Isolation and molecular identification of probiotic bacteria from Cultured shrimps Litopenaeus vannamei in Bushehr province

Abstract

Shrimp culture and rearing industry in particular white leg shrimp species (Litopenaeus vannamei) is one of the major activities in the world, including Iran. The outbreaks of diseases are the main factors limiting production. Due to the adverse effects of abusing antibiotics and chemical disinfectants, disease control and prevention requires novel methods that are affordable, effective and safe for the environment and humans. For this purpose during the five months, sampling was done from three main sites of shrimp culture in Bushehr province and 150 pieces cultured white leg shrimp, 135 water and sediment samples were collected from the ponds, input and output channels by standard methods. Physicochemical parameters of water and biometry of shrimps were done and recorded. Probiotic isolation was performed by culturing samples in Tryptic soy agar and TCBS after incubation in 30º C for 24-48 h. The antimicrobial effects of culture extract of isolates were evaluated against V. harveyi with the well diffusion method and the best isolates were selected. The molecular identification of selected bacteria was performed by 16S ribosomal DNA gene sequence analysis technique. The growth kinetics of selected bacteria and effect of environmental factors on antimicrobial compound production and stability of them in salinity (1.5-5.5%) and (0-50ppt), pH (5-9) and temperature (3040º C) and (35-100º C) were studied respectively. After the determination of antibiotic resistance profiles of them, the In vivo test on the L. vannamei post larvae was performed and effect of these bacteria on growth performance, prevention of vibriosis and water quality include (ammonia, nitrate nitrite, turbidity, total bacteria and Vibrionaceae load were examined. For extraction and identification of bioactive material with anti-bacterial properties produced by selected bacteria, dialysis, SDS-PAGE electrophoresis, ultraviolet spectroscopy and gas chromatography mass was performed. According to the results of this dissertation the most frequency of facultative aerobic and anaerobic heterotrophic bacteria was seen in shrimp digestion tract (3.04 ± 0.75) ×105 CFU/g in September .Genus Vibrio spp. (37.88%) and Bacillus spp. (27.27%) had the most frequency respectively. Of the 198 bacterial isolates, two bacterial strains from the digestive tract and sediment which had the highest inhibition and a stability antagonistic effect on V. harveyi were selected. Based on 16S ribosomal DNA gene sequence analysis, they were identified as Bacillus subtilis subsp. inaquosorum strain IS02 (GenBank: JN856456.1) and Bacillus vallismotis IS03 (GenBank: JQ085958.1) and recorded in the National Center for Biotechnology Information (NCBI). According to results of identifying bioactive material produced by selected bacteria, in SDS-PAGE electrophoresis Bacillus subtilis subsp. inaquosorum strain IS02 did not have any band but Bacillus vallismotis IS03 had two band in 25 kDa and 34-45 kDa molecular weight which probably bacteriocin like substances and group III Bacillus bacteriocin respectively. UV spectra of selected bacteria were in the range (260-265nm) which related to peptide groups. In GC-MS the highest percent of chromatogram was belonged to pyrrolopyrazines in both of selected bacteria, which have antibacterial properties. Overall, according to results both of the selected bacteria were adapted to the ecological conditions of shrimp culture and could be used as appropriate probiotics in this industry. It is hoped that the results of this research is to produce a step towards the realization of national production of indigenous probiotic in Iran.