Biological characteristics of marine Streptomyces SK3 and optimization of cultivation conditions for production of compounds against Vibiriosis pathogen isolated from cultured white shrimp (Litopenaeus vannamei)

Abstract

Antibiotic resistance in shrimp farms has emerged as an extremely serious situation worldwide. The main aim of this study was to optimize the cultural conditions for producing new antibiotic agents from marine Streptomyces species. Streptomyces SK3 was isolated from marine sediment and was identified by its 16S rDNA as well as biochemical characteristics. This microbe produced the highest concentration of bioactive secondary metabolites (BSMs) when cultured in YM medium (YM/2). It produced the maximum total protein (41.8 ± 6.36 mg/ml) during the late lag phase period. The optimum incubation temperature was recorded at 30 °C; BSMs were not produced at ≤10 °C within an incubation period of 3-4 days. The suitable agitation speed was found to be 200 rpm with pH 7.00. The proper carbon, nitrogen, and trace elements supplementation consisted of starch, malt extract, calcium carbonate (CaCO₃), and magnesium sulfate (MgSO₄). The ethyl acetate extract was found to act strongly against three vibriosis pathogens, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vunificus, as indicated by the inhibition zones at 34.5, 35.4, and 34.3 mm, respectively. The extract showed the strongest anti-V. harveyi activity, as indicated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.101 ± 0.02 and 0.610 ± 0.04 mg/ml, respectively. Basic chemical investigation of the crude extract using thin layer chromatography (TLC), bioautography, liquid chromatography tandem mass spectrometry (LC‒MS/MS), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (¹H-NMR) revealed that the active components were the terpenoid and steroid groups of compounds. They showed carboxylic acid and ester functions in their molecules.

Keywords: Anti-vibrio spp; Characterization; Culture; Litopenaeus vannamei; Marine streptomyces; Optimization.

Figure 1. Colony feature of S. hiroshimensis (SK3 strain) and SEM image.

Photographed by Patchara Pedpradab, 8 February, 2024.

Figure 2. Phylogram of phylogenetic relation based on 16S rDNA sequence of S. hiroshimensis.

Figure 3. Inhibition zone of anti-Vibrio spp. screening of the extract from S. hiroshimensis.

(I) and media optimization (II) determined by agar well diffusion method. (A) Tetracycline antibiotic, (B) supernatant, (C) crud extract, (D) pure media, For media optimization process, the supernatant of suspension cell in the media was screened for their anti-V. harveyi property; (E) YM/2, (F) YM, (H) ISP3, (G) ISP3/2. Photographed by Patchara Pedpradab, 1 February, 2024.

Figure 4. Bioautogram of anti-V. harveyi (B), V. vulnificus (C) and V. parahaemolyticus (D).

(A) TLC chromatogram of the extract from S. hiroshimensis under uv light at 366 nm. (E) Reference antibiotic (tetracycline). The brilliant spots on chromatogram (B–E) are the inhibition zones. (H) TLC pattern under UV light at 254 nm. (F and G) TLC patterns sprayed by vanillin and anisaldehyde reagents. Photographed by Patchara Pedpradab, 1 April, 2024.

Figure 5. Effect of media types to BSMs production.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 6. Media optimization to BSMs production.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 7. Protein secretion to liquid medium.

Figure 8. Effect of initial incubation temperature to BSMs production.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 9. Effect of incubation period to BSMs production.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 10. Effect of agitation on BSMs production of S. hiroshimensis.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 11. Effect of initial pH of medium on BSMs production of S. hiroshimensis.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 12. Effect of carbon sources on BSMs production of S. hiroshimensis.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 13. Effect of nitrogen sources on BSMs production of S. hiroshimensis.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05)

Figure 14. Effect of trace elements on BSMs production of S. hiroshimensis.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 15. Effect of salt concentration on BSMs production.

Data are shown as the value of mean ± SE to represent by bars. The difference letters mean statistically significant at 95% confidence interval (p < 0.05).

Figure 16. HPLC chromatograms of the extract of S. hiroshimensis.

Figure 17. ¹H-NMR of the crude extract of S. hiroshimensis.

Figure 18. FTIR spectrum of the crude extract from S. hiroshimensis.