The impact of diet and ethnicity on gut microbiota variation in irritable bowel syndrome: A multi-center study.

BACKGROUND AND AIM: The gut microbiota in irritable bowel syndrome (IBS) is known to vary with diet. We aim to (i) analyze the gut microbiota composition of IBS patients from a multi-ethnic population and (ii) explore the impact of a low FODMAP diet on gastrointestinal symptoms and gut microbiota composition among IBS patients. METHODS: A multi-center study of multi-ethnic Asian patients with IBS was conducted in two phases: (i) an initial cross-sectional gut microbiota composition study of IBS patients and healthy controls, followed by (ii) a single-arm 6-week dietary interventional study of the IBS patients alone, exploring clinical and gut microbiota changes. RESULTS: A total of 34 adult IBS patients (IBS sub-types of IBS-D 44.1%, IBS-C 32.4%, and IBS-M 23.5%) and 15 healthy controls were recruited. A greater abundance of Parabacteroides species with lower levels of bacterial fermenters and short-chain fatty acids producers were found among IBS patients compared with healthy controls. Age and ethnicity were found to be associated with gut microbiota composition. Following a low FODMAP dietary intervention, symptom and quality of life improvement were observed in 24 (70.6%) IBS patients. Symptom improvement was associated with adherence to the low FODMAP diet (46.7% poor adherence vs 92.9% good adherence, P = 0.014), and gut microbiota patterns, particularly with a greater abundance of Bifidobacterium longum, Anaerotignum propionicum, and Blautia species post-intervention. CONCLUSION: Gut microbiota variation in multi-ethnic IBS patients may be related to dietary intake and may be helpful to identify patients who are likely to respond to a low FODMAP diet.

The Association between Hope and Quality of Life among Adolescents with Chronic Diseases: A Systematic Review.

PURPOSE: Hope has been identified as a protective factor that contributes to achieving a better quality to life, especially in patients with chronic disease. The purpose of this review was to synthesize current knowledge about the relationship between hope and quality of life among adolescents living with chronic illnesses. METHODS: We searched major English-language databases (PsycINFO, PubMed, and CINAHL) for studies from January 1, 2002 to July 12, 2019. Studies were included if they provided data on hope and its relationship with quality of life among adolescents with chronic diseases. RESULTS: In total, five articles were selected from the 336 studies that were retrieved. All five studies reported a positive correlation between hope and quality of life, such that people with a higher level of hope had a better quality of life. Hope was found to have direct and indirect effects on quality of life in adolescents with chronic diseases. CONCLUSION: Healthcare professionals should make more efforts to enhance hope in adolescents with chronic diseases in order to improve their quality of life. Future studies exploring how hope develops in adolescents with chronic diseases and the long-term impact of hope on quality of life are necessary.

Potential effects of Psidium sp., Mangifera sp., Mentha sp. and its mixture (PEM) in reducing bacterial populations in biofilms, adherence and acid production of S. sanguinis and S. mutans.

OBJECTIVE: Psidium sp., Mangifera sp. and Mentha sp. and its mixture (PEM) are known to have antimicrobial and anti-adherence effects. DESIGN: Here, we have investigated these individual plant extracts and its synergistic mixture (PEM) for its anti-cariogenic effect to reduce populations of single and mixed-species of Streptococcus sanguinis and Streptococcus mutans in a planktonic or/and biofilm and their others reduced virulence. Bacterial populations in the biofilm after 24 h, hydrophobic cell surface activity to n-hexadecane and pH changes at 5 min' intervals until 90 min of incubation were recorded. Total phenolic content and bioactive compounds in the crude aqueous plant extracts were analysed. Regulatory gene expressions of S. mutans adhesins genes (gtfB, gtfC, gbpB and spaP) upon treatment with PEM were investigated in planktonic and biofilm conditions. RESULTS: All plant extracts strongly reduced S. mutans in the biofilm compared to S. sanguinis in single and mixed-species. PEM reduced S. mutans by 84% with S. sanguinis 87% in the mixed population. Psidium sp. and PEM highly reduced cell-surface hydrophobicity of the two bacteria thus reducing adherence and biofilm formation. PEM and Mangifera sp. lowered initial pH change in the mixed populations of S. sanguinis and S. mutans. PEM downregulated the S. mutans gtfB gene expression in the single species planktonic and mixed-species biofilms. CONCLUSIONS: The effectiveness of PEM in reducing S. mutans within the biofilm, cell-surface hydrophobicity, acid production and adhesin gene (gtfB) expression in mixed-species with S. sanguinis indicates its potential as an antibacterial agent against dental caries. This is attributed to the phenolic content in the PEM.

Purification, characterization, mode of action, and enhanced production of Salivaricin mmaye1, a novel bacteriocin from Lactobacillus salivarius SPW1 of human gut origin

Background: Emergence of antibiotic resistance among pathogenic and food spoilage bacteria such as Staphylococcus aureus, Micrococcus luteus, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mutans, Bacillus cereus, and Listeria monocytogenes triggered the search for alternative antimicrobials. An investigation aimed at purifying, characterizing, elucidating the mode of action, and enhancing the production of salivaricin from Lactobacillus salivarius of human gut origin was conducted. Results: Salivaricin mmaye1 is a novel bacteriocin purified from L. salivarius isolated from human feces. It is potent at micromolar concentrations and has a molecular weight of 1221.074 Da as determined by MALDI-TOF mass spectrometry. It has a broad spectrum of antibacterial activity. Salivaricin mmaye1 showed high thermal and chemical stability and moderate pH stability. The proteinaceous nature of salivaricin mmaye1 was revealed by the complete loss of activity after treatment with pepsin, trypsin, α-chymotrypsin, protease, and proteinase. Salivaricin mmaye1 is cell wall associated, and adsorption­desorption of the bacteriocin from the cell wall of the producer by pH modification proved successful. It exhibited a bactericidal mode of action mediated by pore formation. Its biosynthesis is regulated by a quorum sensing mechanism. Enhanced production of salivaricin mmaye1 was achieved in a newly developed growth medium. Conclusions: A novel, cell wall adhering, highly potent bacteriocin with a broad spectrum of inhibitory activity, membrane-permeabilizing ability, and enhanced production in a newly constituted medium has been isolated. It has a quorum sensing regulatory system and possesses interesting physicochemical characteristics favoring its future use in food biopreservation. These findings pave the way for future evaluation of its medical and food applications.

Characterization, yield optimization, scale up and biopreservative potential of fermencin SA715, a novel bacteriocin from Lactobacillus fermentum GA715 of goat milk origin.

BACKGROUND: Emergence of antibiotic resistance and growing consumer trend towards foods containing biopreservatives stimulated the search for alternative antimicrobials. This research is aimed at characterizing, investigating the mechanism of action, scale up optimization and evaluating the biopreservative potential of a bacteriocin from Lactobacillus fermentum. RESULTS: Fermencin SA715 is a novel, broad-spectrum, non-pore-forming and cell wall-associated bacteriocin isolated from L. fermentum GA715 of goat milk origin. A combination of hydrophobic interaction chromatography, solid-phase extraction and reversed-phase HPLC was necessary for purification of the bacteriocin to homogeneity. It has a molecular weight of 1792.537 Da as revealed by MALDI-TOF mass spectrometry. Fermencin SA715 is potent at micromolar concentration, possesses high thermal and pH stability and inactivated by proteolytic enzymes thereby revealing its proteinaceous nature. Biomass accumulation and production of fermencin SA715 was optimum in a newly synthesized growth medium. Fermencin SA715 did not occur in the absence of manganese(II) sulphate. Tween 80, ascorbic acid, sodium citrate and magnesium sulphate enhanced the production of fermencin SA715. Sucrose is the preferred carbon source for growth and bacteriocin production. Sodium chloride concentration higher than 1% suppressed growth and production of fermencin SA715. Optimum bacteriocin production occurred at 37 °C and pH 6-7. Scale up of fermencin SA715 production involved batch fermentation in a bioreactor at a constant pH of 6.5 which resulted in enhanced production. Fermencin SA715 doubled the shelf life and improved the microbiological safety of fresh banana. Bacteriocin application followed by refrigeration tripled the shell life of banana. CONCLUSIONS: This study reveals the huge potential of fermencin SA715 as a future biopreservative for bananas and reveals other interesting characteristics which can be exploited in the preservation of other foods. Furthermore insights on the factors influencing the production of fermencin SA715 have been revealed and optimized condition for its production has been established facilitating future commercial production.

Pentocin MQ1: A Novel, Broad-Spectrum, Pore-Forming Bacteriocin From Lactobacillus pentosus CS2 With Quorum Sensing Regulatory Mechanism and Biopreservative Potential.

Micrococcus luteus, Listeria monocytogenes, and Bacillus cereus are major food-borne pathogenic and spoilage bacteria. Emergence of antibiotic resistance and consumer demand for foods containing less of chemical preservatives led to a search for natural antimicrobials. A study aimed at characterizing, investigating the mechanism of action and regulation of biosynthesis and evaluating the biopreservative potential of pentocin from Lactobacillus pentosus CS2 was conducted. Pentocin MQ1 is a novel bacteriocin isolated from L. pentosus CS2 of coconut shake origin. The purification strategy involved adsorption-desorption of bacteriocin followed by RP-HPLC. It has a molecular weight of 2110.672 Da as determined by MALDI-TOF mass spectrometry and a molar extinction value of 298.82 M-1 cm-1. Pentocin MQ1 is not plasmid-borne and its biosynthesis is regulated by a quorum sensing mechanism. It has a broad spectrum of antibacterial activity, exhibited high chemical, thermal and pH stability but proved sensitive to proteolytic enzymes. It is potent against M. luteus, B. cereus, and L. monocytogenes at micromolar concentrations. It is quick-acting and exhibited a bactericidal mode of action against its targets. Target killing was mediated by pore formation. We report for the first time membrane permeabilization as a mechanism of action of the pentocin from the study against Gram-positive bacteria. Pentocin MQ1 is a cell wall-associated bacteriocin. Application of pentocin MQ1 improved the microbiological quality and extended the shelf life of fresh banana. This is the first report on the biopreservation of banana using bacteriocin. These findings place pentocin MQ1 as a potential biopreservative for further evaluation in food and medical applications.

Antibacterial and anti-adherence effects of a plant extract mixture (PEM) and its individual constituent extracts (Psidium sp., Mangifera sp., and Mentha sp.) on single- and dual-species biofilms.

BACKGROUND: Plant extracts mixture (PEM) and its individual constituent plant extracts(Psidium sp., Mangifera sp., Mentha sp.) are known to have an anti-adhering effect towards oral bacteria in the single-species biofilm. To date, the adhering ability of the early and late plaque colonisers (Streptococcus sanguinis and Streptococcus mutans) to PEM-treated experimental pellicle have not been investigated in dual-species biofilms. METHODS: Fresh leaves of these plants were used in the preparation of the respective aqueous extract decoctions. The minimum inhibitory concentration (MIC) of the extracts towards S. sanguinis ATCC BAA-1455 and S. mutans ATCC 25175 was determined using a two-fold serial microdilution method. The sum of fractional inhibitory concentration (ΣFIC) index of PEM and its constituent plant extracts was calculated using the MIC values of the plants. The minimum bactericidal concentration (MBC) of the plant extracts was also determined. The anti-adherence effect of the plant extracts (individually and mixed) was carried out by developing simulated S. sanguinis and S. mutans respectively in single- and dual-species of biofilms in the Nordini's Artificial Mouth (NAM) model system in which the experimental pellicle was pretreated with the plant extract before bacterial inoculation. The bacterial population in the respective biofilms was quantified using ten-fold serial dilutions method and expressed as colony forming unit per ml (CFU/ml). The bacterial population was also viewed using Scanning Electron Microscope (SEM). All experiments were done in triplicate. RESULTS: The PEM compared with its respective constituent plants showed the lowest MIC towards S. sanguinis (3.81 mg/ml) and S. mutans (1.91 mg/ml) and exhibited a synergistic effect. The Psidium sp. (15.24 mg/ml) and, PEM and Psidium sp. (30.48 mg/ml) showed the lowest MBC towards S. sanguinis and S. mutans respectively. The anti-adherence effect of the PEM and its respective constituent plants (except Psidium sp.) was different for the two bacteria in the single-species biofilm. In the dual-species biofilms, PEM demonstrated similar anti-adherence effect towards S. sanguinis and S. mutans. The proportions of the bacterial population viewed under SEM appeared to be in agreement with the quantified population. DISCUSSION: The combination of the active constituents of the individual plant extracts in PEM may contribute to its low MIC giving rise to the synergistic effect. The different anti-adherence effect towards S. sanguinis and S. mutans in both single- and dual-species biofilms could be due to the different proportion of the active constituents of the extracts and the interaction between different bacteria. The better adhering ability of S. sanguinis towards the PEM-treated pellicle when present together with S. mutans in the dual-species biofilms may suggest the potential of PEM in controlling the balance between the early and late colonisers in biofilms.

New insights into the mode of action of the lantibiotic salivaricin B.

Salivaricin B is a 25 amino acid polycyclic peptide belonging to the type AII lantibiotics and first shown to be produced by Streptococcus salivarius. In this study we describe the bactericidal mode of action of salivaricin B against susceptible Gram-positive bacteria. The killing action of salivaricin B required micro-molar concentrations of lantibiotic whereas the prototype lantibiotic nisin A was shown to be potent at nano-molar levels. Unlike nisin A, salivaricin B did not induce pore formation or dissipate the membrane potential in susceptible cells. This was established by measuring the fluorescence of the tryptophan residue at position 17 when salivaricin B interacted with bacterial membrane vesicles. The absence of a fluorescence blue shift indicates a failure of salivaricin B to penetrate the membranes. On the other hand, salivaricin B interfered with cell wall biosynthesis, as shown by the accumulation of the final soluble cell wall precursor UDP-MurNAc-pentapeptide which is the backbone of the bacterial peptidoglycan. Transmission electron microscopy of salivaricin B-treated cells showed a reduction in cell wall thickness together with signs of aberrant septum formation in the absence of visible changes to cytoplasmic membrane integrity.

Identification of Gingival Crevicular Fluid Sampling, Analytical Methods, and Oral Biomarkers for the Diagnosis and Monitoring of Periodontal Diseases: A Systematic Review.

Background. Several studies in the last decades have focused on finding a precise method for the diagnosis of periodontal disease in its early stages. Aim. To evaluate from current scientific literature the most common and precise method for gingival crevicular fluid (GCF) sample collection, biomarker analytical methods, and the variability of biomarker quantification, even when using the same analytical technique. Methodology. An electronic search was conducted on in vivo studies that presented clinical data on techniques used for GCF collection and biomarker analysis. Results. The results showed that 71.1%, 24.7%, and 4.1% of the studies used absorption, microcapillary, and washing techniques, respectively, in their gingival crevicular fluid collection. 73.1% of the researchers analyzed their samples by using enzyme-linked immunosorbent assay (ELISA). 22.6%, 19.5%, and 18.5% of the researchers included interleukin-1 beta (IL-1ß), matrix metalloproteinase-8 (MMP-8), and tumor necrosis factor-alpha (TNF-α), respectively, in their studies as biomarkers for periodontal disease. Conclusion. IL-1ß can be considered among the most common biomarkers that give precise results and can be used as an indicator of periodontal disease progression. Furthermore, paper strips are the most convenient and accurate method for gingival crevicular fluid collection, while enzyme-linked immunosorbent assay can be considered the most conventional method for the diagnosis of biofluids.

Purification and Characterization of Bacteriocin Produced by Weissella confusa A3 of Dairy Origin.

A dramatic increase in bacterial resistance towards currently available antibiotics has raised worldwide concerns for public health. Therefore, antimicrobial peptides (AMPs) have emerged as a promisingly new group of therapeutic agents for managing infectious diseases. The present investigation focusses on the isolation and purification of a novel bacteriocin from an indigenous sample of cow milk and it's mode of action. The bacteriocin was isolated from Weissella confusa A3 that was isolated from the sample and was shown to have inhibitory activity towards pathogenic bacteria namely Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Micrococcus luteus. The bacteriocin was shown to be heat stable and functioned well at low pH (2 to 6). Reduction of activity was shown after treatment with proteinase K, trypsin and peptidase that confirmed the proteinaceous nature of the compound. MALDI-TOF analysis of the sample gave a mass approximating 2.7 kDa. The membrane of the bacteria was disrupted by the bacteriocin causing SYTOX® green dye to enter the cell and bind to the bacterial DNA giving fluorescence signal. Bacterial cell treated with the bacteriocin also showed significant morphological changes under transmission electron microscope. No virulence and disease related genes can be detected from the genome of the strain.

Variable characteristics of bacteriocin-producing Streptococcus salivarius strains isolated from Malaysian subjects.

BACKGROUND: Salivaricins are bacteriocins produced by Streptococcus salivarius, some strains of which can have significant probiotic effects. S. salivarius strains were isolated from Malaysian subjects showing variable antimicrobial activity, metabolic profile, antibiotic susceptibility and lantibiotic production. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report new S. salivarius strains isolated from Malaysian subjects with potential as probiotics. Safety assessment of these strains included their antibiotic susceptibility and metabolic profiles. Genome sequencing using Illumina's MiSeq system was performed for both strains NU10 and YU10 and demonstrating the absence of any known streptococcal virulence determinants indicating that these strains are safe for subsequent use as probiotics. Strain NU10 was found to harbour genes encoding salivaricins A and 9 while strain YU10 was shown to harbour genes encoding salivaricins A3, G32, streptin and slnA1 lantibiotic-like protein. Strain GT2 was shown to harbour genes encoding a large non-lantibiotic bacteriocin (salivaricin-MPS). A new medium for maximum biomass production buffered with 2-(N-morpholino)ethanesulfonic acid (MES) was developed and showed better biomass accumulation compared with other commercial media. Furthermore, we extracted and purified salivaricin 9 (by strain NU10) and salivaricin G32 (by strain YU10) from S. salivarius cells grown aerobically in this medium. In addition to bacteriocin production, S. salivarius strains produced levan-sucrase which was detected by a specific ESI-LC-MS/MS method which indicates additional health benefits from the developed strains. CONCLUSION: The current study established the bacteriocin, levan-sucrase production and basic safety features of S. salivarius strains isolated from healthy Malaysian subjects demonstrating their potential for use as probiotics. A new bacteriocin-production medium was developed with potential scale up application for pharmaceuticals and probiotics from S. salivarius generating different lantibiotics. This is relevant for the clinical management of oral cavity and upper respiratory tract in the human population.

Synergy of antibacterial and antioxidant activities from crude extracts and peptides of selected plant mixture.

BACKGROUND: A plant mixture containing indigenous Australian plants was examined for synergistic antimicrobial activity using selected test microorganisms. This study aims to investigate antibacterial activities, antioxidant potential and the content of phenolic compounds in aqueous, ethanolic and peptide extracts of plant mixture. METHODS: Well diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were used to test antibacterial activity against four pathogenic bacteria namely Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Pseudomonas aeruginosa. DPPH (2, 2-diphenyl-1- picrylhydrazyl) and superoxide dismutase (SOD) assays were used to evaluate antioxidant activity. HPLC and gel filtration were used for purification of the peptides. Scanning electron microscope was applied to investigate the mode of attachment of the peptides on target microbial membranes. RESULTS: Aqueous extraction of the mixture showed no inhibition zones against all the test bacteria. Mean diameter of inhibition zones for ethanol extraction of this mixture attained 8.33 mm, 7.33 mm, and 6.33 mm against S. aureus at corresponding concentrations of 500, 250 and 125 mg/ml while E .coli showed inhibition zones of 9.33 mm, 8.00 mm and 6.66 mm at the same concentrations. B. cereus exhibited inhibition zones of 11.33 mm, 10.33 mm and 10.00 mm at concentrations of 500, 250 and 125 mg/ml respectively. The peptide extract demonstrated antibacterial activity against S. aureus, E. coli and B. cereus. The MIC and MBC values for ethanol extracts were determined at 125 mg/ml concentration against S. aureus and E. coli and B. cereus value was 31.5 mg/ml. MIC and MBC values showed that the peptide extract was significantly effective at low concentration of the Australian plant mixture (APM). Phenolic compounds were detected in hot aqueous and ethanolic extracts of the plant mixture. Hot aqueous, ethanol and peptides extracts also exhibited antioxidant activities. CONCLUSIONS: It was concluded that APM possessed good antibacterial and antioxidant activities following extraction with different solvents. The results suggest that APM provide a new source with antibacterial agents and antioxidant activity for nutraceutical or medical applications.

Enhanced production, purification, characterization and mechanism of action of salivaricin 9 lantibiotic produced by Streptococcus salivarius NU10.

BACKGROUND: Lantibiotics are small lanthionine-containing bacteriocins produced by lactic acid bacteria. Salivaricin 9 is a newly discovered lantibiotic produced by Streptococcus salivarius. In this study we present the mechanism of action of salivaricin 9 and some of its properties. Also we developed new methods to produce and purify the lantibiotic from strain NU10. METHODOLOGY/PRINCIPAL FINDINGS: Salivaricin 9 was found to be auto-regulated when an induction assay was applied and this finding was used to develop a successful salivaricin 9 production system in liquid medium. A combination of XAD-16 and cation exchange chromatography was used to purify the secondary metabolite which was shown to have a molecular weight of approximately 3000 Da by SDS-PAGE. MALDI-TOF MS analysis indicated the presence of salivaricin 9, a 2560 Da lantibiotic. Salivaricin 9 is a bactericidal molecule targeting the cytoplasmic membrane of sensitive cells. The membrane permeabilization assay showed that salivaricin 9 penetrated the cytoplasmic membrane and induced pore formation which resulted in cell death. The morphological changes of test bacterial strains incubated with salivaricin 9 were visualized using Scanning Electron Microscopy which confirmed a pore forming mechanism of inhibition. Salivaricin 9 retained biological stability when exposed to high temperature (90-100°C) and stayed bioactive at pH ranging 2 to 10. When treated with proteinase K or peptidase, salivaricin 9 lost all antimicrobial activity, while it remained active when treated with lyticase, catalase and certain detergents. CONCLUSION: The mechanism of antimicrobial action of a newly discovered lantibiotic salivaricin 9 was elucidated in this study. Salivaricin 9 penetrated the cytoplasmic membrane of its targeted cells and induced pore formation. This project has given new insights on lantibiotic peptides produced by S. salivarius isolated from the oral cavities of Malaysian subjects.