Hiroaki Taniai

Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria

Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 μg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 μg/ml) led to breaks of double‐stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single‐stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 μg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.

Method To Detect Only Live Bacteria during PCR Amplification

ABSTRACT Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 108 heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 108 heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 103 to 107 antibiotic-treated L. monocytogenes cells but could detect 102 live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 103 to 107 heat-treated cells but could detect 101 live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.

Longitudinal assessment of liver stiffness by transient elastography for chronic hepatitis B patients treated with nucleoside analog

Aim:  To evaluate the association between liver stiffness measured by transient elastography (FibroScan) and the efficacy of long‐term nucleoside analog (NA) treatment for patients with chronic hepatitis B.

Raloxifene hydrochloride is an adjuvant antiviral treatment of postmenopausal women with chronic hepatitis C: A randomized trial

BACKGROUND & AIMS Early menopause in women with chronic hepatitis C virus (HCV) infection is associated with a low likelihood of a sustained virological response (SVR) in conjunction with their antiviral treatment. This is potentially related to their reduced estrogen secretion. The study was done to determine whether selective estrogen receptor modulator administration might improve the efficacy of the current standard of care (SOC) treatment, pegylated interferon (PegIFN) α2a plus ribavirin (RBV), for postmenopausal women. METHODS One hundred and twenty-three postmenopausal women with genotype 1b chronic hepatitis C were randomly assigned to one of two treatment groups: raloxifene hydrochloride (RLX) (60 mg/day) plus SOC (PegIFNα2a 180 μg/week and RBV 600-1,000 mg/day) (n=62) or SOC only (n=61). Genotyping was performed of the polymorphism in the interleukin-28B (IL28B) gene region (rs8099917) of DNA collected from each patient. RESULTS One RLX-treated patient discontinued RLX because of a systemic rash following 2 weeks of treatment. Twenty-four weeks after treatment, the SVR rate was significantly higher for RLX plus SOC patients (61.3%) than for SOC only patients (34.4%) (p=0.0051). Further, the SVR rate was significantly higher for RLX plus SOC patients with IL28B TT (72.5%) than for SOC only patients with IL28B TT (39.2%) (p=0.0014), but no such relationship was observed in patients carrying the minor IL28B allele. CONCLUSIONS RLX improved the efficacy of SOC in the treatment of postmenopausal women with chronic hepatitis C. RLX shows promise as an adjuvant to the standard antiviral treatment of such patients.

Concerted Action of Lactate Oxidase and Pyruvate Oxidase in Aerobic Growth of Streptococcus pneumoniae : Role of Lactate as an Energy Source

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H(2)O(2)-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.

Insulin resistance undermines the advantages of IL28B polymorphism in the pegylated interferon alpha-2b and ribavirin treatment of chronic hepatitis C patients with genotype 1

BACKGROUND & AIMS Recent studies have suggested that insulin resistance exerts a strong influence on chronic hepatitis C virus (HCV) infection. We analyzed pretreatment factors useful for predicting sustained virological response (SVR), especially interleukin (IL) 28B polymorphism and Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). METHODS This cohort study consisted of 328 chronic hepatitis C patients with HCV genotype 1 who were treated for 48 weeks with pegylated interferon (PegIFN) α-2b and ribavirin (RBV). Genotyping of the polymorphisms in the IL28B gene region (rs8099917) on chromosome 19 was performed on DNA collected from each patient. RESULTS No significant difference in IL28B genotype distribution was found according to HOMA-IR. Multivariate analysis identified the IL28B TT genotype (OR=5.97, 95% CI 2.15-16.55, p=0.0006) and the baseline HOMA-IR (OR=0.65, 95% CI 0.48-0.87, p=0.0044) as significant, independent pretreatment predictors of SVR. Receiver operating characteristic analyses to determine the optimal threshold values of HOMA-IR for predicting SVR showed that the areas under the curve (AUC) were high for both IL28B TT (AUC=0.774, HOMA-IR cut-off value: 2.45) and IL28B TG/GG genotypes (AUC=0.772, HOMA-IR cut-off value: 1.55). CONCLUSIONS For HCV genotype 1, both IL28B and baseline HOMA-IR are independent pretreatment predictors of SVR in patients treated with PegIFNα-2b and RBV. Insulin resistance undermines the advantages of IL28B polymorphism to obtain SVR.

Discrimination of live, anti‐tuberculosis agent‐injured, and dead Mycobacterium tuberculosis using flow cytometry

Flow cytometry (FCM) using propidium iodide (PI)/bis-oxonol (BOX) staining can distinguish live, dead, and sublethally injured Escherichia coli by detecting intact vs. nonintact membranes (PI) and membrane potential (BOX). However, live bacteria, especially Mycobacterium tuberculosis, are not likely to be successfully discriminated from injured bacterium by FCM when utilizing the live/dead staining agents currently on the market. As injured cell membranes have integrity like that of live cells and are regarded as such by FCM, the distinction between live and injured cells has depended on the culture method, where injured bacteria cannot grow in general. We have previously shown that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro. In this study, we found that the chromosomal DNA of antibiotic-injured, but not live, M. tuberculosis could be cleaved within 2 h by EMA, and that the resultant decrease in the spaces of DNA base pairs could greatly inhibit the intercalation of SYTO9 in FCM. The percentage value of SYTO9(+)/PI(-) quadrant from antibiotic-injured M. tuberculosis after EMA treatment decreased by at least 80%, compared with that before EMA, but such a phenomenon did not take place in live cells. FCM (SYTO9/PI) following EMA treatment is a very rapid, simple, and effective method for discriminating live, antibiotic-injured, and dead M. tuberculosis without culture.

Excellent superiority and specificity of COBAS TaqMan HCV assay in an early viral kinetic change during pegylated interferon alpha-2b plus ribavirin treatment

BackgroundAn early virological response (EVR) after the start of interferon (IFN) treatment for chronic hepatitis C leads to a successful virological outcome. To analyze an association between sustained virological response (SVR) and EVR by comparing TaqMan with Amplicor assays in HCV genotype 1-infected patients treated with pegylated (PEG)-IFN alpha-2b plus ribavirin (RBV).MethodsWe retrospectively analyzed a total of 80 HCV genotype 1 patients (39 SVR and 41 non-SVR patients), who received an enough dosage and a complete 48-week treatment of PEG-IFN alpha-2b plus RBV. Serum HCV RNA levels were measured by both TaqMan and Amplicor assays for each patients at Weeks 2, 4, 8 and 12 after the start of the antiviral treatment.ResultsOf the 80 patients with undetectable HCV RNA by Amplicor, 17 (21.3%) patients were positive for HCV RNA by TaqMan at Weeks 12. The quantification results showed that no significant difference in the decline of HCV RNA level between TaqMan and Amplicor 10-fold method assays within the initial 12 weeks of the treatment was found. However, the qualitative analysis showed significant differences of the positive predictive rates for SVR were found between TaqMan (100% at weeks 4 and 100% at weeks 8) and Amplicor (80.0% and 69.6%, respectively).ConclusionsThe COBAS TaqMan HCV assay is very useful for monitoring HCV viremia during antiviral treatment to predict a SVR in HCV genotype 1 patients.

Long-term effects of lamivudine treatment in Japanese chronic hepatitis B patients

AIM To analyze the association between the emergence of tyrosine-methionine-asparatate-asparatate (YMDD) mutants (reverse transcription; rtM204I/V) and deterioration of liver function during long-term lamivudine treatment of Japanese patients with chronic hepatitis B virus (HBV) infection. METHODS The data of 61 consecutive Japanese patients with chronic hepatitis B who underwent continuous lamivudine treatment for more than 24 mo and had a virological response were analyzed. Analysis of YMDD mutants was done by real-time polymerase chain reaction with LightCycler probe hybridization assay for up to 90 mo (mean, 50.8 mo; range, 24-90 mo). RESULTS A mixed mutant-type (YMDD + tyrosine-isoleucine-asparatate-asparatate: YIDD or tyrosine-valine-asparatate-asparatate: YVDD) or a mutant-type (YIDD or YVDD) were found in 57.4% of 61 patients at 1 year, 78.7% of 61 patients at 2 years, 79.6% of 49 patients at 3 years, 70.5% of 34 patients at 4 years, 68.4% of 19 patients at 5 years, 57.1% of 14 patients at 6 years, and 33.3% of 6 patients at 7 years. Of the 61 patients, 56 (92%) had mixed mutant- or a mutant-type. Only 5 (8%) had no mutants at each observation point. Virological breakthrough was found in 26 (46.4%) of 56 patients with YMDD mutants, 20 of whom had a hepatitis flare-up: the remaining 30 (53.6%) had neither a virological breakthrough nor a flare-up. All 20 patients who developed a hepatitis flare-up had a biochemical and virological response after adefovir was added to the lamivudine treatment. CONCLUSION Our results suggest that it is possible to continue lamivudine treatment, even after the emergence of YMDD mutants, up to the time that the patients develop a hepatitis flare-up.

Electron microscopic examination of uncultured soil-dwelling bacteria

Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze‐substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram‐positive type; (iii) Gram‐negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram‐negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram‐negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil‐dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low‐temperature, low‐nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages.