Integrated Multi-Omics Analysis Reveals Differential Effects of Fructo-Oligosaccharides (FOS) Supplementation on the Human Gut Ecosystem.

Changes in the gut ecosystem, including the microbiome and the metabolome, and the host immune system after fructo-oligosaccharide (FOS) supplementation were evaluated. The supplementation of FOS showed large inter-individual variability in the absolute numbers of fecal bacteria and an increase in Bifidobacterium. The fecal metabolome analysis revealed individual variability in fructose utilization in response to FOS supplementation. In addition, immunoglobulin A(IgA) tended to increase upon FOS intake, and peripheral blood monocytes significantly decreased upon FOS intake and kept decreasing in the post-FOS phase. Further analysis using a metagenomic approach showed that the differences could be at least in part due to the differences in gene expressions of enzymes that are involved in the fructose metabolism pathway. While the study showed individual differences in the expected health benefits of FOS supplementation, the accumulation of "personalized" knowledge of the gut ecosystem with its genetic expression may enable effective instructions on prebiotic consumption to optimize health benefits for individuals in the future.

Quorum Sensing Inhibitors against Chromobacterium violaceum CV026 Derived from an Actinomycete Metabolite Library.

Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No. 29-2: pimprinine [5-(1H-indol-3-yl)-2-methyloxazole]; No. 48: (2E,4E)-2-methyl-5-phenyl-2,4-pentadienoic acid; No. 74: (3E,5E)-5-methyl-6-(4-nitrophenyl)-hexa-3,5-dien-2-ol; and No. 130: methyphenazine-1-carboxylate-derived from an actinomycete metabolite library. These compounds were confirmed to be QS inhibitors that reduced violacein production in Chromobacterium violaceum CV026. Additionally, compounds No. 1, No. 74, and No. 130 significantly reduced fluorescent pigment production in Pseudomonas aeruginosa ATCC 27853.

Expression of a cryptic secondary sigma factor gene unveils natural competence for DNA transformation in Staphylococcus aureus.

It has long been a question whether Staphylococcus aureus, a major human pathogen, is able to develop natural competence for transformation by DNA. We previously showed that a novel staphylococcal secondary sigma factor, SigH, was a likely key component for competence development, but the corresponding gene appeared to be cryptic as its expression could not be detected during growth under standard laboratory conditions. Here, we have uncovered two distinct mechanisms allowing activation of SigH production in a minor fraction of the bacterial cell population. The first is a chromosomal gene duplication rearrangement occurring spontaneously at a low frequency [≤10(-5)], generating expression of a new chimeric sigH gene. The second involves post-transcriptional regulation through an upstream inverted repeat sequence, effectively suppressing expression of the sigH gene. Importantly, we have demonstrated for the first time that S. aureus cells producing active SigH become competent for transformation by plasmid or chromosomal DNA, which requires the expression of SigH-controlled competence genes. Additionally, using DNA from the N315 MRSA strain, we successfully transferred the full length SCCmecII element through natural transformation to a methicillin-sensitive strain, conferring methicillin resistance to the resulting S. aureus transformants. Taken together, we propose a unique model for staphylococcal competence regulation by SigH that could help explain the acquisition of antibiotic resistance genes through horizontal gene transfer in this important pathogen.

Staphylococcus aureus requires cardiolipin for survival under conditions of high salinity.

BACKGROUND: The ability of staphylococci to grow in a wide range of salt concentrations is well documented. In this study, we aimed to clarify the role of cardiolipin (CL) in the adaptation of Staphylococcus aureus to high salinity. RESULTS: Using an improved extraction method, the analysis of phospholipid composition suggested that CL levels increased slightly toward stationary phase, but that this was not induced by high salinity. Deletion of the two CL synthase genes, SA1155 (cls1) and SA1891 (cls2), abolished CL synthesis. The cls2 gene encoded the dominant CL synthase. In a cls2 deletion mutant, Cls1 functioned under stress conditions, including high salinity. Using these mutants, CL was shown to be unnecessary for growth in either basal or high-salt conditions, but it was critical for prolonged survival in high-salt conditions and for generation of the L-form. CONCLUSIONS: CL is not essential for S. aureus growth under conditions of high salinity, but is necessary for survival under prolonged high-salt stress and for the generation of L-form variants.

A helical string of alternately connected three-helix bundles for the cell wall-associated adhesion protein Ebh from Staphylococcus aureus.

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.

Tracking intermediate performance of vigilant attention using multiple eye metrics.

Vigilance deficits account for a substantial number of accidents and errors. Current techniques to detect vigilance impairment measure only the most severe level evident in eyelid closure and falling asleep, which is often too late to avoid an accident or error. The present study sought to identify ocular biometrics of intermediate impairment of vigilance and develop a new technique that could detect a range of deficits in vigilant attention (VA). Sixteen healthy adults performed well-validated Psychomotor Vigilance Test (PVT) for tracking vigilance attention while undergoing simultaneous recording of eye metrics every 2 hours during 38 hours of continuous wakefulness. A novel marker was found that measured VA when the eyes were open-the prevalence of microsaccades. Notably, the prevalence of microsaccades decreased in response to sleep deprivation and time-on-task. In addition, a novel algorithm for detecting multilevel VA was developed, which estimated performance on the PVT by integrating the novel marker with other eye-related indices. The novel algorithm also tracked changes in intermediate level of VA (specific reaction times in the PVT, i.e. 300-500 ms) during prolonged time-on-task and sleep deprivation, which had not been tracked previously by conventional techniques. The implication of the findings is that this novel algorithm, named "eye-metrical estimation version of the PVT: PVT-E," can be used to reduce human-error-related accidents caused by vigilance impairment even when its level is intermediate.

Collagen adhesion gene is associated with bloodstream infections caused by methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. METHODS: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. RESULTS: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. CONCLUSIONS: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

Dietary intervention of mice using an improved Multiple Artificial-gravity Research System (MARS) under artificial 1 g.

Japan Aerospace Exploration Agency (JAXA) has developed mouse habitat cage units equipped with an artificial gravity-producing centrifuge, called the Multiple Artificial-gravity Research System (MARS), that enables single housing of a mouse under artificial gravity (AG) in orbit. This is a report on a hardware evaluation. The MARS underwent improvement in water leakage under microgravity (MG), and was used in the second JAXA mouse mission to evaluate the effect of AG and diet on mouse biological system simultaneously. Twelve mice were divided into four groups of three, with each group fed a diet either with or without fructo-oligosaccharide and housed singly either at 1 g AG or MG for 30 days on the International Space Station, then safely returned to the Earth. Body weight tended to increase in AG mice and decrease in MG mice after spaceflight, but these differences were not significant. This indicates that the improved MARS may be useful in evaluating AG and dietary intervention for space flown mice.

Methicillin-resistant Staphylococcus saprophyticus isolates carrying staphylococcal cassette chromosome mec have emerged in urogenital tract infections.

Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed beta-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

Staphylococcus aureus giant protein Ebh is involved in tolerance to transient hyperosmotic pressure.

Staphylococcus aureus is well known to colonize on human skin where the physiological condition is characterized by hypervariable water activity, i.e., repeated dehydration or rehydration. To determine the facilitating factors for the colonization under hypervariable water activity, we studied the giant protein Ebh (extracellular matrix (ECM)-binding protein homologue). The ebh mutant RAM8 showed invaginated vacuoles along the septum, similar to that found in partial plasmolysis, and the cells burst under osmotic upshift. RAM8 was also relatively susceptible to abrupt hyperosmotic upshift, teicoplanin, and Triton X-100. By using the green fluorescent protein (GFP) as a reporter, Ebh was localized over the entire cell surface. This suggests that Ebh might contribute to structural homeostasis by forming a bridge between the cell-wall and cytoplasmic membrane to avoid plasmolysis under hyperosmotic condition.

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus.

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.

Electron microscopy and computational studies of Ebh, a giant cell-wall-associated protein from Staphylococcus aureus.

Ebh, a giant protein found in staphylococci, contains several domains, including a large central region with 52 imperfect repeats of a domain composed of 126 amino acids. We used electron microscopy to observe the rod-like structure of a partial Ebh protein containing 10 repeating units. This is the first report of the direct observation of an Ebh structure containing a large number of repeating units, although structures containing one, two, or four repeating units have been reported. The observed structure of the partial Ebh protein was distorted and had a length of ca. 520A and a width of ca. 21A. The observed structures were consistent with those deduced from crystal structure analysis, suggesting that the Ebh domains are connected to form a rod-like structure. The crystal structure data revealed distorted, string-like features in the simulated structure of the whole-length Ebh protein. Superposition of fragments of the simulated whole-length structure of the Ebh protein onto each electron micrograph showed a high level of correlation between the observed and calculated structures. These results suggest that Ebh is composed of highly flexible filate molecules. The highly repetitive structure and the associated unique structural flexibility of Ebh support the proposed function of this protein, i.e. binding to sugars in the cell wall. This binding might result in intra-cell-wall cross-linking that contributes to the rigidity of bacterial cells.

The sigH gene sequence can subspeciate staphylococci.

In an evolutionarily conserved gene organization (syntenic region), the sigH gene shares exceptionally low homology among staphylococcal species. We analyzed the "positionally cloned" sigH sequences of 39 staphylococcal species. The topology of the SigH phylogenetic tree was consistent with that of 16S rRNA. Certain clinical isolates were successfully differentiated at the species level with the sigH sequence data set. We propose that the sigH gene is a promising molecular target in genotypic identification because it is highly discriminative in differentiating closely related staphylococcal species.

Expression, purification, crystallization and preliminary diffraction analysis of CapF, a capsular polysaccharide-synthesis enzyme from Staphylococcus aureus.

Capsular polysaccharides (CPs) are important virulence factors of Staphylococcus aureus. The biosynthesis of type 5 and type 8 CPs (CP5 and CP8), which are produced by most clinical isolates of S. aureus, is catalyzed by 16 CP-assembling proteins. One of these proteins is the enzyme CapF, which catalyzes the synthesis of UDP-N-acetyl-L-fucosamine, a component of both CP5 and CP8. Here, the cloning, expression, purification, crystallization and diffraction analysis of CapF are reported. Optimization of the crystallization conditions by differential scanning calorimetry afforded a crystal of selenomethionine-substituted CapF that diffracted to a resolution of 2.80 A. The crystal belongs to space group P3(2)21, with unit-cell parameters a = b = 119.6, c = 129.5 A.

Probiotics into outer space: feasibility assessments of encapsulated freeze-dried probiotics during 1 month's storage on the International Space Station.

Suppression of immune function during long spaceflights is an issue that needs to be overcome. The well-established probiotic Lactobacillus casei strain Shirota (LcS) could be a promising countermeasure, and we have launched a project to investigate the efficacy of its use on the International Space Station (ISS). As a first step, we developed a specialist probiotic product for space experiments, containing freeze-dried LcS in capsule form (Probiotics Package), and tested its stability through 1 month of storage on the ISS. The temperature inside the ISS ranged from 20.0 to 24.5 °C. The absorbed dose rate of the flight sample was 0.26 mGy/day and the dose equivalent rate was 0.52 mSv/day. The number of live LcS was 1.05 × 1011 colony-forming units/g powder (49.5% of the initial value) 6 months after the start of the study; this value was comparable to those in the two ground controls. Profiles of randomly amplified polymorphic DNA, sequence variant frequency, carbohydrate fermentation, reactivity to LcS-specific antibody, and the cytokine-inducing ability of LcS in the flight sample did not differ from those of the ground controls. We can therefore maintain the viability and basic probiotic properties of LcS stored as a Probiotics Package on the ISS.

Sesquiterpene farnesol as a competitive inhibitor of lipase activity of Staphylococcus aureus.

Staphylococcus aureus lipase (SAL) is known to possess broad substrate specificity for triacylglycerides. We found that a sub-minimum inhibitory concentration of farnesol (1000 mg L(-1)) inhibits this lipase activity on a Mueller-Hinton agar containing 1% Tween substrates. A quantitative lipase assay using p-nitrophenyl palmitate (pNPP) revealed that the inhibitory action of farnesol appears to be the result of the inhibition of lipase activity rather than of its secretion into the culture medium. The inhibition was observed in all the tested 8 methicillin-susceptible S. aureus and 31 methicillin-resistant S. aureus clinical isolates. Using homogeneous lipase purified by hydrophobic interaction chromatography, it was revealed that farnesol could competitively inhibit the lipase activity against the substrate pNPP.

Evaluation of a cefoxitin disk diffusion test for the detection of mecA-positive methicillin-resistant Staphylococcus saprophyticus.

In order to validate the current Clinical and Laboratory Standards Institute (CLSI) criteria for the detection of mecA-mediated resistance in Staphylococcus saprophyticus, 101 clinical isolates, including 8 mecA-positive isolates, were investigated. All the isolates were in the range of the resistant category for coagulase-negative staphylococci with the 1 microg oxacillin disk diffusion method and agar dilution method, despite 93 isolates (92%) being mecA-negative. On the other hand, the 30 microg cefoxitin disk diffusion method showed clearly distinguishable zone diameters between the mecA-positive and -negative isolates. However, four of the mecA-negative isolates that would be considered resistant were false positive, and the current interpretive criteria of the CLSI may thus require reconsideration. This study suggests that the cefoxitin disk diffusion method could be more suitable than the oxacillin disk diffusion method for detecting mecA-mediated resistance in S. saprophyticus.

Genetic characterization of the natural SigB variants found in clinical isolates of Staphylococcus aureus.

The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.

Morphological significance of cladosporium contaminants on materials and utensils in contact with food.

Cladosporium contaminants on materials and utensils that come into contact with food were morphologically investigated. The most common contaminants, C. cladosporioides and C. sphaerospermum, were detected on the samples. The morphological changes of the Cladosporium species were investigated by using stereoscopic, optical light, fluorescent, and scanning electron microscopes. Microscopically the Cladosporium contaminants were observed as aggregated dark brown spots, strongly pigmented, irregularly swollen, and in long chains. Using fluorescent microscopy, the Cladosporium mycelia were clearly stained with fluorescein diacetate as viable cells, but the old cells were mostly non-viable, as shown by staining with propidium iodide. The dynamics of the morphological changes showed that the penetrating mycelia were closely attached to the surface of the materials and utensils under investigation. These results provide information about the significance of Cladosporium contamination on materials and utensils in contact with food and may contribute to the control of fungal contamination.

[Educational system for medical sciences at the University of Tsukuba--with special reference to medical technology].

Three-year colleges for nursing, medical technology, and so on, have all been reorganized into four-year educational institutions in national universities. Since the reform, universities are not responsible for educating medical scientists except nurses. The new College of Nursing and Medical Technology in the University of Tsukuba has been developed along these lines. Here introduce some of its attempts and provide an opportunity to a better system. The Department has the following three characteristics: 1. Medical scientists are educated in the new Department, and the Department is closely cooperating with the School of Medicine. 2. There are courses for medical researchers concerning Molecular Pathology, Pathological Engineering and Environmental Pathology. 3. The qualification to apply to a national test for medical technologists is given to the students. Unfortunately, the system is not fully understood by the faculty or the students and does not work well because the Medical Technology Department and the Nursing Department are grouped together as a single institution. Moreover, this flaw in the system prevents the Medical Technology Department from actively promoting highly advanced medical sciences, such as organ transplantation, artificial organs, gene therapy, reproductive medicines, and so forth (Fig. 1). Few specialists exist who can bridge achievements in basic or advanced sciences and clinical application. Serious social problems about food safety, care systems, post-genome medicine, the youth, and so on, have to be dealt with, too. We are thus planning to separate the Department as the College of Medical Science (Fig. 2) and link it to the educational system in the master's and doctoral programs (Fig. 3). This model will successfully educate a new type of medical specialists.