organ-on-a-Chip (OoC) devices monitoring growth, viability, and metabolism of cells. Despite ISO 10993 approval, systematic investigation of bacteria grown onto COC is a still not documented issue. This study discusses biofilm formations of the canonical wild type BB120 Vibrio campbellii strain on a native COC substrate and addresses the impact of the physico-chemical properties of COC compared to conventional hydroxyapatite (HA) and poly(dimethylsiloxane) (PDMS) surfaces. An interdisciplinary approach combining bacterial colony counting, light microscopy imaging and advanced digital image processing remarks interesting results. First, COC can reduce biomass adhesion with respect to common biopolymers, that is suitable for tuning biofilm formations in the biological and medical areas. Second, remarkably different biofilm morphology (dendritic complex patterns only in the case of COC) was observed among the examined substrates. Third, the observed biofilm morphogenesis was related to the interaction of COC with the conditioning layer of the planktonic biological medium. Fourth, Level Co-occurrence Matrix (CGLM)-based analysis enabled quantitative assessment of the biomass textural fractal development under different coverage conditions. All of this is of key practical relevance in searching innovative biocompatible materials for pharmaceutical, implantable and medical products.

Whole-genome sequencing of Pseudoalteromonas piscicida 2515 revealed its antibacterial potency against Vibrio anguillarum: a preliminary invitro study.

Pseudoalteromonas piscicida 2515, isolated from Litopenaeus vannamei culture water, is a potential marine probiotic with broad anti-Vibrio properties. However, genomic information on P. piscicida 2515 is scarce. In this study, the general genomic characteristics and probiotic properties of the P. piscicida 2515 strain were analysed. In addition, we determined the antibacterial mechanism of this bacterial strain by scanning electron microscopy (SEM). The results indicated that the whole-genome sequence of P. piscicida 2515 contained one chromosome and one plasmid, including a total length of 5,541,406 bp with a G + C content of 43.24%, and 4679 protein-coding genes were predicted. Various adhesion-related genes, amino acid and vitamin metabolism and biosynthesis genes, and stress-responsive genes were found with genome mining tools. The presence of genes encoding chitin, bromocyclic peptides, lantibiotics, and sactipeptides showed the strong antibacterial activity of the P. piscicida 2515 strain. Moreover, in coculture with Vibrio anguillarum, P. piscicida 2515 displayed vesicle/pilus-like structures located on its surface that possibly participated in its bactericidal activity, representing an antibacterial mechanism. Additionally, 16 haemolytic genes and 3 antibiotic resistance genes, including tetracycline, fluoroquinolone, and carbapenem were annotated, but virulence genes encoding enterotoxin FM (entFM), cereulide (ces), and cytotoxin K were not detected. Further tests should be conducted to confirm the safety characteristics of P. piscicida 2515, including long-term toxicology tests, ecotoxicological assessment, and antibiotic resistance transfer risk assessment. Our results here revealed a new understanding of the probiotic properties and antibacterial mechanism of P. piscicida 2515, in addition to theoretical information for its application in aquaculture.

Probiotic potential of a novel endophytic Streptomyces griseorubens CIBA-NS1 isolated from Salicornia sp. against Vibrio campbellii infection in shrimp.

A novel endophytic Streptomyces griseorubens CIBA-NS1 was isolated from a salt marsh plant Salicornia sp. The antagonistic effect of S. griseorubens against Vibrio campbellii, was studied both in vitro and in vivo. The strain was validated for its endophytic nature and characterized through scanning electron microscopy, morphological and biochemical studies and 16SrDNA sequencing. The salinity tolerance experiment has shown that highest antibacterial activity was at 40‰ (16 ± 1.4 mm) and lowest was at 10 ‰ salinity (6.94 ± 0.51 mm). In vivo exclusion of Vibrio by S. griseorubens CIBA-NS1 was studied in Penaeus indicus post larvae and evaluated for its ability to improve growth and survival of P. indicus. After 20 days administration of S. griseorubens CIBA-NS1, shrimps were challenged with V. campbellii. The S. griseorubens CIBA-NS1 reduced Vibrio population in test group when compared to control, improved survival (60.5 ± 6.4%) and growth, as indicated by weight gain (1.8 ± 0.05g). In control group survival and growth were 48.4 ± 3.5% and 1.4 ± 0.03 g respectively. On challenge with V. campbellii, the S. griseorubens CIBA-NS1 administered group showed better survival (85.6 ± 10%) than positive control (64.3 ± 10%). The results suggested that S. griseorubens CIBA-NS1 is antagonistic to V. campbellii, reduce Vibrio population in the culture system and improve growth and survival. This is the first report on antagonistic activity of S. griseorubens isolated from salt marsh plant Salicornia sp, as a probiotic candidate to prevent V. campbellii infection in shrimps.

Antagonistic and enzymatic activities of Bacillus species isolated from the fish gastrointestinal tract as potential probiotics use in Artemia culture.

Probiotics have been used successfully in aquaculture to enhance disease resistance, nutrition, and/or growth of cultured organisms. Six strains of Bacillus were isolated from the intestinal tracts of fish and recognised by conventional biochemical traits. The six isolated strains were Bacillus cereus and Bacillus subtilis using MALDI-TOF-MS technique. The probiotic properties of these Bacillus strains were studied. The tested bacillus strains exhibit antibacterial activity against the different pathogens. The strain S5 gave the important inhibition zones against most pathogens (20.5, 20.33, 23, and 21 mm against Vibrio alginolyticus, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella typhimurium, respectively). According to our results, all Bacillus strains have extracellular components that can stop pathogenic bacteria from growing. The enzymatic characterization showed that the tested strains can produce several biotechnological enzymes such as α-glucosidase, naphtol-AS-BI-Phosphohydrolase, esterase lipase, acid phosphatase, alkaline phosphatase, amylase, lipase, caseinase, and lecithinase. All Bacillus strains were adhesive to polystyrene. The adding Bacillus strains to the Artemia culture exerted significantly greater effects on the survival of Artemia. The challenge test on Artemia culture showed that the protection against pathogenic Vibrio was improved. These findings allow us to recommend the examined strains as prospective probiotic options for the Artemia culture, which will be used as food additives to improve the culture conditions of crustacean larvae and marine fish.

Characterization of tryptanthrin as an antibacterial reagent inhibiting Vibrio splendidus.

Isolates of Vibrio splendidus are ubiquitously presented in various marine environments, and they can infect diverse marine culture animals, leading to high mortality and economic loss. Therefore, a control strategy of the infection caused by V. splendidus is urgently recommended. Tryptanthrin is a naturally extracted bioactive chemical with antimicrobial activity to other bacteria. In this study, the effects of tryptanthrin on the bacterial growth and virulence-related factors of one pathogenic strain V. splendidus AJ01 were determined. Tryptanthrin (10 µg/mL) could completely inhibit the growth of V. splendidus AJ01. The virulence-related factors of V. splendidus AJ01 were affected in the presence of tryptanthrin. Tryptanthrin resulted an increase in biofilm formation, but lead to reduction in the motility and hemolytic activity of V. splendidus cells. In the cells treated with tryptanthrin, two distinctly differentially expressed extracellular proteins, proteases and flagellum, were identified using SDS-PAGE combined with LC-MS. Real-time reverse transcriptase PCR confirmed that the genes involved in the flagellar formation and hemolysin decreased, whereas specific extracellular proteases and the genes involved in the biofilm formation were upregulated. Two previously annotated luxOVs genes were cloned, and their expression levels were analyzed at different cell densities. Molecular docking was performed to predict the interaction between LuxOVs and ATP/tryptanthrin. The two sigma-54-dependent transcriptional regulators showed similar ATP or tryptanthrin binding capacity but with different sites, and the direct competitive binding between ATP and tryptanthrin was present only in their binding to LuxO1. These results indicated that tryptanthrin can be used as a bactericide of V. splendidus by inhibiting the growth, bacterial flagella, and extracellular proteases, but increasing the biofilm. Sigma-54-dependent transcriptional regulator, especially the quorum sensing regulatory protein LuxO1, was determined to be the potential target of tryptanthrin. KEY POINTS: • Tryptanthrin inhibited the growth of V. splendidus in a dose-dependent manner. • The effect of tryptanthrin on the virulence factors of V. splendidus was characterized. • LuxO was the potential target for tryptanthrin based on molecular docking.

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.

Prevalence and antimicrobial resistance of major foodborne pathogens isolated from pangas and tilapia fish sold in retail markets of Dhaka city, Bangladesh.

Fish sold at retail markets are often contaminated with harmful bacterial pathogens, posing significant health risks. Despite the growing aquaculture industry in Bangladesh to meet high demand, little attention has been paid to ensuring the safety of fish. The objective of this study was to evaluate the microbiological quality of tilapia and pangas fish sold in retail markets across Dhaka city, Bangladesh. Specifically, the study aimed to compare the quality of fish from traditional wet markets and modern supermarkets, as well as fish samples collected during morning and evening hours. A total of 500 raw cut-fish samples (250 tilapia and 250 pangas) were collected at the point of sale from 32 wet markets and 25 supermarkets. All samples were tested for Escherichia coli, extended-spectrum ß-lactamase-producing E. coli (ESBL-Ec), along with the foodborne pathogens Salmonella, Shigella, Vibrio, and Cryptosporidium spp. Bacterial isolates were characterized using antibiotic susceptibility tests (AST) and the presence of common virulence and antibiotic-resistant genes. Fish samples from retail markets had higher prevalence of tested bacteria including E. coli (92 %), V. cholerae (62 %), ESBL-Ec (48 %), and Salmonella spp. (24 %). There was a significant difference in the prevalence of E. coli (97 % vs. 71 %), ESBL-Ec (58 % vs. 8 %) and Salmonella spp. (28 % vs. 8 %) on the wet market samples compared to supermarket samples (p < 0.005). The mean concentration of E. coli on fish from the wet market was 3.0 ± 0.9 log10 CFU/g, while that from supermarkets was 1.6 ± 0.9 log10 CFU/g. The mean concentration of ESBL-Ec in fish from wet markets and supermarkets were 2.3 ± 0.8 log10 CFU/g and 1.6 ± 0.5 log10 CFU/g, respectively. AST revealed that 46 % of E. coli isolates were multi-drug resistant (MDR), while 4 %, 2 % and 5 % of E. coli, Salmonella spp. and Vibrio spp. isolates, respectively, were resistant to carbapenems. At least 3 % of total E. coli isolates were found to be diarrheagenic, while 40 % of Salmonella isolates harbored pathogenic genes (stn, bcfC, ssaQ, avrA and sodC1), and none of the V. cholerae isolates harbored ctxA and tcpA. Our research shows that raw-cut fish samples from retail markets are contaminated with pathogenic and antibiotic-resistant bacteria, which could be a significant food safety concern. Public health interventions should be implemented to improve food safety and hygiene practices in the retail fish markets.

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".

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.

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.

Long- and short-term dietary ß-glucan improves intestinal health and disease resistance in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

ß-Glucans are immunostimulants and are widely used in aquaculture industry. The present study was conducted to evaluate the effects of different periods of ß-glucan management on growth performance, intestinal health, and disease resistance in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). A commercial feed was used as control diet (CD), and the ß-glucan diet (ßD) was based on CD and further supplemented with 0.1% ß-glucan. Grouper in control and long-term ß-glucan diet (LGD) groups were fed with CD and ßD for 8 weeks, respectively. Groupers in short-term ß-glucan diet (SGD) group were fed with CD for the first 4 weeks and ßD for the last 4 weeks. We found that LGD and SGD had no effect on growth performance but reduced the mortalities of grouper after challenging with Vibrio harveyi. In addition, both LGD and SGD increased intestinal morphology, enhanced antioxidant capacity, enhanced immunity, inhibited apoptosis, altered the transcriptional profile, and activated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway in the intestine of grouper. Furthermore, the effect of LGD on most of the above parameters was comparable to that of SGD. In conclusion, LGD and SGD did not affect growth rate parameters but enhanced the intestinal health and disease resistance of pearl gentian grouper.

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.

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).

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.