Isolation of bacteria
The bacterial strains were isolated from the intestinal tract of healthy C. auratus (n = 10, 200–220 g) and cultured on de Man-Rogosa-Sharpe (MRS) agar plates (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) at 37 °C for 24 h. Colonies differing in morphological characteristics were selected and subcultured in MRS broth. A total of eight bacterial strains from different colonies were isolated, purified and stored in sterile glycerol (15% v/v) at − 80 °C.
Four indicator pathogens were used in this study, including Aeromonas veronii, Staphylococcus haemolyticus, Vibrio parahaemolyticus and Vibrio vulnificus, stored in the Key Lab of Aqua-ecology and Aquaculture, Tianjin Agricultural University. The concentrations of the eight isolated strains and the indicator pathogens were adjusted to 1 × 106 CFU/mL and 1 × 107 CFU/mL, respectively. Antagonistic activities of the eight isolated bacterial strains against these indicator pathogens were determined according to the Oxford cup method (Vincent et al. 1944). In brief, 100 μL of bacterial suspension of each indicator pathogen was spread evenly on a Luria–Bertani (LB) agar plates (tryptone 10 g/L, yeast extract powder 5 g/L, agar 15 g/L and Nacl 10 g/L; Beijing Aoboxing Bio-Technology Co., Ltd., Beijing, China) and allowed to absorb, then equal volume of bacterial suspension of the isolate to be tested was placed in the Oxford cups. The LB agar plate was incubated at 37 °C for 24 h, then the antagonistic activity was examined according to the diameters of inhibition zones appearing around the cups. A strain named R8 with stronger inhibitory activity against the pathogenic bacteria was selected for the subsequent experiments.
Biochemical characteristics tests
The Gram-staining method was used for the morphological investigation. The commercial microtest systems (Hangzhou Tianhe Microorganism Reagent Co., Ltd., Hangzhou, China) were used to perform the biochemical tests, including oxidative/fermentative, methyl red test, urea, Voges Proskauer test, gluconate, catalase, oxidase, arginine, NO3− reductase, amylum, sorbitol, mannitol, saligenin, sucrose, raffinose, glucose, xylose, lactose, bile esculin and arabinose. And it was also studied for the growth condition at 0–10% of NaCl (w/v) and temperature of 4–42 °C. The incubation was performed at 37 °C for 48 h and the results were observed with reference to the manual of systematic and determinative bacteriology (Dong and Cai 2001).
The boiling method was used to extract total genomic DNA of the isolate (Chen et al. 2015). The 16S rRNA gene was amplified with a pair of universal primers, 27F: 5′-AGAGTTTGATCATGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (Cao et al. 2007). The PCR reaction of the samples underwent an initial denaturation of 4 min at 95 °C, and then 30 cycles of 45 s at 94 °C, 45 s at 55 °C and 1 min at 72 °C, followed by 10 min at 72 °C. Reaction products were purified and cloned according to the report of Han et al. (2017). The nucleotide sequences were compared with known sequences in the NCBI database by using the Blast tool (NCBI, http://www.ncbi.nih.gov/BLAST/). The neighbor-joining algorithm of MEGA 5.22 software was used to construct the phylogenetic trees, with 1000 bootstrap replicates.
According to the method of Yang et al. (2013), the haemolytic analysis of the bacterial isolate R8 was performed on a blood agar plate.
Sixty healthy C. auratus with an average weight of 74 ± 10 g and length of 14 ± 0.5 cm were purchased from a large aquatic wholesale market in Tianjin, China. Fish were transferred back to the Tianjin Agricultural University and acclimatized for 2 weeks, with water temperature adjusted to 28 °C and pH 7.5. Aeration was provided to maintain optimal DO and fish were fed with commercial feed pellets twice daily. All the fish were randomly divided into five groups with twelve fish in each group. Four groups were injected intraperitoneally with 0.2 mL of the suspension of R8 strain at a concentration of 1 × 105 CFU/mL, 1 × 106 CFU/mL, 1 × 107 CFU/mL and 1 × 108 CFU/mL, respectively. The last group used as control was injected with the same dose of 0.85% physiological saline. The health condition and mortality of C. auratus were observed within 14 days after injection. The protocol was approved by the Animal Experimentation Ethical Committee of the Tianjin Agricultural University.
Tolerant ability against acid, bile and temperature
The tolerant abilities against various pH value, bile conditions and temperature were determined. The bacterial isolate R8 stored in glycerol-cryopreservative medium was resuscitated in MRS broth at 37 °C until arriving at stationary phase. Then the bacterial suspension was adjusted to a concentration of 1 × 108 CFU/mL. To determine the acid tolerance of this bacterial isolate, 0.5 mL of the bacterial suspension was inoculated into 10 mL of LB broth with pH values of 2, 3 or 4, and cultured for 2 h, 4 h and 8 h, respectively at 37 °C, and the value of optical density (OD) was read at 600 nm with a ultraviolet and visible spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). The pH value of LB broth was adjusted with the sterile solution of 1 mol/L NaOH or 1 mol/L HCl.
For determination of the tolerance against bile, 0.5 mL of the R8 bacterial suspension (1 × 108 CFU/mL) was inoculated into 10 mL of LB broth supplemented with 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2% bile salts or without (control), and incubated for 24 h at 37 °C. Then, the value of OD of the bacterial suspension at 600 nm was recorded.
To test the tolerance against various temperatures, 0.5 mL of the R8 bacterial suspension was inoculated into 10 mL of LB broth and the temperature was controlled at 50 °C, 60 °C, 70 °C, 80 °C and 90 °C. Growth was checked at 2 min, 5 min, 10 min, 20 min and 30 min, respectively and the value of OD of the bacterial suspension at 600 nm was recorded.
Growth characteristics were tested according to the method of Han et al. (2017). The temperature, pH value and NaCl concentration were adjusted to various conditions based on LB broth (Beijing Aoboxing Bio-Technology Co., Ltd., Beijing, China). The impact of pH value of 4, 5, 6, 7, 8 and 9 on the growth of the bacterial isolate R8 was studied in LB broth with 30 ppt at 37 °C; the impact of salinity of 20 ppt, 30 ppt and 40 ppt was studied in LB broth with pH value of 7.0 at 37 °C; the impact of temperature at 27 °C, 32 °C, 37 °C and 42 °C was studied in LB broth with the pH value of 7.0 at 30 ppt. The LB broth for bacterial cultivation was inoculated with 200 μL bacterial suspension with a concentration of 1 × 108 CFU/mL, and incubated at 180 rpm for 28 h. The OD values at 600 nm of the bacterial suspensions were measured every 2 h.