The Use of Selected Bacteria and Yeasts to Control Vibrio spp. in Live Food.

Vibrio species are a significant causative of mass mortality in mariculture worldwide, which can quickly accumulate in live food and transmit into the larval gut. With restrictions on the use of antibiotics in aquaculture, finding a proper solution to reduce the risk of Vibriosis is vital. This study aimed to evaluate the susceptibility of Vibrio harveyi, V. campbellii, V. anguillarum, and V. parahaemolyticus to twenty-six bacterial and yeast strains and use the beneficial ones to enrich live food (Branchiopod, Artemia franciscana, rotifer, Brachionus plicatilis and copepod, Tigriopus japonicus). Thus, a modified disk diffusion method was applied. After a susceptibility assay, the bacteria and yeast beneficial in suppressing the Vibrio species were labeled by fluorescent stain and used to measure the accumulation potential in different live foods. Also, the beneficial bacteria and yeast were used to enrich live foods, and then the count of loaded Vibrio was estimated after 5, 10, 15, and 20 hours by the serial dilution method. From the total bacteria and yeast strains that were used, Candida parapsilosis, Pseudoalteromonas flavipulchra, Lactobacillus sakei, Bacillus natto, and B. amyloliquefaciens inhibited all four Vibrio species. The results of microbial labeling showed that L. sakei in Artemia, C. parapsilosis in rotifers, and V. harveyi in copepods had the highest accumulation rate. The results of the estimation of loaded Vibrio in different live foods also showed that the use of beneficial bacteria and yeast each significantly reduced the count of Vibrio. Application of bacteria and yeast to suppress pathogenic Vibrio maybe a sustainable method for preventing this pathogen from harmfully invading aquaculture and may also aid in reducing the chances of antibiotic resistance in pathogenic Vibrio.

Huaishuia halophila gen. nov., sp. nov., isolated from coastal seawater.

A novel Gram-negative, non-motile bacterium, designated ZXM137(T), was isolated from seawater collected from a coastal region of Qingdao, China, during a massive green algae (Enteromorpha prolifera) bloom. Strain ZXM137(T) was strictly aerobic and did not accumulate poly-ß-hydroxybutyrate. Growth occurred with 0.5-11.0% (w/v) NaCl, at pH 6-9 (optimum of pH 7) and at 4-45 °C (optimum at 28 °C). It contained Q-10 as the predominant ubiquinone and the major polar lipids were phosphatidylglycerol, phospholipids, and an unidentified aminolipid and lipid. The major cellular fatty acids of strain ZXM137(T) were C(18:1)ω7c, C(18:1)ω6c and 11-methyl C(18:1)ω7c. The DNA G+C content was 60.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ZXM137(T) formed a distinct evolutionary lineage within the Roseobacter group in the class Alphaproteobacteria. On the basis of phenotypic, chemotaxonomic and phylogenetic evidence, strain ZXM137(T) represents a novel species in a new genus, for which the name Huaishuia halophila gen. nov., sp. nov. is proposed; the type strain is ZXM137(T) (=CGMCC 1.8891(T)=LMG 24854(T)).

Algoriphagus faecimaris sp. nov., isolated from coastal sediment.

A Gram-negative, non-motile, non-sporulating bacterial strain, designated LYX05(T), was isolated from coastal sediment of Qingdao, China, on the coast of the Yellow Sea. Strain LYX05(T) was aerobic and heterotrophic. The strain grew optimally at 37 °C and pH 7.5 and in the presence of 2% (w/v) NaCl. Colonies were 1-2 mm in diameter, circular, reddish orange and shiny with entire edges on marine agar medium. Cells were rods (0.3-0.5 µm wide and 0.8-1.6 µm long). The dominant fatty acids were iso-C(15:0) (40.82%) and C(16:0) (10.45%). The DNA G+C content was 42.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LYX05(T) was phylogenetically related to the members of the genus Algoriphagus and the closest relative was Algoriphagus hitonicola 7-UAH(T) (95.8% 16S rRNA gene sequence similarity). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain LYX05(T) was considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus faecimaris sp. nov. is proposed. The type strain is LYX05(T) (=JCM 16561(T) =DSM 23095(T) =LMG 25474(T)).

Mutation of tryptophanase gene tnaA in Edwardsiella tarda reduces lipopolysaccharide production, antibiotic resistance and virulence.

A fish pathogen Edwardsiella tarda LTB-4 produced various indole alkaloids, including indole, 2-(1H-indol-3-yl)ethanol, 4-di(1H-indol-3-yl)methylphenol, tri(1H-indol-3-yl)methane and 2-[2,2-bis(1H-indol-3-yl)]ethylphenylamine. Indole was the most abundant among these indole alkaloids. E. tarda LTB-4 produced indoles during its whole growth phase and maintained a high level (around 35.5 µM) during the stationary phase. The relevant tryptophanase (TnaA) gene tnaA was cloned from LTB-4 and conditionally expressed in Escherichia coli; the recombinant TnaA catalysed L-tryptophan to indole. A tnaA in-frame deletion mutant ΔtnaA was constructed through double cross-over allelic exchange by means of the suicide vector pRE118; deletion of tnaA caused some phenotypic changes including decreased swarming and twitching motility, lipopolysaccharide production and multiple antibiotic resistances. Also, subtherapeutic doses of chloromycetin, carbenicillin and tetracyline could cause the decrease of bacterial growth, but greatly induce the production of indole by E. tarda. Most importantly, attenuated virulence of the ΔtnaA mutant to zebra fish by increasing the LD50 for about 55-fold indicated that TnaA involved in the virulence of E. tarda.