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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 µ 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 µ 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 µ 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.

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.The gram-positive bacterium Streptococcus pneumoniae, also known as pneumococcus, is often a commensal resident of the human upper respiratory tract but also represents an important human pathogen, causing invasive diseases such as pneumonia, otitis media, and meningitis (18,20,22). Recently, a sharp rise in the incidence of drug resistance among clinical isolates of S. pneumoniae has been posing serious problems (13).A member of lactic acid bacteria, S. pneumoniae is aerotolerant but lacks the cytochromes necessary for aerobic respiration. Under anaerobic conditions, it is believed to be totally dependent on homolactic fermentation for the acquisition of energy required for growth, in which glucose is metabolized to pyruvate and then to the final product lactate. Under aerobiosis, however, pyruvate is also known to be converted to acetate, with acetyl phosphate being the intermediate capable of phosphorylating ADP to yield ATP by the action of acetate kinase (30). The H 2 O 2 -forming flavoprotein pyruvate oxidase (EC 1.2.3.3, the product of the spxB gene), which catalyzes the formation of acetyl phosphate, CO 2 and H 2 O 2 from pyruvate, orthophosphate, and O 2 , has been shown to be involved in this pathway and also to account for most of the H 2 O 2 produced by aerobically growing S. pneumoniae cells (23, 30). Massive production of H 2 O 2 has been a well-known hallmark of this bacterium since the time of Oswald Avery (2, 19), usually necessitating the addition of catalase to the culture medium to obtain full growth under aerobic conditions. S. pneumoniae possesses another H 2 O 2 -forming flavoprotein, L-lactate oxidase (the product of the lox gene; formerly EC 1.1.3.2 but now sharing the EC number 1.13.12.4 with lactate monooxigenase) catalyzing the ...

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Liver stiffness, measured by transient elastography, of chronic hepatitis B patients treated with NA showed a rapid decline in the first 3 years followed by a more steady transition for from 3 to 5 years irrespective of long term virological effect.