Construction of a versatile in vitro cultivation screening platform using human oral microbiota.

We developed a simulation model of human oral microbiota using Bio Palette oral medium (BPOM) containing 0.02% glucose and lower bacterial nitrogen sources, derived from saliva and dental plaque. By decreasing the concentration of Gifu anaerobic medium (GAM) from 30 to 10 g L-1 , we observed increased ratios of target pathogenic genera, Porphyromonas and Fusobacterium from 0.5% and 1.7% to 1.2% and 3.5%, respectively, in the biofilm on hydroxyapatite (HA) discs. BPOM exhibited the higher ratios of Porphyromonas and Fusobacterium, and amplicon sequence variant number on HA, compared with GAM, modified GAM and basal medium mucin. Mixing glycerol stocks of BPOM culture solutions from four human subjects resulted in comparable ratios of these bacteria to the original saliva. In this simulation model, sitafloxacin showed higher inhibitory effects on P. gingivalis than minocycline hydrochloride at a low dosage of 0.1 µg mL-1 . Probiotics such as Streptococcus salivarius and Limosilactobacillus fermentum also showed significant decreases in Porphyromonas and Fusobacterium ratios on HA, respectively. Overall, the study suggests that BPOM with low carbon and nutrients could be a versatile platform for assessing the efficacy of antibiotics and live biotherapeutics in treating oral diseases caused by Porphyromonas and Fusobacterium.

The Effect of Increasing Nickel Content on the Microstructure, Hardness, and Corrosion Resistance of the CuFeTiZrNix High-Entropy Alloys.

In recent years, high-entropy alloys (HEAs) that contain fine grains of intermetallic compounds (IMCs) have gained increasing attention as they have been shown to exhibit both high mechanical strength and strong corrosion resistance. One such class of HEAs is that of CuFeTiZrNi alloys. In this study, we have investigated the effect of increasing Ni content on the microstructure, hardness, and corrosion resistance of the CuFeTiZrNix alloys (where x = 0.1, 0.3, 0.5, 0.8, 1.0 in a molar ratio). The alloys used in this study were prepared in an arc melting furnace and then annealed at 900 °C. First-principles calculations of the bulk modulus were also performed for each alloy. The results revealed that increasing the Ni content had several effects. Firstly, the microstructure of the CuFeTiZrNix alloys changed from B2_BCC and Laves_C14 in the CuFeTiZrNi0.1 and CuFeTiZrNi0.3 alloys to FCC, B2_BCC, and Laves_C14 in the CuFeTiZrNi0.5 alloys; and to FCC, B2_BCC, Cu51Zr14, and Laves_C14 in the CuFeTiZrNi0.8 and CuFeTiZrNi1.0 alloys. Secondly, IMCs arising from a combination of the refractory elements (Ti and Zr) and atomic size differences were found in the interdendritic region. Thirdly, as the Ni content in the CuFeTiZrNix alloys increased, the hardness decreased, but the corrosion resistance increased.

The first case of congenital syphilis diagnosed by 16S ribosome-RNA gene sequence analysis.

Syphilis infection during pregnancy causes perinatal complications and mother-to-child transmission if untreated. A newborn was delivered by emergent cesarean section due to non-reassuring fetal status at 34 weeks of gestation. The mother tested negative for rapid plasma reagin (RPR) and Treponema pallidum hemagglutination (TPHA) in early pregnancy. The newborn had a severe inflammatory reaction, thrombocytopenia, and elevated IgM as well as disseminated intravascular coagulation and multiple organ failure. The 16S ribosomal RNA gene sequence analysis of the amniotic fluid detected Treponema pallidum. The newborn tested positive for RPR, TPHA, and IgM fluorescent treponemal antibody-absorption in the blood, and thus, congenital syphilis was diagnosed. This is the first case that 16S ribosomal RNA gene sequencing of the amniotic fluid led to an early diagnosis of congenital syphilis in a newborn. The 16S rRNA gene sequencing may be a useful method for the early detection of the primary causative microbe of congenital infection.

Gene expression analysis in the potent bactericidal activity of sitafloxacin against Streptococcus pneumoniae.

We investigated the degree of expression of Streptococcus pneumoniae genes associated with bacteriolysis and cell death in relation to the rapid bactericidal activity of sitafloxacin. S. pneumoniae ATCC 49619 was added to brain heart infusion containing sitafloxacin and garenoxacin concentrations equivalent to the Cmax achieved with the usual single dose and 4 h post-Cmax concentration. RNA was extracted and cDNA was prepared using reverse transcriptase. Following RNA extraction and cDNA synthesis, quantitative PCR was performed to determine the amount of gene expression for 13 genes associated with cell death. Of the 13 genes analyzed, S. pneumoniae exposed for 10 min to a sitafloxacin concentration of 4 h post-Cmax showed 3.9 times increased expression of lytA compared to the control strain. Furthermore, we observed a slightly increased expression for cibA encoding a competence induced bacteriocin. Our study suggests that the induction of a lytic enzyme and bacteriocin may reflect gene expression in response to sitafloxacin accounting for part of its rapid bactericidal activity against S. pneumoniae.

Visual Detection of Amplified DNA by Polymerase Chain Reaction Using a Genetic Alphabet Expansion System.

Visual DNA amplification using a simple polymerase chain reaction (PCR) device is useful for field tests to detect target DNA and RNA. We hereby describe a detection system involving PCR amplification visualized with the naked eye, by genetic alphabet expansion. The system employs fluorescence resonance energy transfer (FRET) between unnatural base combinations: self-quenched dinucleotides of 2-amino-6-(2-thienyl)purine (s) as a donor and Cy3-conjugated 2-nitro-4-propynylpyrrole (Cy3-hx-Px) as an acceptor. During PCR, the triphosphate substrate of Cy3-hx-Px (Cy3-hx-dPxTP) is incorporated into DNA opposite its pairing partner, 7-(2-thienyl)-imidazo[4,5- b]pyridine (Ds), in the primer, which also contains the dinucleotides of s. Thus, the amplified DNA can be visualized by the Cy3 fluorescence resulting from the FRET between the s-dinucleotides and the incorporated Cy3-hx-Px upon 365 nm irradiation. Using this system, we demonstrated the visual single nucleotide polymorphism detection of a series of quinolone-resistant bacteria genes.

Genes and Proteins Involved in qnrS1 Induction.

Expression of the quinolone resistance gene qnrS1 is increased by quinolones, but unlike induction of some other qnr genes, the bacterial SOS system is not involved and no lexA box is found upstream. Nonetheless, at least 205 bp of upstream sequence is required for induction to take place. An upstream sequence bound to beads trapped potential binding proteins from cell extracts that were identified by mass spectrometry as Dps, Fis, Ihf, Lrp, CysB, and YjhU. To further elucidate their role, a reporter plasmid linking the qnrS1 upstream sequence to lacZ was introduced into cells of the Keio collection with single-gene deletions and screened for lacZ expression. Mutants in ihfA and ihfB had decreased lacZ induction, while induction in a cysB mutant was increased and dps, fis, lrp, yjhU, and other mutants showed no change. The essential upstream sequence contains potential binding sites for Ihf and DnaA. A dnaA deletion could not be tested because it provides essential functions in cell replication; however, increased dnaA expression decreased qnrS1 induction while decreased dnaA expression enhanced it, implying a role for DnaA as a repressor. In a mobility shift assay, purified IhfA, IhfB, and DnaA proteins (but not CysB) were shown to bind to the upstream segment. Induction decreased in a gyrA quinolone-resistant mutant, indicating that GyrA also has a role. Thus, quinolones acting through proteins DnaA, GyrA, IhfA, and IhfB regulate expression of qnrS1.

In Vitro Activity of Sitafloxacin and Additional Newer Generation Fluoroquinolones Against Ciprofloxacin-Resistant Neisseria gonorrhoeae Isolates.

Emergence of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally, and new antimicrobials for treatment of gonorrhea are imperative. In this study, the in vitro activity of sitafloxacin, a fluoroquinolone mainly used for respiratory tract or urogenital infections in Japan, and additional newer generation fluoroquinolones were determined against ciprofloxacin-resistant N. gonorrhoeae isolates. Minimum inhibitory concentrations (MICs) of ciprofloxacin, levofloxacin, moxifloxacin, sitafloxacin, pazufloxacin, and tosufloxacin against 47 N. gonorrhoeae isolates cultured in 2009 in Japan were determined by agar dilution method. The quinolone resistance-determining region (QRDR) of gyrA and parC was sequenced. The in vitro potency of sitafloxacin was substantially higher compared with all other tested fluoroquinolones. The MICs of sitafloxacin ranged from 0.03 to 0.5 mg/L for 35 ciprofloxacin-resistant N. gonorrhoeae isolates (ciprofloxacin MICs from 2 to 32 mg/L). No identified mutations in GyrA and ParC QRDR resulted in higher sitafloxacin MIC than 0.5 mg/L. Sitafloxacin had a high activity against N. gonorrhoeae isolates, including strains with mutations in DNA gyrase and topoisomerase IV, resulting in high-level resistance to ciprofloxacin and all other newer generation fluoroquinolones examined. However, it was still to a lower extent affected by GyrA and ParC QRDR mutations resulting in sitafloxacin MICs of up to 0.5 mg/L. This indicates that sitafloxacin should not be considered for empirical first-line monotherapy of gonorrhea. However, sitafloxacin could be valuable in a dual antimicrobial therapy and for cases with ceftriaxone resistance or allergy.

Lifestyle Changes and Oxidative Stress in a High-incidence Area of Amyotrophic Lateral Sclerosis in the Southwestern Kii Peninsula, Japan.

Objective Lifestyle changes may play an important role in the incidence reduction and delay of onset age of amyotrophic lateral sclerosis (ALS) in the Koza/Kozagawa/Kushimoto (K) area. The aim of this study was to evaluate recent lifestyle changes in the K area and to investigate the relationships between lifestyle and oxidative stress among the residents. Methods We conducted a medical checkup for elderly residents in the K area and the control area and evaluated the urinary 8-OHdG levels, cognitive function test scores and metal contents in serum and scalp hair, coupled with a lifestyle questionnaire survey between 2010 and 2015. Results Recent lifestyle changes among the K residents, including a decrease in the Japanese pickle consumption, increase in fresh vegetable consumption and decrease in farm work, were evaluated in this study. Low consumption of Japanese pickles, high consumption of fresh vegetables, rare farm work and low levels of 8-OHdG/creatinine were all associated with high scores in the cognitive function tests. Frequent farm work and consumption of Japanese pickles was associated with high contents of transition metals, such as Mn, Al and V, in the scalp hair. Conclusion These lifestyle changes among residents in the K area may be associated with their oxidative stress.

Interactions between QnrB, QnrB mutants, and DNA gyrase.

Plasmid-encoded protein QnrB1 protects DNA gyrase from ciprofloxacin inhibition. Using a bacterial two-hybrid system, we evaluated the physical interactions between wild-type and mutant QnrB1, the GyrA and GyrB gyrase subunits, and a GyrBA fusion protein. The interaction of QnrB1 with GyrB and GyrBA was approximately 10-fold higher than that with GyrA, suggesting that domains of GyrB are important for stabilizing QnrB1 interaction with the holoenzyme. Sub-MICs of ciprofloxacin or nalidixic acid reduced the interactions between QnrB1 and GyrA or GyrBA but produced no reduction in the interaction with GyrB or a quinolone-resistant GyrA:S83L (GyrA with S83L substitution) mutant, suggesting that quinolones and QnrB1 compete for binding to gyrase. Of QnrB1 mutants that reduced quinolone resistance, deletions in the C or N terminus of QnrB1 resulted in a marked decrease in interactions with GyrA but limited or no effect on interactions with GyrB and an intermediate effect on interactions with GyrBA. While deletion of loop B and both loops moderately reduced the interaction signal with GyrA, deletion of loop A resulted in only a small reduction in the interaction with GyrB. The loop A deletion also caused a substantial reduction in interaction with GyrBA, with little effect of loop B and dual-loop deletions. Single-amino-acid loop mutations had little effect on physical interactions except for a Δ105I mutant. Therefore, loops A and B may play key roles in the proper positioning of QnrB1 rather than as determinants of the physical interaction of QnrB1 with gyrase.

Neutron activation analysis of scalp hair from ALS patients and residents in the Kii Peninsula, Japan.

The aim of this study was to evaluate the accumulation of transition metals in the scalp hair of amyotrophic lateral sclerosis (ALS) patients in the Koza/Kozagawa/Kushimoto (K) area (K-ALS) in the Kii Peninsula, Japan. Metal contents were measured in the unpermed, undyed hair samples of 88 K-residents, 20 controls, 7 K-ALS patients, and 10 sporadic ALS patients using neutron activation analysis at the Research Reactor Institute, Kyoto University. A human hair standard and elemental standards were used as comparative standards. The contents of Zn, Mn, and V were higher, while that of S was lower in K-ALS patients than in the controls. The content of Mn in K-ALS patients negatively correlated with clinical durations. The content of Al was significantly higher in K-residents than in the controls, with 15.9 % of K-residents having high Mn contents over the 75th percentile of the controls. The contents of Zn, Mn, and V were high in the scalp hair of K-ALS patients and correlated with the content of Al. The accumulation of these transition metals may chronically increase metal-induced oxidative stress, which may, in turn, trigger the neuronal degeneration associated with K-ALS.

Measurement of air dose rates over a wide area around the Fukushima Dai-ichi Nuclear Power Plant through a series of car-borne surveys.

A series of car-borne surveys using the Kyoto University RAdiation MApping (KURAMA) and KURAMA-II survey systems has been conducted over a wide area in eastern Japan since June 2011 to evaluate the distribution of air dose rates around the Fukushima Dai-ichi Nuclear Power Plant and to evaluate the time-dependent trend of decrease in air dose rates. An automated data processing system for the KURAMA-II system was established, which enabled rapid analysis of large amounts of data obtained using about 100 KURAMA-II units. The initial data used for evaluating the migration status of radioactive cesium were obtained in the first survey, followed by other car-borne surveys conducted over more extensive and wider measurement ranges. By comparing the measured air dose rates obtained in each survey (until December 2012), the decreasing trend of air dose rates measured through car-borne surveys was found to be more pronounced than those expected on the basis of the physical decay of radioactive cesium and of the air dose rates measured using NaI (Tl) survey meters in the areas surrounding the roadways. In addition, it was found that the extent of decrease in air dose rates depended on land use, wherein it decreased faster for land used as building sites than for forested areas.

Rapid bactericidal activity of sitafloxacin against Streptococcus pneumoniae.

The initial bactericidal activity of quinolones against Streptococcus pneumoniae at the concentration equivalent to their respective peak serum concentration (C(max)) and free drug fraction of C(max) (fC(max)) were investigated. The bactericidal activity of sitafloxacin (STFX), levofloxacin (LVFX), moxifloxacin (MFLX), and garenoxacin (GRNX) were compared by determining the actual killing of bacteria at C(max) and fC(max) for 1 and 2 hours based on the Japanese maximum dose per administration (100, 500, 400, and 400 mg, respectively). Against 4 quinolone-susceptible clinical isolates (wild-type), STFX with C(max) and fC(max) exhibited the most rapid bactericidal activity resulting in an average reduction of > or = 3.0 log10 colony forming units (CFU)/ mL in 1 hour. STFX with C(max) and fC(max) also showed the most rapid and potent bactericidal activity against 9 clinical isolates with single par (C/E) mutation, resulting in > or = 3.0 log10 CFU/mL average reduction in viable cells in 1 hour. STFX showed a statistically significant advantage in initial bactericidal activity over other quinolones for single mutants (P < 0.001). The propensity that the difference in the initial bactericidal activity between STFX and other quinolones was higher in single mutants than wild-type strains, was confirmed using S. pneumoniae ATCC49619 (wild-type) and its laboratory single parC mutant. As a result, STFX showed a similar rapid and potent initial bactericidal activity against both strains, while initial bactericidal activity for other quinolones was significantly reduced in the single mutant (P < 0.05). In conclusion, STFX has the most rapid and potent initial bactericidal activity against wild-type and single mutants of S. pneumoniae and its bactericidal activity is not affected by the presence of a single par mutation compared to LVFX, MFLX, and GRNX.

[Antimicrobial and rapid bactericidal activities of sitafloxacin and other agents against Streptococcus pyogenes].

We evaluated the in vitro activity of sitafloxacin against Japanese clinical isolates of Streptococcus pyogenes by broth microdilution susceptibility testing and time-kill studies to elucidate its eradication potential against S. pyogenes. One hundred and nineteen clinical isolates of S. pyogenes isolated from pharynx were tested to sitafloxacin and seven other agents in the susceptibility testing. The time-kill studies were conducted with five strains, one of which was resistant to clarithromycin, one resistant to levofloxacin and one type strain of S. pyogenes. In the time-kill studies, sitafloxacin, garenoxacin, amoxicillin and clarithromycin were assessed at static concentrations of their respective peak concentrations in plasma (C(max)) when administered as oral single doses for adult patients with S. pyogenes infections. We found the rank order of antimicrobial activity against S. pyogenes isolates was: cefcapene (MIC90, 0.015 microg/mL) > amoxicillin (0.03 microg/mL) > sitafloxacin (0.12 microg/mL) > garenoxacin (0.25 microg/mL) > levofloxacin (4 microg/mL) > minocycline (16 microg/mL). Macrolide-resistant isolates accounted for 72 (60.5%), resulting in clarithromycin and azithromycin MIC90s of > 32 and > 128 microg/mL, respectively. Sitafloxacin exhibited the most rapid bactericidal activity (> or = log reduction from the initial inoculum) within 2h against all tested strains, including even one levofloxacin-resistant strain. For garenoxacin, bactericidal activity was achieved between 2 and 6 h. Amoxicillin revealed no significant bactericidal activity up to 6 h. Clarithromycin showed no bactericidal activity and did not inhibit growth of a clarithromycin-resistant strain. These data indicate the potential usefulness of sitafloxacin for the treatment of S. pyogenes eradication.

Quinolone induction of qnrVS1 in Vibrio splendidus and plasmid-carried qnrS1 in Escherichia coli, a mechanism independent of the SOS system.

Plasmid-carried qnrS1 is derived from Vibrio splendidus chromosomal qnrVS1. qnrVS1 transcripts increased 21- to 34-fold with subinhibitory concentrations of ciprofloxacin but much less with mitomycin. No LexA binding sites were upstream of qnrS1 or qnrVS1, and similar induction levels were observed in lexA-positive and lexA-negative Escherichia coli strains with native qnrS1 plasmid pMG306 but not with pUC18-cloned qnrS1 or qnrVS1. Thus, qnrS1 induction by quinolones is independent of the SOS system and requires sequence besides that of the structural gene.

Implication of the NorB efflux pump in the adaptation of Staphylococcus aureus to growth at acid pH and in resistance to moxifloxacin.

Staphylococcus aureus is an important pathogen that adapts and survives in low-pH environments. One component of this adaptation involves the regulation of genes encoding bacterial transporters that could affect response to antibiotics under these conditions. We previously demonstrated that the transcriptional regulator MgrA in its phosphorylated form (MgrA-P) represses the expression of norB, encoding the NorB multidrug resistance efflux pump. In this study, we focused on changes in the expression of mgrA at the transcriptional and posttranslational levels, following a shift from pH 7.0 to pH 4.5. We then correlated those changes with modifications in transcript levels of norB and to resistance to moxifloxacin, a substrate of NorB. At pH 4.5, S. aureus MgrA increased 2-fold and MgrA-P decreased 4-fold, associated with an 8-fold increase in norB transcripts and a 6-fold reduction in bacterial killing by moxifloxacin, and the phenomenon was dependent on intact mgrA. Taken together, these new data showed that phosphoregulation of MgrA at low pH reverses its repression of norB expression, conferring resistance to moxifloxacin.

In vitro antifungal activities of D11-2040, a beta-1,6-glucan inhibitor, with or without currently available antifungal drugs.

We recently reported our discovery of small molecule beta-1,6-glucan inhibitor named D75-4590 and the anti-Candida activity of its derivatives D11-2040 and D21-6076. In this study, we further evaluated the antifungal profile of D11-2040. It alone strongly inhibited the vegetative growth and/or hyphal development of various Candida species, but no significant activity was observed against Cryptococcus neoformans or any of the filamentous fungi tested. Synergism was detected for C. albicans in the interaction of D11-2040 and caspofungin by the chequerboard method and in that of D11-2040 and fluconazole by the time-kill method. Slight but positive interactions were observed in several combinations for C. neoformans and Aspergillus fumigatus as well. These results suggested that beta-1,6-glucan inhibitors have promising potential as single drugs as well as concomitants.

Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell research.

To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining "fluorescent activated cell sorting (FACS) index sorting" analysis and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT-PCR. The results were plotted on the FACS sorting profile using the "index sorting" function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2/M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates.

Quinolones with enhanced bactericidal activity induce autolysis in Streptococcus pneumoniae.

OBJECTIVES: DC-159a and sitafloxacin show greater bactericidal activity against Streptococcus pneumoniae than garenoxacin and other quinolones. We investigated whether the autolysis induced by these quinolones contributes to their rapid bactericidal activity. METHODS: Time-kill studies were conducted against a S. pneumoniae clinical isolate in broth with choline chloride, which is known to inhibit autolytic amidases, and lytA mutants. Western blot analysis was performed to examine LytA production. Scanning electron microscopy (SEM) was used to investigate morphological differences after exposure to quinolone. RESULTS: Bactericidal activity of DC-159a and sitafloxacin against S. pneumoniae at 2 h of exposure to twice the minimum inhibitory concentration (MIC) was found to decrease by approximately 1 log CFU/ml when autolytic amidases were blocked. Time-kill studies using lytA mutants showed that DC-159a exhibited slower killing than that against the lytA-positive strains. On exposure to the MIC and twice the MIC of DC-159a and sitafloxacin, R6 and a clinical isolate overexpressed LytA, while garenoxacin caused a less significant increase in LytA than DC-159a and sitafloxacin. Scanning electron microscopy images revealed that R6 treated with DC-159a underwent distinct morphological changes, while the lytA mutant did not. CONCLUSIONS: The present study demonstrated that quinolone-induced autolysis may provide quinolones more powerful bactericidal activity against S. pneumoniae.

Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae.

OBJECTIVES: DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. METHODS: MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. RESULTS: DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. CONCLUSIONS: DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants.