Identification and characterization of probiotics isolated from indigenous chicken (Gallus domesticus) of Nepal.

BACKGROUND: Excessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). METHODS: Selective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37°C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. RESULTS: A total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16S rDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. CONCLUSION: Our study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

Detection of Bacillus Species with Arsenic Resistance and Plant Growth Promoting Efficacy from Agricultural Soils of Nepal.

Arsenic contamination in soil and water is one of the major environmental problems in multiple countries including Nepal imposing a serious threat to the ecosystem and public health. Many soil bacteria can detoxify arsenic, including genus Bacillus. With an objective to gauge the plant growth-promoting activities of arsenic-resistant Bacillus species, 36 samples (soil, rice, cauliflower, and beans) were collected from the Terai region of Nepal. For selective isolation of Bacillus species, each sample was heated at 80°C for 15 min before the inoculation into nutrient agar (NA). Following the standard protocol, arsenic-resistant Bacillus species were screened using NA supplemented with 100 ppm sodium arsenate and sodium arsenite. Among 158 randomly selected isolates, only five isolates were able to tolerate sodium arsenite concentration up to 600 ppm. Notably, all five isolates were able to produce indole acetic acid (IAA), a plant hormone, and solubilize phosphate. Based on biochemical analysis and 16S rRNA gene sequencing, isolates N4-1, RW, KR7-12, Bhw1-4, and BW2-2 were identified as B. subtilis subsp. stercosis, B. flexus, B. licheniformis, B. cereus, and B. flexus, respectively. To the best of our knowledge, this is the first study showing the presence of arsenic-resistant B. flexus in Nepalese soil with plant growth-promoting traits. Possible utilization of these Bacillus strains could facilitate the novel bioremediation pathway to reduce the toxic effect of arsenic from the soil and water in the Terai region of Nepal.