In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Antimicrobial properties of the novel bacterial isolate Paenibacilllus sp. SMB1 from a halo-alkaline lake in India.

Antibiotic-resistance is ever growing burden on our society for the past many years. Many synthetic chemistry approaches and rational drug-design have been unable to pace up and tackle this problem. Natural resources, more specifically, the microbial diversity, on the other hand, make a traditional and still the best platform to search for new chemical scaffolds and compounds. Here, we report the antimicrobial characteristics of novel bacterial isolate from a salt lake in India. We screened the bacterial isolates for their inhibitory activity against indicator bacteria and found that four novel species were able to prevent the growth of test strains studied in vitro. Further, we characterized one novel species (SMB1T = SL4-2) using polyphasic taxonomic approaches and also purified the active ingredient from this bacterium. We successfully characterized the antimicrobial compound using mass spectroscopy and amino acid analysis. We also allocated two novel biosynthetic gene clusters for putative bacteriocins and one novel non-ribosomal peptide gene cluster in its whole genome. We concluded that the strain SMB1T belonged to the genus Paenibacilllus with the pairwise sequence similarity of 98.67% with Paenibacillus tarimensis DSM 19409T and we proposed the name Paenibacillus sambharensis sp. nov. The type strain is SMB1T (=MTCC 12884 = KCTC 33895T).

Pseudomonas koreensis Recovered From Raw Yak Milk Synthesizes a ß-Carboline Derivative With Antimicrobial Properties.

Natural evolution in microbes exposed to antibiotics causes inevitable selection of resistant mutants. This turns out to be a vicious cycle which requires the continuous discovery of new and effective antibiotics. For the last six decades, we have been relying on semisynthetic derivatives of natural products discovered in "Golden Era" from microbes, especially Streptomyces sp. Low success rates of rational drug-design sparked a resurgence in the invention of novel natural products or scaffolds from untapped or uncommon microbial niches. Therefore, in this study, we examined the microbial diversity inhabiting the yak milk for their ability to produce antimicrobial compounds. We prepared the crude fermentation extracts of fifty isolates from yak milk and screened them against indicator strains for the inhibitory activity. Later, with the aid of gel filtration chromatography followed by reversed-phase HPLC, we isolated one antimicrobial compound Y5-P1 from the strain Y5 (Pseudomonas koreensis) which showed bioactivity against Gram-positive and Gram-negative bacteria. The compound was chemically characterized using HRMS, FTIR, and NMR spectroscopy and identified as 1-acetyl-9H-ß-carboline-3-carboxylic acid. It showed minimum inhibitory activity (MIC) in the range of 62.5-250 µg /ml. The cytotoxicity results revealed that IC50 against two mammalian cell lines i.e., HepG2 and HEK293T was 500 and 750 µg/ml, respectively. This is the first report on the production of this derivative of ß-carboline by the microorganism. Also, the study enlightens the importance of microbes residing in uncommon environments or unexplored habitats in the discovery of a diverse array of natural products which could be designed further as drug candidates against highly resistant pathogens.

Salibacterium nitratireducens sp. nov., a haloalkalitolerant bacterium isolated from a water sample from Sambhar salt lake, India.

A strictly aerobic, haloalkali-tolerant, Gram-stain-positive, non-motile, rod-shaped bacterium, designated strain SMB4T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. Growth occurred at 25-50 °C, 4-12 % (w/v) NaCl and pH of 5-9. Strain SMB4T was positive for ß-galactosidase, oxidase, catalase and urease activities. The fatty acids were dominated by branched forms of fatty acids with iso- and anteiso-saturated fatty acids, with a high abundance of anteiso-C15 : 0, anteiso-C17 : 0 and C18 : 0. The cell-wall peptidoglycan of strain SMB4T contained meso-diaminopimelic acid, while the polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and three unidentified lipids. The DNA G+C content of strain SMB4T was 49.1 mol%. A blast sequence similarity search based on 16S rRNA gene sequence indicated that Salibacterium halochares, Salibacterium halotolerans and Salibacterium qingdaonense were the nearest phylogenetic neighbours, with a pair-wise sequence similarities of 98.4, 98.2 and 97.0 % respectively. Phylogenetic analysis showed that strain SMB4T was clustered with S. halochares and together clustered with S. halotolerans and S. qingdaonense. DNA-DNA hybridization of strain SMB4T with S. halochares DSM 21373T, S. halotolerans S7T and S. quigdaonense DSM 21621T showed a relatedness values of only 39.8, 26.3 and 42.8 %, respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain SMB4T represents a novel species of the genus Salibacterium, for which the name Salibacterium nitratireducens sp. nov. is proposed. The type strain is SMB4T (=MTCC 12633T=KCTC 33876T=JCM 32187T).

Bacillus shivajii sp. nov., isolated from a water sample of Sambhar salt lake, India.

A novel Gram-stain-positive, rod-shaped, motile, spore-forming, strictly aerobic, alkali- and halo- tolerant bacterium, designated strain AK72T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. The colony appears circular, shiny, smooth, translucent or slightly pale in colour and convex with an entire margin after 48 h incubation at 37 °C with pH 9. Growth of the bacterium occurred at 10-42 °C (optimum, 25-37 °C), at salinities of 0.5-10 % (w/v) NaCl (optimum 3-5 % NaCl) and pH of 6-10 (optimum pH 9). Strain AK72T was positive for oxidase, catalase, nitrate reductase, phenylalanine deaminase, ornithine decarboxylase, aesculinase, lipase and urease activities. The predominant fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0 and the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The major polar lipids of the strain were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, three unidentified phospholipids and three unidentified lipids. The genomic DNA G+C content of the strain AK72T was 36.8 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain AK72T was closely related to Bacillus cellulosilyticus (96.5 %) and Bacillus vedderi (96.3 %), but the novel strain AK72T formed a separate clade with Bacillus aurantiacus whereas B. cellulosilyticus and B. vedderi were clustered in a separate clade. The above data in combination with the phenotypic characteristics and phylogenetic data inferred that strain AK72T represents a novel species of the genus Bacillus, for which the name Bacillusshivajii sp. nov. is proposed. The type strain is AK72T (=MTCC 12636T=KCTC 33981T=JCM 32183T).

Bacillus alkalilacus sp. nov., isolated from a sediment sample from a lake in India.

An aerobic, endospore-forming, haloalkali-tolerant, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK73T, was isolated from a sediment sample collected from Sambhar lake, Jaipur, Rajasthan, India. Colonies were circular, 1-2 mm in diameter, glossy, smooth, yellowish and convex with an entire margin after 48 h growth on marine agar at pH 9 and 37 °C. Growth occurred at 15-42 °C, 0-10 % (w/v) NaCl and at a pH range of 7-12. Strain AK73T was positive for catalase and arginine dihydrolase 2 activities, hydrolysis of Tweens 20, 40 and 80, and negative for esculinase, caseinase, gelatinase, ß-galactosidase, lipase (Tween 60) and urease activities. The fatty acids were dominated by branched iso-, anteiso-, saturated fatty acids with a high abundance of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0; MK-7 was the major menaquinone. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, four unidentified phospholipids and three unidentified lipids. The DNA G+C content of strain AK73T was 54 mol%. Analysis based on comparative 16S rRNA gene sequence analysis indicated that Bacillus alcalophilus was the nearest phylogenetic neighbour, with a pair-wise sequence similarity of 96.0 %. Phylogenetic analysis showed that strain AK73T formed a separate lineage but was loosely associated with a peripheral cluster of organisms that contained Bacillus gibsonii, Bacillus murimartini and Bacillus plakortidis with similarity values of 93.6, 93.5 and 93.4 %, respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain AK73T represents a novel species of the genus Bacillus, for which the name Bacillus alkalilacus sp. nov. is proposed. The type strain is AK73T (=JCM 32184T=MTCC 12637T=KCTC 33880T).

Sphingobacterium bovisgrunnientis sp. nov., isolated from yak milk.

A novel Gram-negative, rod shaped, non-motile bacterium, designated strain YK2T, was isolated from yak milk from Leh, India. The strain was positive for oxidase- and catalase-activities and negative for starch hydrolysis, nitrate reduction, citrate utilization, urease, lysine decarboxylase and ornithine decarboxylase activities. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH (summed feature 3). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and six unidentified lipids. The DNA G+C content of the strain was 38.9 mol%. The 16S rRNA gene sequence analysis indicated that strain YK2T was a member of the genus Sphingobacterium and closely related to Sphingobacterium alimentarium and Sphingobacterium composti with pair-wise sequence similarity of 98.3 and 97.9 %, respectively. The sequence similarity to other members of the genus Sphingobacterium was between 92.6 to 96.3 %. Phylogenetic analysis showed that strain YK2T clustered with Sphingobacterium alimentarium and together clustered with Sphingobacterium composti. DNA-DNA hybridization of strain YK2T with Sphingobacterium alimentarium WCC 4521T and Sphingobacterium composti T5-12T showed a relatedness of only 38 and 54 %, respectively. Based on the phenotypic characteristics and on phylogenetic inference, it appears that strain YK2T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium bovisgrunnientis sp. nov. is proposed. The type strain of Sphingobacterium bovisgrunnientis sp. nov. is YK2T (=MTCC 12631T=KCTC 52685T=JCM 31951T).

Bacillus lacus sp. nov., isolated from a water sample of a salt lake in India.

A strictly aerobic, alkaliphilic, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK74T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. Colonies were circular, 1.2 mm in diameter, shiny, smooth, whitish and convex with an entire margin after 48 h growth at 37 °C with pH 9.0. Growth occurred at 25-42 °C, 0-4 % (w/v) NaCl and at a pH of 7-12. Strain AK74T was positive for aesculinase, caseinase, lipase activities and negative for oxidase, catalase, amylase, cellulase, DNase, gelatinase and urease activities. The fatty acids were dominated by branched iso-, anteiso- and saturated fatty acids with a high abundance of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and C16 : 1 and the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The DNA G+C content of strain AK74T was 51.6 mol%. A blast sequence similarity search based on 16S rRNA gene sequences indicated that Bacillus niabensis, Bacillus idriensisand Bacillus halosaccharovorans were the nearest phylogenetic neighbours, with a pair-wise sequence similarities of 96.6, 96.6 and 96.5%, respectively. Phylogenetic analysis showed that strain AK74T clustered with Bacillus mangrove and together clustered with Bacillus idriensisand Bacillus indicus. Based on its phenotypic characteristics and on phylogenetic inference, strain AK74T represents a novel species of the genus Bacillus, for which the name Bacilluslacus sp. nov. is proposed. The type strain is AK74T (=MTCC 12638T=KCTC 33946T=JCM 32185T).

Lactic acid bacteria isolated from yak milk show probiotic potential.

Probiotic industries strive for new, efficient and promising probiotic strains that impart a positive impact on consumer health. Challenges are persisting in isolation, screening, and selection of the new indigenous probiotic strains. In the present research, we explored the probiotic potential of 17 lactic acid bacteria isolated from Yak milk in a series of in vitro tests. We also demonstrated their health benefits, i.e., cholesterol degradation, lactose digestion, antimicrobial activity, antioxidant, and anticancer activities. Principal component analysis revealed that more than 50% of the strains fulfilled the examined criteria, e.g., survival in acidic pH, bile concentrations, and adherent property. Approximately all the strains produced antimicrobial substances against the maximum number of tested strains including clinical strains. Most strains degraded cholesterol in comparison to the reference probiotic strain whereas strain Yc showed 1.5 times higher the degradation efficiency of the control strain. Lan4 strain exhibited remarkable anticancer activity and induced the maximum apoptosis (87%) in the Hela cells and was non-toxic to the non-cancerous HEK293 cells. Around ten strains showed positive lactose digestion. Overall, this can be concluded that selected lactic acid bacteria revealed excellent probiotic properties along with desirable health benefits. These strains need to be further investigated in details for their application in the development of novel probiotic preparations for the improvement of public health.

Beighton scores and cut-offs across the lifespan: cross-sectional study of an Australian population.

Objectives: The primary aim of this study was to evaluate generalized joint hypermobility (GJH) according to the Beighton scoring system in an Australian population. Secondary aims were to identify whether the commonly used Beighton score cut-off of ⩾4 is appropriate, and to suggest age- and sex-specific Beighton score cut-offs across the lifespan. Methods: A thousand individuals aged 3-101 years were assessed for GJH with the Beighton scoring system. Differences between age, sex and ethnicity were investigated. The appropriateness of the ⩾4 cut-off was investigated with use of a binary logistic regression. Each Beighton score cut-off was established as the nearest Beighton score that delineated the uppermost 5% of the population. Results: Overall, females and non-Caucasians had higher Beighton scores across the lifespan (P < 0.001). Based on a binary logistic regression model, if a cut-off of ⩾4 was utilized, the Beighton scoring system demonstrated a sensitivity of 0.8% and a specificity of 99.3% (P < 0.001). A cut-off of ⩾4 was only found to be appropriate for females aged 40-59 years and males aged 8-39 years. Conclusion: Beighton scores varied across the lifespan and were significantly influenced by age, sex and ethnicity. Assessing GJH using the Beighton scoring system required age- and sex-specific cut-off scores based on the uppermost 5% values. This was confirmed by the low sensitivity, high specificity and 60% false-positive rate if a cut-off of ⩾4 was used for both sexes across the lifespan. To lower the risk of a false-positive diagnosis of GJH, further tests of hypermobility need to be utilized.