New investigation of encoding secondary metabolites gene by genome mining of a marine bacterium, Pseudoalteromonas viridis BBR56.

Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA TM050. DNA purity and quantity were assessed using NanoDrop spectrophotometers and Qubit fluorometers. The DNA library and sequencing were prepared using Oxford Nanopore Technology GridION MinKNOW 20.06.9 with long read, direct, and comprehensive analysis. High accuracy base calling was assessed with Guppy version 4.0.11. Filtlong and NanoPlot were used for filtering and visualizing the FASTQ data. Flye (2.8.1) was used for de novo assembly analysis. Variant calls and consensus sequences were created using Medaka. The annotation of the genome was elaborated by DFAST. The assembled genome and annotation were tested using Busco and CheckM. Herein, we found that the highest similarity of the BBR56 isolate was 98.37% with the 16 S rRNA gene sequence of P. viridis G-1387. The genome size was 5.5 Mb and included chromosome 1 (4.2 Mbp) and chromosome 2 (1.3 Mbp), which encoded 61 pseudogenes, 4 noncoding RNAs, 113 tRNAs, 31 rRNAs, 4,505 coding DNA sequences, 4 clustered regularly interspaced short palindromic repeats, 4,444 coding genes, and a GC content of 49.5%. The sequence of the whole genome of P. viridis BBR56 was uploaded to GenBank under the accession numbers CP072425-CP072426, biosample number SAMN18435505, and bioproject number PRJNA716373. The sequence read archive (SRR14179986) was successfully obtained from NCBI for BBR56 raw sequencing reads. Digital DNA-DNA hybridization results showed that the genome of BBR56 had the potential to be a new species because no other bacterial genomes were similar to the sample. Biosynthetic gene clusters (BGCs) were assessed using BAGEL4 and the antiSMASH bacterial version. The genome harbored diverse BGCs, including genes that encoded polyketide synthase, nonribosomal peptide synthase, RiPP-like, NRP-metallophore, hydrogen cyanide, betalactone, thioamide-NRP, Lant class I, sactipeptide, and prodigiosin. Thus, BBR56 has considerable potential for further exploration regarding the use of its secondary metabolite products in the human and fisheries sectors.

Isolation, genetic characterization, and virulence profiling of different Aeromonas species recovered from moribund hybrid catfish (Clarias spp.).

Background and Aim: The high diversity of Aeromonas spp. results in various pathogenicity levels. This group of bacteria causes a serious disease named motile Aeromonas septicemia (MAS) in catfish (Clarias spp.). This study aimed to characterize the species and virulence gene diversity of Aeromonas spp. isolated from diseased catfish. Materials and Methods: Nine Aeromonas spp. were isolated from infected catfish cultivated in Java, Indonesia, and they were identified at the phenotypic and molecular levels (16S rDNA). The virulence genes assessed included aer/haem, alt, ast, flaA, lafA, and fstA. Results: Phylogenetic analysis identified nine isolates of Aeromonas spp.: Aeromonas hydrophila (11.11%), Aeromonas caviae (11.11%), Aeromonas veronii bv. veronii (44.44%), and Aeromonas dhakensis (33.33%). Virulence genes, such as aer/haem, alt, ast, flaA, lafA, and fstA, were detected in all isolates at frequencies of approximately 100%, 66.67%, 88.89%, 100%, 55.56%, and 66.67%, respectively. This study is the first report on A. dhakensis recovered from an Indonesian catfish culture. Furthermore, our study revealed the presence of A. veronii bv veronii, a biovar that has not been reported before in Indonesia. Conclusion: This finding confirms that MAS was caused by multiple species of Aeromonas, notably A. dhakensis and A. veronii bv veronii, within Indonesian fish culture. The presence of these Aeromonas species with multiple virulence genes poses a significant threat to the freshwater aquaculture industry.

Characteristics and Antioxidant Activity of Fucoidan from Sargassum hystrix: Effect of Extraction Method.

Fucoidan is a bioactive compound of brown seaweed with antioxidant characteristics. This study examined the aftermath of the extraction method on the yield, fucose content, xylose content, sulfate content, total sugar, antioxidant activity, and functional groups of fucoidan from Sargassum hystrix. The brown seaweed was extracted using 4 methods, namely, A (0.1 N HCl, room temperature, 24 h), B (2% CaCl2, 85°C, 4 h), C (85% ethanol, room temperature, 12 h), and D (0.5% EDTA, 70°C, 3 h). The antioxidant activity testing was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ferric-Reducing Antioxidant Power (FRAP), and Hydroxyl Radical Scavenging Activity (HRSA). The yield for methods of A, B, C, and D was 2.46 ± 0.30, 0.68 ± 0.34, 1.18 ± 0.15, and 0.62 ± 0.25%, with fucose content of 39.97 ± 4.82, 26.72 ± 3.38, 41.08 ± 9.49, and 40.62 ± 8.59%, xylose content of 8.07 ± 0.92, 5.63 ± 0.40, 6.80 ± 0.83, and 7.83 ± 1.83%, and the sulfate content of 11.47 ± 2.20, 15.31 ± 2.47, 30.62 ± 2.76, and 27.80 ± 3.59%. The result indicated the occurrence of a sulfate ester group in the functional group analysis with numerous similarities with the commercial fucoidan. The highest antioxidant activity of fucoidan from S. hystrix was found in method C, which was influenced by sulfate levels. Therefore, the extraction method of fucoidan from S. hystrix affects the characteristics and antioxidant activity.

New Report: Genome Mining Untaps the Antibiotics Biosynthetic Gene Cluster of Pseudoalteromonas xiamenensis STKMTI.2 from a Mangrove Soil Sediment.

The marine bacterium Pseudoalteromonas xiamenensis STKMTI.2 was isolated from a mangrove soil sediment on Setokok Island, Batam, Indonesia. The genome of this bacterium consisted of 4,563,326 bp (GC content: 43.2%) with 1 chromosome, 2 circular plasmids, 2 linear plasmids, 4,824 protein-coding sequences, 25 rRNAs, 104 tRNAs, 4 ncRNAs, and 1 clustered, regularly interspaced, short palindromic repeated (CRISPR). This strain possessed cluster genes which are responsible for the production of brominated marine pyrroles/phenols (bmp), namely, bmp8 and bmp9. Other gene clusters responsible for the synthesis of secondary metabolites were identified using antiSMASH and BAGEL4, which yielded five results, namely, non-ribosomal peptides, polyketide-like butyrolactone, Lant class I, and RiPP-like, detected in chromosome 1, while prodigiosin was detected in the unnamed plasmid 5. This suggests that these whole genome data will be of remarkable importance for the improved understanding of the biosynthesis of industrially important bioactive and antibacterial compounds produced by P. xiamenensis STKMTI.2.

Sargassum hystrix as a Source of Functional Food to Improve Blood Biochemistry Profiles of Rats under Stress.

This study was conducted to determine the influence of Sargassum hystrix powder (SHP) as an alternative source of functional food for treating in vivo stress by measuring levels of glucose, triacylglycerol, total cholesterol, and cortisol, and liver histopathology. Wistar rats aged 3 months and weighing 150~200 g were divided into 7 groups: normal control, fasting control, negative control (stress without adaptogen), and 4 experimental conditions (stress+0.18 mg/kg diazepam, stress+450 mg/kg pellet, stress+mixture of pellet with SHP 450 mg/kg, and stress+450 mg/kg of SHP). Intake of liquids and and body weight were measured daily. Blood samples were collected on day 0 (baseline), day 5, and day 10 to analyze levels of glucose, triacylglycerol, cholesterol, and cortisol. On day 10, rats were euthanized and livers were collected to observe the severity of inflammation. The results indicated that rats receiving SHP 450 mg/kg and the mixture of pellet with SHP 450 mg/kg showed a similar ability as those receiving diazepam 0.18 mg/kg to cope with stress, indicated by an improvement in all blood biochemistry parameters. Supplementation with SHP 450 mg/kg can be used as an alternative source of functional food for overcoming oxidative stress, as indicated by its ability to improve levels of blood glucose, triacylglycerol, total cholesterol, and cortisol, and to improve liver histology by decreasing severity of liver inflammation.

Alginate from Sargassum siliquosum Simultaneously Stimulates Innate Immunity, Upregulates Immune Genes, and Enhances Resistance of Pacific White Shrimp (Litopenaeus vannamei) Against White Spot Syndrome Virus (WSSV).

Although alginate is known as an immunostimulant in shrimp, the comprehensive and simultaneous study on its activity to resolve the relationship of the hematological parameters, upregulation of immune-related gene expression, and resistance to pathogen has not been found in shrimp. We performed experiments to evaluate the effect and mechanism of alginate from S. siliquosum on Pacific white shrimp immune system. Hematological parameters were examined after oral administration of Na alginate in the shrimp. White spot syndrome virus (WSSV) was injected to the shrimp at 14 days, and its copy number was examined quantitatively (qRT-PCR). Immune-related gene expression was evaluated by qRT-PCR. Alginate increased some hematological immune parameters of shrimp. Before WSSV infection, expression levels of Toll and lectin genes were upregulated. The lectin gene were upregulated post infection, and the Toll gene in all the treatments were downregulated, except the shrimps fed with alginate at 6.0 g kg-1 at 48 h post infection (hpi). The shrimps fed with alginate at 6.0 g kg-1 were the most resistant and gave the least WSSV copy number at 48 hpi. Resistance of shrimps fed the alginate-supplemented diets against WSSV was significantly higher compared to that of the control treatment with 56% and 10% of survival rates, respectively. Oral administration of alginate did not affect the growth and total protein plasma. At 120 h post challenge, alginate treatment at 6.0 g kg-1 exhibited the highest survival rate. It is concluded that oral administration of alginate enhanced the innate immunity by upregulating immune-related gene expression. Consequently, the enhancement of the shrimp innate immunity improves the resistance against WSSV infection.

Antibacterial activity of Indonesian red algae Gracilaria edulis against bacterial fish pathogens and characterization of active fractions.

In this study the crude ethyl acetate extracts and fractions from red algae Gracilaria edulis were studied against fish pathogens. G. edulis was extracted with ethyl acetate, fractionated in silica gel coloum chromatography and gradient hexane-ethyl acetate. The extract and fractions were subjected for bioassay guided isolation against fish pathogen Vibrio spp and Aeromonas hydrophila. Bioassays were accomplished by diffusion agar, bioauthography, and 96 well microplates format. Profiling chemical constituent was done by thin layer chromatography and GC-MS. Metabolites analysis using GC-MS yielded chemical constituents of active fractions as 9-hexadecenoic acid, hexadecanoic acid, 13-octadecenoic acid, 10-octadecenoic acid, eicosanoic acid, cholest-8-en-3-ol. The results provide in vitro scientific support to the possible application of Gracilaria edulis as antimicrobial agent that may contribute for treatment of bacterial infections in aquaculture.

Simultaneous Administration of Boesenbergia pandurata Extract and Vaccination to Stimulate Immune Response in Tilapia, Oreochromis niloticus.

BACKGROUND AND OBJECTIVE: The use of adjuvants or immunostimulants is often necessary to increase vaccine efficacy, in this study we evaluated the improvement of the immune response in tilapia treated by either oral and immersion administration with vaccine and Boesenbergia pandurata extract (BPE). MATERIALS AND METHODS: The initial concentration of BPE and the cell density of vaccine were 900 mg L-1 and 104 CFU mL-1 for oral administration while 106 CFU mL-1 for immersion, respectively. The extract and vaccine were mixed homogeneously in a ratio of 1:1. Further, the mixture was supplemented to feed at 1 mL g-1 feed. Tilapia with average initial body weight of 15 g were fed containing vaccine and BPE 3 times a day. The other group of fish was immersed with vaccine and BPE for 20 min. After 7th (d7), 14th (d14) and21th (d21) days of treatment, a challenge test was conducted by intramuscularly injection of 0.1 mL of Aeromonas hydrophila and Pseudomonas fluorescens mixture (1:1) at a density of 105 CFU mL-1. Antibody levels, total white blood cell (WBC) and phagocytic activity (PA) were evaluated to determine the immune improvement of the fish. Furthermore, relative percent survival (RPS) and the survival rate (SR) were evaluated at week 2 and 4 after challenge test. RESULT: Results indicated that the all parameters of tilapia immune system were increased (p<0.05) after 2-4 weeks of both administration methods. Meanwhile, the efficacy of the vaccine has increased by combining BPE treatment using immersion method better than oral method. The RPS of vaccination plus extract by immersion was 83-100% and by oral administration was 83-87%. CONCLUSION: The present results implied that B. pandurata extract boost the efficacy of the Pseudomonas sp. vaccine by increasing the immune system and diseases resistance in tilapia.

Innate immune-stimulating and immune genes up-regulating activities of three types of alginate from Sargassum siliquosum in Pacific white shrimp, Litopenaeus vannamei.

The Total Haemocyte Count (THC), phenoloxidase (PO), Superoxide Dismutase (SOD) activity, Phagocytic Activity/Index and Total Protein Plasma (TPP) were examined after feeding the white shrimp Litopenaeus vannamei with diets supplemented with three different types of alginates (acid, calcium and sodium alginates). Immune-related genes expression was evaluated by quantitative Real Time PCR (qRT-PCR). Results indicated that the immune parameters directly increased according to the doses of alginates and time. The 2.0 g kg(-1) of acid and sodium alginate treatments were gave better results. Four immune-related genes expression i.e. LGBP, Toll, Lectin, proPO were up regulated. It is therefore concluded that the supplementation of alginate of Sargassum siliquosum on the diet of L. vannamei enhanced the innate immunity as well as the expression of immune-related genes. It is the first report on the simultaneous evaluation of three alginate types to enhance innate immune parameters and immune-related genes expression in L. vannamei.

Analysis of hypoxia in the western interior parts of the Ariake Sea, Japan, using a box model.

To clarify the mechanism of hypoxia in the western interior parts of the Ariake Sea (WIAS), field observation data collected in the period of 1972-2004 were analyzed using a two-layer box model. Monthly averages of advection velocity, vertical diffusion coefficient (K(z)), and biochemical oxygen consumption rate (R) in WIAS were evaluated quantitatively during the above period. The estimated advection velocity comparatively corresponded to the observed residual flow pattern of bay head in summer and winter. The estimated K(z) was relatively high (0.6-5.3 cm(2) s( -1)) from September to March but lower (0.2-0.4 cm(2) s( -1)) from April to August. The estimated R ranged from 0.30 to 0.46 mg L( -1) day( -1) during May to August. In summer, the temporal variation of dissolved oxygen (DO) concentration in the lower layer was controlled largely by K ( z ) and R. Monthly variations of K(z), R, and degree of density stratification (P) in the 1970s, the 1980s, and the 1990s-early 2000s were analyzed. P, K ( z ), and R were not significantly different among the calculated periods (p = 0.93, 0.23, and 0.49). However, the variations of R in summer between the 1970s and the other calculated periods changed. DO consumption period was longer in the 1980s and the 1990s-early 2000s than in the 1970s. R in the 1980s was highest among the calculated periods. The increase in R in the 1980s was caused by the increase in organic matter load originating from red tide phytoplankton due to a decrease in the suspension feeders.

Bioactive substances produced by marine isolates of Pseudomonas.

Pseudomonas is a genus of non-fermentative gram-negative Gammaproteobacteria found both on land and in the water. Many terrestrial isolates of this genus have been studied extensively. While many produce bioactive substances, enzymes, and biosurfactants, other Pseudomonas isolates are used for biological control of plant diseases and bioremediation. In contrast, only a few marine isolates of this genus have been described that produce novel bioactive substances. The chemical structures of the bioactive substances from marine Pseudomonas are diverse, including pyroles, pseudopeptide pyrrolidinedione, phloroglucinol, phenazine, benzaldehyde, quinoline, quinolone, phenanthren, phthalate, andrimid, moiramides, zafrin and bushrin. Some of these bioactive compounds are antimicrobial agents, and dibutyl phthalate and di-(2-ethylhexyl) phthalate have been reported to be cathepsin B inhibitors. In addition to being heterogeneous in terms of their structures, the antibacterial substances produced by Pseudomonas also have diverse mechanisms of action: some affect the bacterial cell membrane, causing bacterial cell lysis, whereas others act as acetyl-CoA carboxylase and nitrous oxide synthesis inhibitors. Marine Pseudomonas spp. have been isolated from a wide range of marine environments and are a potential untapped source for medically relevant bioactive substances.

Anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of MC21-B, an antibacterial compound produced by the marine bacterium Pseudoalteromonas phenolica O-BC30T.

The aim of this study was to purify, characterise and evaluate the in vitro activity of MC21-B, an antibiotic produced by the marine bacterium Pseudoalteromonas phenolica O-BC30(T). MC21-B was purified by sequential silica and Cosmosil chromatography followed by high-performance liquid chromatography (HPLC). The chemical structure of MC21-B was determined by ultraviolet, infrared, electron impact mass and nuclear magnetic resonance spectrometric analyses. To evaluate its antibacterial activity, minimum inhibitory concentrations (MICs) against 10 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) as well as kill times were determined. Antifungal activity was determined by the paper disk diffusion method. Cytotoxicity against human cells was determined with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Based on spectrophotometric analyses, MC21-B was predicted to be a novel substance, 2,2',3-tribromobiphenyl-4,4'-dicarboxylic acid. MC21-B exhibited anti-MRSA activity against all 10 clinical isolates of MRSA, with MICs between 1 microg/mL and 4 microg/mL. MC21-B was highly active against Bacillus subtilis and Enterococcus serolicida but was inactive against Gram-negative bacteria and fungi. Furthermore, MC21-B exhibited cytotoxic activity against human normal dermal fibroblasts and human leukaemic (MOLT) cells at 3-12-fold higher concentrations than required for its antibacterial activity. These results demonstrated that MC21-B has high in vitro activity against MRSA and might be useful as a lead compound in developing new anti-MRSA substances.

Antibacterial activity of 2,4-diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga, against vancomycin-resistant Staphylococcus aureus.

Antibacterial activity of 2,4-diacetylphloroglucinol (DAPG) was evaluated against 23 vancomycin-resistant Staphylococcus aureus (VRSA) strains isolated from several Asian and European countries, Brazil, South Africa and USA, and against vancomycin-resistant Enterococcus spp (VRE) genotypes A, B and C. DAPG was active against a wide range of VRSA isolates as well as vancomycin hetero-resistant S. aureus (h-VRSA) at MIC 4 mg/l. This substance also had moderate activity against both VRE-A and -B at MIC 8 mg/l, but not against VRE-C at up to 16 mg/l. The activity of DAPG did not directly correlate with levels of vancomycin resistance in VRSA and VRE. These results suggest that DAPG might be useful in developing new antibiotics against VRSA.

Pseudoalteromonas phenolica sp. nov., a novel marine bacterium that produces phenolic anti-methicillin-resistant Staphylococcus aureus substances.

Four strains of aerobic, Gram-negative rods, motile by means of a single polar flagellum, that produced phenolic anti-methicillin-resistant Staphylococcus aureus (MRSA) substances and brown-pigmented colonies, were isolated from sea water. The G + C content of the DNA ranged from 39.9 to 40.6 mol%. The isolates grew at 18-37 degrees C and pH 6.5-9.5 (optimal pH 7.5-9) and in medium containing 1-5% (w/v) NaCl (optimal NaCl concentration 2-3.5%). The isolates grew optimally in medium dissolved in 40-100% artificial sea water. Based on 16S rDNA similarities, the novel strains were closely related to Pseudoalteromonas luteoviolacea and Pseudoalteromonas piscicida, with 96.3 and 95.7% sequence similarity, respectively. However, the strains could be differentiated from P. lutioviolacea by seven traits and from P. piscicida by 10 traits. Analysis of DNA-DNA relatedness to these related species revealed low levels of DNA hybridization (19.6% to P. luteoviolacea and 22.4% to P. piscicida). However, the type strain, O-BC30T, and the other three bacterial isolates showed high DNA relatedness to each other, ranging from 84.8 to 93.7%. Based on the results of phenotypic characterization, phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization, it is concluded that these isolates represent a novel species in the genus Pseudoalteromonas. Because the type strain, O-BC30T (=IAM 14989T =KCTC 12086T), produces phenolic anti-MRSA substances, the name proposed for this novel species is Pseudoalteromonas phenolica sp. nov.

MC21-A, a bactericidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30(T), against methicillin-resistant Staphylococcus aureus.

We previously reported a new marine bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30(T), which produced a bactericidal antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we purified an anti-MRSA substance (MC21-A) from the methanol extract of the cells of P. phenolica O-BC30(T) and analyzed its chemical structure. MC21-A was determined to be 3,3',5,5'-tetrabromo-2,2'-biphenyldiol by spectrometric analyses. Its anti-MRSA activity against 10 clinical isolates of MRSA was comparable to that of vancomycin (MC21-A MICs, 1 to 2 micro g/ml; vancomycin MICs, <0.25 to 2 micro g/ml). This substance was also high active against Enterococcus serolicida, Enterococcus faecium, and Enterococcus faecalis but was less active against Streptococcus spp. A time-kill study also demonstrated that MC21-A was bactericidal and that its killing rate was much higher than that of vancomycin. The postantibiotic effect (PAE) of MC21-A against a clinical MRSA isolate, strain E 31243, was also comparable to that of vancomycin (MC21-A PAEs, 1.46 to 1.65 h; vancomycin PAEs, 0.84 to 1.43 h). However, a lysis experiment demonstrated that this substance failed to lyse MRSA cells. This substance also did not lyse human erythrocytes. A SYTOX Green staining experiment implied that this substance permeabilized the cell membrane of MRSA as its mode of action. When its activities against a hypersensitive Escherichia coli mutant (KO 1489) and wild-type strains were tested, MC21-A exhibited higher levels of activity against the former. Furthermore, MC21-A was not cytotoxic to human normal fibroblast, rat pheochromocytoma, and Vero cells at concentrations up to 50 micro g/ml. These results suggest that MC21-A might be useful as a lead compound in the development of new types of anti-MRSA substances with modes of action different from those of vancomycin and teicoplanin.

Lysis of methicillin-resistant Staphylococcus aureus by 2,4-diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga.

Previously 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas sp. AMSN isolated from a marine alga, had demonstrated a high level of anti-methicillin-resistant Staphylococcus aureus (MRSA) comparable with that of vancomycin. In this study, this substance had bacteriolytic activity against MRSA at 1 microg/ml as well as similar activity against Vibrio parahaemolyticus at 24 microg/ml that suggests a novel antibacterial mode of action by this substance. Heat and pH stability tests showed it to be stable at temperatures ranging from 35 to 70 degrees C and at pHs ranging from 2-7. It was not acutely toxic to mice at levels up to 100 mg/kg.