Symbiotic influence of endophytic Bacillus pumilus on growth promotion and probiotic potential of the medicinal plant Ocimum sanctum

Bacillus pumilus was isolated from surface-sterilized tissues of the medicinal plant Ocimum sanctum. Scanning electron microscopic (SEM) imaging confirmed the presence of a rod shaped bacterium within the plant tissues. The bacterium was identified as B. pumilus by biochemical analyses and 16S rRNA gene sequencing. In vitro analyses indicate that the isolated strain of B. pumilus was endowed with multiple plant growth promotion (PGP) traits such as phosphate solubilization and the production of indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN). Phosphate solubilization (37.3 μg ml^?1) and IAA production (36.7 μg ml^?1) by the isolate was found to reach a maximum after 60 h of incubation. Siderophore mediated iron sequestration by B. pumilus may confer a competitive advantage to the host with respect to pathogen inhibition. Siderophore produced by the isolate was found to be of a trihydroxamate type with hexadentate nature. The B. pumilus isolate also exhibited cellulolytic, proteolytic and chitinolytic activity. Cell free supernatant, culture filtrates of the isolate were found to suppress the growth of fungal phytopathogens. The culture filtrate retained its antifungal activity even after exposure to heat. In addition to PGP, the isolate exhibited probiotic properties such as acid tolerance (pH2), bile salt tolerance (2 %), auto-aggregation, antibiotic resistance and the absence of haemolytic activity. These finding suggest the possibility of utilizing this endophytic strain of B. pumilus as a bioinoculant to enhance plant growth and also as a probiotic.     

Evaluation of probiotic properties of Lactobacillus plantarum strains isolated from Chinese sauerkraut

Lactobacillus plantarum strains isolated and identified from naturally-fermented Chinese sauerkraut were examined in vitro for potential probiotic properties and in vivo for cholesterol-lowering effect in mice. Among 7 isolated L. plantarum strains, strains S2-5 and S4-1 were found to possess desirable probiotic properties including ability to survive at pH 2.0 for 60 min, tolerate pancreatin and bile salts, adhere to Caco-2 cells, produce high β-galactosidase activity and antimicrobial activity against Escherichia coli O157 and Shigella flexneri CMCC(B). In addition, strains S2-5 and S4-1 were susceptible to several antibiotics, and capable of reducing cholesterol level in MRS medium by assimilation of cholesterol at 20.39 and 22.28 μg ml^?1, respectively. The in vivo study with L. plantarum S4-1 showed that feeding with fermented milk containing this strain was able to effectively reduce serum cholesterol level in mice, demonstrating its potential as an excellent probiotic candidate for applications in functional products.     

Effect of bacteriocin and exopolysaccharides isolated from probiotic on <Emphasis Type="Italic">P. aeruginosa</Emphasis> PAO1 biofilm

Microorganisms develop biofilms on indwelling medical devices and are associated with biofilm-related infections, resulting in substantial morbidity and mortality. Therefore, to prevent and control biofilm-associated infections, the present study was designed to assess the anti-biofilm potential of postbiotics derived from probiotic organisms against most prevalent biofilm-forming Pseudomonas aeruginosa PAO1. Eighty lactic acid bacteria isolated from eight neonatal fecal samples possessed antibacterial activity against P. aeruginosa PAO1. Among these, only four lactic acid bacteria produced both bacteriocin and exopolysaccharides but only one isolate was found to maximally attenuate the P. aeruginosa PAO1 biofilm. More specifically, the phenotypic and probiotic characterization showed that the isolated lactic acid bacteria were gram positive, non-motile, and catalase and oxidase negative; tolerated acidic and alkaline pH; has bile salt concentration; showed 53% hydrophobicity; and was found to be non-hemolytic. Phylogenetically, the organism was found to be probiotic Lactobacillus fermentum with accession no. KT998657. Interestingly, pre-coating of a microtiter plate either with bacteriocin or with exopolysaccharides as well as their combination significantly (p < 0.05) reduced the number of viable cells forming biofilms to 41.7% compared with simultaneous coating of postbiotics that had 72.4% biofilm-forming viable cells as observed by flow cytometry and confocal laser scanning microscopy. Therefore, it can be anticipated that postbiotics as the natural biointerventions can be employed as the prophylactic agents for medical devices used to treat gastrointestinal and urinary tract infections.     

Semi-industrial Scale Production of a New Yeast with Probiotic Traits, <Emphasis Type="Italic">Cryptococcus</Emphasis> sp. YMHS,Isolated from the Red Sea

A new yeast strain with promising probiotic traits was isolated from the Red Sea water samples. The isolate (YMHS) was subjected to genetic characterization and identified as Cryptococcus sp. Nucleotide sequence analysis of the rRNA gene internal transcribed spacer regions showed 95% sequence similarity between the isolate and Cryptococcus albidus. Cryptococcus sp. YMHS exhibited desirable characteristics of probiotic microorganisms; it has tolerance to low pH in simulated gastric juice, resistance to bile salts, hydrophobic characteristics, broad antimicrobial activity, and in vitro ability to degrade cholesterol. The isolate grew well in a semi-defined medium composed of yeast extract, glucose, KH₂PO₄, (NH₄)₂SO₄, and MgSO₄, yielding cell mass of 2.32 and 5.82 g/l in shake flask and in bioreactor cultures, respectively. Fed-batch cultivation, with controlled pH, increased the biomass gradually in culture, reaching 28.5 g/l after 32 h cultivation. Beside the feasible use as a probiotic, the new strain also could be beneficial in the development of functional foods or novel food preservatives. To our knowledge, this is the first report of yeast with probiotic properties isolated from the Red Sea.     

Screening and molecular identification of potential probiotic lactic acid bacteria in effluents generated during ogi production

Screening and molecular identification of probiotic lactic acid bacteria (LAB) in effluents generated during the production of ogi, a fermented cereal (maize, millet, and sorghum) were done. LAB were isolated from effluents generated during the first and second fermentation stages in ogi production. Bacterial strains isolated were identified microscopically and phenotypically using standard methods. Probiotic potential properties of the isolated LAB were investigated in terms of their resistance to pH 1.5 and 0.3% bile salt concentration for 4 h. The potential LAB isolates ability to inhibit the growth of pathogenic organisms (Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium) was evaluated in vitro. The pH and LAB count in the effluents ranged from 3.31 to 4.49 and 3.67 to 4.72 log cfu/ml, respectively. A total of 88 LAB isolates were obtained from the effluents and only 10 LAB isolates remained viable at pH 1.5 and 0.3% bile salt. The zones of inhibition of the LAB isolates with probiotic potential ranged from 7.00 to 24.70 mm against test organsisms. Probiotic potential LAB isolates were molecularly identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus brevis. Survival and proliferation of LAB isolates at low pH, 0.3% bile salt condition, and their inhibition against some test pathogens showed that these LAB isolates could be a potential probiotics for research and commercial purposes.     

Evaluation of Potential Probiotics Isolated from Human Milk and Colostrum

Several studies have demonstrated a diversity of bacterial species in human milk, even in aseptically collected samples. The present study evaluated potential probiotic bacteria isolated from human milk and associated maternal variables. Milk samples were collected from 47 healthy women and cultured on selective and universal agar media under aerobic and anaerobic conditions. Bacterial isolates were counted and identified by Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight mass spectrometry and then tested for probiotic properties. Total bacteria in human milk ranged from 1.5 to 4.0 log₁₀ CFU/mL. The higher bacterial counts were found in colostrum (mean = 3.9 log₁₀ CFU/mL, 95% CI 3.14–4.22, p = 0.00001). The most abundant species was Staphylococcus epidermidis (n = 76). The potential probiotic candidates were Lactobacillus gasseri (n = 4), Bifidobacterium breve (n = 1), and Streptococcus salivarius (n = 4). Despite the small sample size, L. gasseri was isolated only in breast milk from mothers classified into a normal weight range and after a vaginally delivered partum. No potential probiotics showed antagonism against pathogens, but all of them agglutinated different pathogens. Nine bacterial isolates belonging to the species L. gasseri, B. breve, and S. salivarius were selected as potential probiotics. The present study confirms the presence in breast milk of a bacterial microbiota that could be the source of potential probiotic candidates to be used in the formula of simulated maternal milk.     

Antibacterial,anti-inflammatory and probiotic potential of Enterococcus hirae isolated from the rumen of Bos primigenius

In the present study bacterial strains were isolated from the rumen fluids of Bos primigenius and investigated their in vitro probiotic properties with potent antibacterial activity and anti-inflammatory effects. 9 g positive bacterial isolates were obtained and three isolates could able to tolerate gastric conditions, high bile salt concentrations and exhibited significant bactericidal effect against the enteric pathogens Vibrio cholera, Enterococcus faecalis, Enterobacter aerogens, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi. Moreover it showed above 70 % cell surface hydrophobicity, significant low-invasion ability and potential adherence capacity in Caco-2 cells when compared with the control. The proinflammatory cytokines (TNF-α) was greatly reduced in rumen bacteria treatment and ARBS-1 modulate the immune response by activating the IL-4 secretion in parallel to TNF-α suppression. The 16s rRNA gene sequence of the active isolates were identified as Enterococcus hirae (ARBS-1), Pediococcus acidilactici (ARBS-4) and Bacillus licheniformis (ARBS-7). This study revealed the probiotic bactericidal properties of E. hirae obtained from the rumen of B. primigenius with potential antibacterial and anti-inflammatory effects. Future studies with the strains may yield some novel probiotic product for livestock’s.     

Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198

Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.     

Hoeflea halophila sp. nov., a novel bacterium isolated from marine sediment of the East Sea, Korea

A Gram-negative, aerobic, motile, straight or curved rod-shaped marine bacterium was isolated from marine sediment of the East Sea, Korea. The isolated strain, JG120-1^T, grows with 0–5 % (w/v) NaCl and at 15–30 °C and pH 6–9. α-galactosidase activity test was positive. Comparative 16S rRNA gene sequence studies showed that this strain belonged to the Alphaproteobacteria and was the most closely related to Hoeflea alexandrii AM1 V30^T, Hoeflea phototrophica DFL-43^T and Hoeflea marina LMG 128^T (98.9, 97.9 and 97.0 % 16S rRNA gene sequence similarities, respectively). Strain JG120-1^T was found to possess summed feature 8 (C18:1ω7c/C18:1ω6c, 71.11 %) as the major cellular fatty acid. The major ubiquinone was determined to be Q-10. Polar lipids include phosphatidylglycerol, phosphatidylethanolamine, sulfoquinovosyl diacylglycerol, phosphatidylcholine and phosphatidylmonomethylethanolamine. The G+C content of the genomic DNA of strain JG120-1^T was determined to be 57.8 mol %. DNA–DNA relatedness data indicated that strain JG120-1^T represents a distinct species that is separate from H. phototrophica DFL-43^T, H. marina LMG128^T and H. alexandrii AM1 V30^T. On the basis of polyphasic evidences, it is proposed that strain JG120-1^T (= KCTC 23107^T = JCM 16715^T) represents the type strain of a novel species, Hoeflea halophila sp. nov.     

Alteromonas australica sp. nov., isolated from the Tasman Sea

A non-pigmented, motile, Gram-negative bacterium designated H 17^T was isolated from a seawater sample collected in Port Phillip Bay (the Tasman Sea, Pacific Ocean). The new organism displayed optimal growth between 4 and 37 °C, was found to be neutrophilic and slightly halophilic, tolerating salt water environments up to 10 % NaCl. Strain H 17^T was found to be able to degrade starch and Tween 80 but unable to degrade gelatin or agar. Phosphatidylglycerol (27.7 %) and phosphatidylethanolamine (72.3 %) were found to be the only associated phospholipids. The major fatty acids identified are typical for the genus Alteromonas and include C_16:0, C_16:1ω7, C_17:1ω8 and C_18:1ω7. The G+C content of the DNA was found to be 43.4 mol%. A phylogenetic study, based on the 16S rRNA gene sequence analysis and Multilocus Phylogenetic Analysis, clearly indicated that strain H 17^T belongs to the genus Alteromonas. The DNA?DNA relatedness between strain H 17^T and the validly named Alteromonas species was between 30.7 and 46.4 mol%. Based on these results, a new species, Alteromonas australica, is proposed. The type strain is H 17^T (= KMM 6016^T = CIP 109921^T).     

Prebiotic effects of bovine lactoferrin on specific probiotic bacteria

Bovine lactoferrin (bLf) is a natural iron-binding protein and it has been suggested to be a prebiotic agent, but this finding remains inconclusive. This study explores the prebiotic potential of bLf in 14 probiotics. Initially, bLf (1–32 mg/mL) treatment showed occasional and slight prebiotic activity in several probiotics only during the late experimental period (48, 78 h) at 37 °C. We subsequently supposed that bLf exerts stronger prebiotic effects when probiotic growth has been temperately retarded. Therefore, we incubated the probiotics at different temperatures, namely 37 °C, 28 °C, room temperature (approximately 22–24 °C), and 22 °C, to retard or inhibit their growth. As expected, bLf showed more favorable prebiotic activity in several probiotics when their growth was partially retarded at room temperature. Furthermore, at 22 °C, the growth of Bifidobacterium breve, Lactobacillus coryniformis, L. delbrueckii, L. acidophilus, B. angulatum, B. catenulatum, and L. paraplantarum were completely blocked. Notably, these probiotics started regrowing in the presence of bLf (1–32 mg/mL) in a significant and dose-dependent manner. Accordingly, bLf significantly increased the growth of Pediococcus pentosaceus, L. rhamnosus, and L. paracasei (BCRC 17483; a locally isolated strain) when their growth was retarded by incubation at 22 °C. In conclusion, bLf showed inconsistent prebiotic activity in the 14 probiotics at 37 °C, but revealed strong prebiotic activity in 10 probiotic strains at 22 °C. Therefore, this study enables determining additional roles of Lf in probiotic strains, which can facilitate developing novel combinational approaches by simultaneously using Lf and specific probiotics.     

Coralslurrinella hongkonensis gen. nov., sp. nov., a novel bacterium in the family Psychromonadaceae, isolated from the coral Platygyra carnosus

A novel bacterial strain, JLT2006^T, was isolated from the scleractinian coral Platygyra carnosus, located in Hong Kong, China. Cells of this strain were Gram-negative, rod-shaped or oval-shaped and motile by the means of polar flagella. They formed faint-yellow, round colonies on marine agar medium. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the strain JLT2006^T belonged to the class Gammaproteobacteria and was most closely related to Alteromonas-like bacteria of the genera Psychromonas, Pseudoalteromonas, Moritella, Shewanella and Ferrimonas, with less than 93 % sequence similarity. The predominant fatty acids were identified as C18:1ω7c/C18:1ω6c (23.0 %), C16:1ω7c/C16:1ω6c (18.2 %) and C16:0 (16.4 %). The quinone was menaquinone-7 (100 %). The polar lipids were determined to be phosphatidylglycerol, phosphatidylethanolamine, phospholipid, glycolipid and lipid. The genomic DNA G+C content was 40.3 mol%. Based on the 16S rRNA gene sequence as well as the physiological and biochemical features that separate the strain JLT2006^T from other recognized bacteria, a novel species of a new genus with the name Coralslurrinella hongkonensis gen. nov., sp. nov. is proposed. The type strain is JLT2006^T (=JCM 18796^T = CGMCC 1.10992^T).     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Desulfotomaculum tongense sp. nov., a moderately thermophilic sulfate-reducing bacterium isolated from a hydrothermal vent sediment collected from the Tofua Arc in the Tonga Trench

A novel, strictly anaerobic, moderately thermophilic, endospore-forming, sulfate-reducing bacterium, designated TGB60-1^T, was isolated from a hydrothermal sediment vent collected from the Tofua Arc in the Tonga Trench. The strain was characterized phenotypically and phylogenetically. The isolated strain was observed to be Gram-positive, with slightly curved rod-shaped cells and a polar flagellum. Strain TGB60-1^T was found to grow anaerobically at 37–60 °C (optimum, 50 °C), at pH 6.0–8.5 (optimum, pH 7.0) and with 1.0–4.0 % (w/v) NaCl (optimum, 3.0 %). The electron acceptors utilised were determined to be sulfate, sulfite, and thiosulfate. Strain TGB60-1^T was found to utilise pyruvate and H₂ as electron donors. Strain TGB60-1^T was determined to be related to representatives of the genus Desulfotomaculum and the closest relatives within this genus were identified as Desulfotomaculum halophilum SEBR 3139^T, Desulfotomaculum alkaliphilum S1^T and Desulfotomaculum peckii LINDBHT1^T (92.7, 92.1, and 91.8 % 16S rRNA gene sequence similarity, respectively). The major fatty acids (>20 %) were identified as C_16:0 and C_18:1 ω7c. The G+C content of the genomic DNA of this novel bacterium was determined to be 53.9 mol%. Based on this polyphasic taxonomic study, strain TGB60-1^T is considered to represent a novel species in the genus Desulfotomaculum, for which the name Desulfotomaculum tongense sp. nov. is proposed. The type strain of D. tongense is strain TGB60-1^T (= KTCT 4534^T = JCM 18733^T).     

Halomonas nanhaiensis sp. nov., a halophilic bacterium isolated from a sediment sample from the South China Sea

A novel Gram-negative, aerobic, slightly halophilic, yellow-pigmented, oxidase-negative, Voges–Proskauer positive, non-spore-forming bacterium, designated YIM M 13059^T, was isolated from a sediment sample collected from the South China Sea at a depth of 310 m. Optimal growth was found to occur at 28–30 °C, pH 7.0 and in the presence of 3–4 % (w/v) NaCl. Cells were observed to be rod-shaped and motile by peritrichous flagella. The polar lipids of strain YIM M 13059^T were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a ninhydrin-positive phospholipid, one glycolipid and two unknown phospholipids. The predominant respiratory quinone was determined to be Q-9. The major fatty acids were identified as C_18:1 ω7c, C_16:1 ω6c/C_16:1 ω7c, C_16:0 and C_12:0 3-OH. The genomic DNA G+C content was determined to be 54.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belongs to the genus Halomonas in the family Halomonadaceae. The 16S rRNA gene sequence similarities between strain YIM M 13059 ^T and the type strains of members of the genus Halomonas were in the range 93.3–98.3 %. However, the levels of DNA–DNA relatedness values between YIM M 13059 and the type strains of the most closely related species, Halomonas zhangjiangensis, Halomonas variabilis, Halomonas neptunia, Halomonas boliviensis and Halomonas sulfadieris were 50.2 ± 0.68 %, 46.8 ± 1.9 %, 28.5 ± 0.74 %, 42.9 ± 0.55 % and 37.1 ± 0.68 %, respectively. Based on phylogenetic, chemotaxonomic and phenotypic data, the strain YIM M 13059^T is proposed to represent a novel member of the genus Halomonas, with the name Halomonas nanhaiensis sp. nov. The type strain is YIM M 13059^T (=JCM 18142^T =CCTCC AB 2012911^T).     

Therapeutic potential of Lactobacillus ingluviei ADK10, a newly established probiotic organism against acetaminophen induced uremic rats

In the present study, Lactobacillus ingluviei ADK10 (Acc. No. JQ395039) from intestinal origin was tested for its probiotic characteristic as well as uremia ameliorating activity on acetaminophen induced uremic rats. The results revealed that L. ingluviei ADK10 was able to tolerate pH 3.0–9.0 and 0.5% bile salt along with good hydrophobicity (67%) and adherence index with Ht-29 cell line on 258/100 cells. It was susceptible to 20 antibiotics. The organism was able to degrade food ingredients, like starch and milk proteins. The strain showed significant growth inhibition of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Shigella dysentery, Pseudomonas aeruginosa, and Klebsiella pneumoniae (average diameter of 10 mm). The therapeutic potentiality of this probiotic bacterium was tested against acetaminophen induced uremic rats. It was found that supplementation of L. ingluviei ADK10 for 14 days with food reduced severe increase of uremic profiles, such as blood urea (85%), creatinine (68%) and uric acid (41%) in comparison to the uremic rats. Moreover, during the feeding of rats with probiotic strain at a dose of 1×10⁹ bacteria, reduction of enterobacteria in faeces was observed. Our studies indicated that L. ingluviei ADK10 could be used as a health-promoting probiotic along with antiuremic efficacy.     

Isolation and characterization of a novel violacein-like pigment producing psychrotrophic bacterial species Janthinobacterium svalbardensis sp. nov

A bacterial strain designated JA-1, related to Janthinobacterium lividum, was isolated from glacier ice samples from the island Spitsbergen in the Arctic. The strain was tested for phenotypic traits and the most prominent appeared to be the dark red brown to black pigmentation different from the violet pigment of Janthinobacterium, Chromobacterium and Iodobacter. Phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization tests showed that strain JA-1 belongs to the genus Janthinobacterium but represents a novel lineage distinct from the two known species of this genus, J. lividum and Janthinobacterium agaricidamnosum. The DNA G + C content of strain JA-1 was determined to be 62.3 mol %. The isolate is a psychrotrophic Gram negative bacterium, rod-shaped with rounded ends, containing intracellular inclusions and one polar flagellum. On the basis of the presented results strain JA-1 is proposed as the type strain of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium svalbardensis sp. nov. is proposed (JA-1^T = DSM 25734, ZIM B637).