The antimicrobial activity and probiotic properties of Bacillus subtilis strain KKU213, isolated from local soil, were investigated. The cell-free supernatant (CFS) of a KKU213 culture containing crude bacteriocins exhibited inhibitory effects on Gram-positive bacteria, including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. The antibacterial activity of the CFS precipitated with 40% ammonium sulfate (AS) remained even after treatment at 60 and 100 °C, at pH 4 and 10 and with proteolytic enzymes, detergents and heavy metals. When analyzed by SDS-PAGE and overlaid with the indicator strains B. cereus and S. aureus, the 40% AS precipitate exhibited inhibitory activity on proteins smaller than 10 kDa. However, proteins larger than 25 kDa and smaller than 10 kDa were still observed on a native protein gel. Purified subtilosin A was prepared by Amberlite XAD-16 bead extraction and HPLC and analyzed by Nano-LC-QTOF-MS. Its molecular mass was found to be 3.4 kDa, and it retained its antibacterial activity. These results are consistent with the detection of the anti-listerial subtilosin A gene of the sbo/alb cluster in the KKU213 strain, which is 100% identical to that of B. subtilis subsp. subtilis 168. In addition to stable and cyclic subtilosin A, a mixture of many extracellular antibacterial peptides was also detected in the KKU213 culture. The KKU213 strain produced extracellular amylase, cellulase, lipase and protease, is highly acid-resistant (pH 2) when cultured in inulin and promotes health and reduces infection of intestinally colonized broiler chickens. Therefore, we propose that bacteriocin-producing B. subtilis KKU213 could be used as a potential probiotic strain or protective culture.

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In this study, the phenotypic and pathogenic properties in freshwater host were characterized in 14 strains of halophilic Vibrio harveyi isolated from infected marine black tiger shrimp, Penaeus monodon. The lysogenic phenotype was assayed via prophage excision and mitomycin C induction. The bacteria were grouped into two types, corresponding to lysogenic and non-lysogenic strains. The pathogenicity was determined via direct injection of bacterial cultures into post-larval juvenile giant freshwater prawns, Macrobrachium rosenbergii De Man. All of the infected prawns showed similar symptoms and inflamed hepatopancreas. The V. harveyi isolates derived from the firstinjected infected prawns were re-isolated and re-injected into healthy giant freshwater prawns, in which they retained similar infectivity. Both lysogenic and non-lysogenic Vibrio spp. showed identical virulence associated with 100% mortality within one day post-injection. TEM micrographs showed hepatopancreatic nuclear deformation and lipid breakdown caused by lysogenic c-hemolytic VL19 and non-lysogenic b-hemolytic V33. However, the V33 strain was associated with severely disrupted mitochondria. None of the V. harveyi strains was able to produce a biofilm. Together, our findings indicate that the lysogenic and nonlysogenic halophilic V. harveyi isolated from marine shrimps may use different virulence factors that are responsible for their pathogenicity in freshwater prawns.

Aim: The aim of the study was to determine the potentials and effects of Bacillus subtilis and lactic acid bacteria (LAB) as probiotics on broiler growth, health, and Salmonella infection. Materials and Methods: To evaluate the inoculum size applicable for broilers, 1-day-old broilers were orally fed fresh cultures of single strains and a B. subtilis KKU213/Pediococcus pentosaceus NP6 mixture at 108 and 1012 colony-forming unit (CFUs)/mL/chick. The body weight gain (BWG), Salmonella contamination level and total Bacillus and LAB abundances in the crop and intestine were measured. Subsequently, 1-day-old broilers were orally fed of KKU213, CH403, and Pediococcus acidilactici SH8 at 1010 CFUs/mL, followed by inulin. After 35 days, the BWG, Bacillus and LAB abundances in the cecum, blood parameters, and KKU213 colonization were assessed. Results: The broilers fed single strains or KKU213+NP6 exhibited a higher BWG and a higher crop LAB abundance than the controls (p<0.05). Probiotic feeding decreased the intestinal Salmonella abundance and correspondingly increased the LAB abundance. The broilers fed the mixed culture (KKU213+CH403+SH8) followed by prebiotics showed lower mortality, higher blood high-density lipoprotein levels, and lower blood uric acid levels than the controls (p<0.0004). Probiotic feeding significantly increased the Bacillus and LAB counts (p<0.05). A CE330 isolate obtained from the cecum after 35 days of KKU213 feeding was closely related to B. subtilis KKU213. Conclusion: B. subtilis KKU213 is a potent probiotic strain that can survive, colonize and reduce Salmonella infection in broilers and improve their growth and health. This strain, combined with different LAB can act synergistically in the gut and promote broiler growth.

Cupriavidus taiwanensis KKU2500-3 is a cadmium (Cd)-tolerant bacterial strain that was previously isolated from rice fields contaminated with high levels of Cd. In 500 μmol/L CdCl2, the KKU2500-3 strain grew slower and with a more prolonged lag-phase than when grown in the absence of Cd. A proteomic approach was used to characterize the protein expression in the Cd-tolerant bacteria C. taiwanensis KKU2500-3 during growth under Cd stress. When compared with the untreated cells, a total of 982 differentially expressed protein spots were observed in the CdCl2-treated cells, and 59 and 10 spots exhibited >2- and >4-fold changes, respectively. The level of up- and downregulation varied from 2.01- to 11.26-fold and from 2.01- to 5.34-fold, respectively. Of the 33 differentially expressed protein spots analyzed by MALDI TOF MS/MS, 19 spots were successfully identified, many of which were involved in stress responses. The most highly upregulated protein (+7.95-fold) identified was the chaperone GroEL, which indicated that this factor likely contributed to the bacterial survival and growth in response to Cd toxicity. Detection of the downregulated protein flagellin (-3.52-fold) was consistent with the less effective ATP-mediated and flagella-driven motility. The flagella-losing cells were also observed in the Cd-treated bacteria when analyzed by scanning electron microscopy. Thus, the Cd-stressed cells may downregulate pathways involving ATP utilization in favor of other mechanisms in response to Cd toxicity. When the KKU2500-3 strain was grown in the presence of Cd, H2S was not detected, suggesting a possible role of the sulfur in precipitation with Cd. Apart from a general response, no specific process could be determined using the present proteomic approach. However, the potential role of protein folding-mediated GroEL, flagella-mediated motility and CdS biotransformation in Cd toxicity response observed in this study as well as the extent of Cd-tolerant mechanisms using other methods could facilitate the future application of this strain in addressing Cd environmental contamination.

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