Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus)

Data analysis The Bray-Curtis analysis [40] was performed using SPSS and the diversity factor of the Bray- Curtis analysis was estimated, basing on the presence/absence matrix of intestinal bacteria obtained from fish in the five groups [41, 42]. Determination of bioactivity (antibacterial activity) The bioactivity (antibacterial activity) of 35 bacterial representative isolates recovered in this study was determined by a double-layer technique [43, 44]. The selected bacterial representative isolates were grown on LB media that was allowed to grow for 1–2 days depending upon the growth rate of the various isolates. The agar blocks containing the cells were the excised and placed on the assay plates spread with 4 indicator microorganisms (four fish pathogens) includ- ing Streptococcus agalactiae (SA), Streptococcus iniae (SI), Micrococcus luteus (ML), Vibrio para- hemolyticus (VP) [45–48]. The assay plates with agar blocks and indicator microorganisms were cultivated at 37˚C for 18–36 hours. The antimicrobial activity was expressed as the diameter of the growth inhibition zone (in milliliter). Each test was performed three times. All the indicator microorganisms were from the Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, China. After the initial evaluation for anti-bacterial activity, the minimum inhibitory concentrations (MICs) of the active extracts were further determined by a microtitre plate assay [49]. First, connect fresh Bacillus altitudinis,

Bacillus amyloliquefaciens, Bacillus pumilus to the sterilized 200 mL LB liquid medium (tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/l and sea salt 30 g/l) at 1% inoculation volume, and incubate at 30˚C, 180 r / min for 18h, then ferment the broth at 10000 r / min. After centrifugation for 5 min, the supernatant was filtered with a 0.22 μm microporous filter membrane to obtain a sterile filtrate. Sterile filtrate and n-butanol were subjected to ultrasonic extraction at a ratio of 1: 7 for 0.5h. The extract layer was collected and concentrated under reduced pressure using a rotary evaporator at 45˚C. The resulting crude extract was dissolved in 1 mL of methanol and stored at 4˚C until use [50]. And, Staphylococcus sp., Streptomyces rutgersensis strain was activated, cul- tured at 28˚C, and seed liquid was prepared. Choose a 250 mL Erlenmeyer flask with the same specifications and fill it with 50 mL RA medium (soluble starch 20 g (boiling and dissolving), glu- cose 10g, maltose 10g, malt extract powder 10g, corn flour 5 g, CaCO3 2 g, pH 7.5), The seed solution was inoculated into three kinds of sterilized fermentation medium, and cultured with shaking at 200 r / min at 28˚C for 7 days. The fermentation supernatant was mixed with an equal volume of butanone for ultrasonic extraction, the butanone extract layer was collected, concen- trated under reduced pressure at 45˚C on a rotary evaporator, and the crude extract was dis- solved in 1 mL of methanol, and stored at 4˚C until use [51]. Finally, the overnight culture of

bacteria was adjusted to an optical density of 0.6 at 600 nm in the corresponding medium. Then, transfer the active extracts of different concentrations (50, 25, 12.5, 6.2, 3.1, and 1.6 μg / ml) to the wells of a 96-well polystyrene microtiter plate (100 μl per well) at 30˚C Incubate for 24h. The MIC was recorded as the lowest concentration of active extract at which no bacterial growth was observed. Each extract was performed in triplicate with the positive control (streptomycin). Results BLAST searches and analysis of 16S rDNA gene sequences The data obtained from the 16S rDNA sequences of 38 representative bacterial isolates were deposited in GenBank. The numbers are MK281515-MK281549. The 16S rDNA sequences of the 133 selected bacterial isolates were sequenced and com- pared with other sequences in GenBank. The results showed that 128 isolates shared 95–100% PLOS ONE Effects of wood forages on gut bacterial biodiversity and bioactivity in tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0235560 July 2, 2020 5 / 17