Complete Genome of Bacillus velezensis CMT-6 and Comparative Genome Analysis Reveals Lipopeptide Diversity.

The complete genome sequence of Bacillus velezensis type strain CMT-6 is presented for the first time. A comparative analysis between the genome sequences of CMT-6 with the genome of Bacillus amyloliquefaciens DSM7T, B. velezensis FZB42, and Bacillus subtilis 168 revealed major differences in the lipopeptide synthesis genes. Of the above, only the CMT-6 strain possessed an integrated synthetase gene for synthesizing surfactin, iturin, and fengycin. However, CMT-6 shared 14, 12, and 10 other lipopeptide-producing genes with FZB42, DSM7T, and 168 respectively. The largest numbers of non-synonymous mutations were detected in 205 gene sequences that produced these three lipopeptides in CMT-6 and 168. Comparing CMT-6 with DSM7T, 58 non-synonymous mutations were detected in gene sequences that contributed to produce lipopeptides. In addition, InDels were identified in yczE and glnR genes. CMT-6 and FZB42 had the lowest number of non-synonymous mutations with 8 lipopeptide-related gene sequences. And InDels were identified in only yczE. The numbers of core genes, InDels, and non-synonymous mutations in genes were the main reasons for the differences in yield and variety of lipopeptides. These results will enrich the genomic resources available for B. velezensis and provide fundamental information to construct strains that can produce specific lipopeptides.

Biotransformation enzyme activities and phase I metabolites analysis in Litopenaeus vannamei following intramuscular administration of T-2 toxin.

T-2 toxin (T-2) is a type-A trichothecene produced by Fusarium that causes toxicity to animals. T-2 contamination of grain-based aquatic feed is a concern for the industries related to edible aquatic crustacean species such as the shrimp industry because it can lead to serious food safety issues. T-2, its metabolites, and selected phase I (EROD, CarE) and phase II (GST, UGT, SULT) detoxification enzymes in hemolymph and tissues were monitored at 0, 5, 10 15, 30, 45, and 60 min following T-2 intramuscular administration (3 mg/kg bw) in shrimp (Litopenaeus vannamei). Marked increases of EROD activity in hepatopancreas and CarE activity in hemolymph, gill, hepatopancreas and intestine were observed followed by increases in phase II enzymes (GST, UGT, SULT) in hepatopancreas, hemolymph, intestine and gill, which remained elevated for an extended period. Time-dependent decrease in shrimp tissue T-2 concentration was observed. HT-2 increased up to 15 min. Most other T-2 metabolites were detected but not T-2 tetraol. Enzyme responses on exposure to T-2 were tissue-specific and time-dependent. Detection results indicated that HT-2 may not be the only important metabolite in aquatic crustacean species. Further investigation into T-2 metabolite toxicity is needed to fully understand the food safety issues related to T-2.

A sensitive method for simultaneous quantitative determination of surfactin and iturin by LC-MS/MS.

Surfactin and iturin are antimicrobial lipopeptides produced from Bacillus spp. and have significant prospective applications in many fields. Therefore, accurate analysis of these lipopeptides in the fermented product of some Bacillus strains is important. A sensitive method for simultaneous quantitative determination of surfactin and iturin fermented by Bacillus natto NT-6 was developed and validated using liquid chromatography-tandem mass spectrometry. Crude extracts of antimicrobial lipopeptide samples were dissolved in a mixture of acetonitrile/water (7:3, v/v) in 0.1 % (v/v) formic acid and eluted with acetonitrile/water (7:3, v/v) containing 5 mmol L-1 ammonium acetate and 0.1 % (v/v) formic acid. The target compounds were detected by mass spectrometry (ESI+) using selective ion monitoring. A good linear regression in the range of 0.20-10.0 mg L-1 for both surfactin and iturin (R 2 ≥ 0.9995) was observed with spiked recoveries of 93.3-108.2 %, RSD values less than 15 %, precision 4.14-13.30 %, and a detection limit of 0.374 mg L-1. This method has a simple preprocessing operation, good repeatability, and provides an accurate quantitative analysis of surfactin and iturin. Graphical Abstract Surfactin and iturin from Bacillus natto NT-6 extraction and detection procedure.

A sensitive and validated immunomagnetic-bead based enzyme-linked immunosorbent assay for analyzing total T-2 (free and modified) toxins in shrimp tissues.

Accurate analyses of total T-2 (free and modified) in aquatic organisms including shrimp are important as the hazard caused by T-2 has been caught increasing attention. Therefore, acurate analysis of free T-2 especially of modified T-2 in shrimp tissues is important. A rapid, sensitive, and validated method for quantitative determination of free T-2 and modified T-2 toxin was developed using immunomagnetic-bead based enzyme-linked immunosorbent assay (IMB-ELISA). Super paramagnetic particles with a carboxyl group activated by an ester method coupled with envelope antigen 3- acetylneosolaniol- hemisuccinate - ovalbumin (3-Ac-NEOS-HS-OVA) was used to form immunomagnetic beads which could bind to T-2 skeletal structure antibodies. The conditions for magnetic bead coating of T-2 skeletal structure antibodies, and the concentrations of the polyclonal antibody and HRP-labeled goat anti-rabbit antibody were optimized. A good linear relationship with T-2 concentrations ranging from 5-75ng/mL (R2 =0.9965) was observed. The detection limit of different shrimp tissues of the IMB-ELISA ranged from 2.53 to 3.20ng/mL. And the IC50 was 63ng/mL. The recovery varied from 86% to 99% with a standard deviation of 2.8-5.8%. The application of this method to study the distribution in tissues showed that the total T-2 concentration in hepatopancreas was 26.7µg/kg > blood > head > muscle in the highest dose group of 12.2mg/kg. Our research showed a combination of ELISA and immunomagnetic bead technology provide a new, convenient approach to significantly improve the accuracy and sensitivity of total T-2 measurement in shrimp tissues.

Selection and Evaluation of Indexes Commonly Used to Determine Contamination with T-2 Toxin in Pacific White Shrimp Litopenaeus vannamei by the Grey Relational Method.

The objectives of the present study were to evaluate the effects of different concentrations of the mycotoxin T-2 toxin in feed on muscle performance in the Pacific white shrimp Litopenaeus vannamei, evaluate indexes of physiological variables that indicate T-2 toxin contamination in the shrimp using the grey relational method, and determine the dose-response relationships between T-2 toxin and the indexes. Of the 6 physical, 7 biochemical, and 17 nutritional indexes examined, the values of the grey relational coefficients were highest for the hepatopancreas: body weight ratio (HBR), alanine aminotransferase (ALT) activity, and serine (SER) content (0.83, 0.68, and 0.82, respectively). Therefore, the HBR, ALT activity, and SER content were selected as appropriate indexes for contamination of Pacific white shrimp muscle with T-2 toxin. Based on their dose-response relationship curves, mean effective doses of 1.45, 1.69, and 1.33 mg of T-2 toxin/kg of feed were obtained for the HBR, ALT activity, and SER content, respectively. These results offer technical reference points for the evaluation and control of T-2 toxin in shrimp feed. Received April 28, 2016; accepted April 9, 2017.

[Diversity of antimicrobial bacteria associated with Siganus fuscescens from Naozhou island in Zhanjiang, China].

OBJECTIVE: Marine bacteria are a rich source of potentially useful antimicrobial molecules. The purpose of the study is to explore the diversity of bacteria with antimicrobial activity isolated from Siganus fuscescens gastrointestinal tract collected from Naozhou Island (20 degrees 52' N-20 degrees 56' N 110 degrees 33' E-110 degrees 38' E), Leizhou Bay, South China Sea. METHODS: We isolated bacteria from the gastrointestinal tract of fish sample using classical culturing technique, and determined antimicrobial activities of the isolates by Oxford cup method. We investigated diversity of antimicrobial isolates using phylogenetic comparative analysis of 16S rRNA gene sequences. RESULTS: According to the results of morphological observation and part of physiological and biochemical experiments, we isolated 68 strains from fish gastrointestinal tract. Among them, 19 strains with antimicrobial activities were acquired (27.9% of the isolates) and represented 19 different species, belonging to 12 genera (Rothia, Micrococcu, Brachybacterium, Brevibacterium, Psychrobacter, Paracoccus, Cobetia, Citrobacter, Pseudomonas, Bacillus, Lactobacillus and Staphylococcus) of 11 families (Microbacteriaceae, Dermabacteraceae, Brevibacteriaceae, Moraxellaceae, Rhodobacteraceae, Halomonadaceae, Enterobacteriaceae, Pseudomonadaceae, Bacillaceae, Lactobacillaceae and Staphylococcaceae) in three phyla (Actinobacteria, Proteobacteria and Firmicutes). Eight strains (42.1%), 7 strains (36.8%) and 4 strains (21.1%) were belonging to the phylum Firmicutes, Proteobacteria and Actinobacteria, respectively. The phylogenetic distance matrix results suggested that there were obvious genetic divergences between the majority of strains with antimicrobial activity and their phylogenetically most closely related typical strain, due to 16S rRNA gene sequences similarities ranging from 96.2 to 99.9%. In addition, 4 strains (ZJHD2-31, ZJHD5-23, ZJHD2-58 and M26) could represent potential new species, and identification of the novel strain M26 has been published in Antonie van Leeuwenhoek. CONCLUSION: There are abundant diversity for bacteria with antimicrobial activity and potentially more new species of microorganism in Siganus fuscescens gastrointestinal tract collected from Naozhou Island.

Antimicrobial peptide AMPNT-6 from Bacillus subtilis inhibits biofilm formation by Shewanella putrefaciens and disrupts its preformed biofilms on both abiotic and shrimp shell surfaces.

Shewanella putrefaciens biofilm formation is of great concern for the shrimp industry because it adheres easily to food and food-contact surfaces and is a source of persistent and unseen contamination that causes shrimp spoilage and economic losses to the shrimp industry. Different concentrations of an antimicrobial lipopeptide, the fermentation product of Bacillus subtilis, AMPNT-6, were tested for the ability to reduce adhesion and disrupt S. putrefaciens preformed biofilms on two different contact surfaces (shrimp shell, stainless steel sheet). AMPNT-6 displayed a marked dose- and time-dependent anti-adhesive effect>biofilm removal. 3MIC AMPNT-6 was able both to remove biofilm and prevent bacteria from forming biofilm in a 96-well polystyrene microplate used as the model surface. 2MIC AMPNT-6 prevented bacteria from adhering to the microplate surface to form biofilm for 3h and removed already existing biofilm within 24h. Secretion of extracellular polymeric substances incubated in LB broth for 24h by S. putrefaciens was minimal at 3× MIC AMPNT-6. Scanning electron microscopy showed that damage to S. putrefaciens bacteria by AMPNT-6 possibly contributed to the non-adherence to the surfaces. Disruption of the mature biofilm structure by AMPNT-6 contributed to biofilm removal. It is concluded that AMPNT-6 can be used effectively to prevent attachment and also detach S. putrefaciens biofilms from shrimp shells, stainless steel sheets and polystyrene surfaces.

Pathogenicity of Vibrio parahaemolyticus in Different Food Matrices.

The pathogenicity and virulence factors of Vibrio parahaemolyticus in four food matrices--shrimp, freshwater fish, pork, and egg-fried rice--were compared by measuring the thermostable direct hemolysin activity and total hemolytic titer. Significantly high thermostable direct hemolysin and also hemolytic titers (P < 0.05) were produced by V. parahaemolyticus in egg-fried rice > shrimp > freshwater fish > pork. Filtrates of V. parahaemolyticus in shrimp given intraperitoneally induced marked liver and kidney damage and were highly lethal to adult mice compared with filtrates of V. parahaemolyticus in freshwater fish > egg-fried rice > pork. From in vitro and in vivo pathogenicity tests, it seems the type of food matrix has a significant impact on the virulence of V. parahaemolyticus. These results suggest that hemolysin may not necessarily be the only virulence factor for pathogenicity of V. parahaemolyticus. This is the first report that shows that virulence factors produced by V. parahaemolyticus in seafood such as shrimp are more toxic in vivo than in nonseafood.