The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system

Controlled oxidation, such as disulfide bond formation in sperm nuclei and during ovulation, plays a fundamental role in mammalian reproduction. Excess oxidation, however, causes oxidative stress, resulting in the dysfunction of the reproductive process. Antioxidation reactions that reduce the levels of reactive oxygen species are of prime importance in reproductive systems in maintaining the quality of gametes and support reproduction. While anti-oxidative enzymes, such as superoxide dismutase and peroxidase, play a central role in eliminating oxidative stress, reduction-oxidation (redox) systems, comprised of mainly glutathione and thioredoxin, function to reduce the levels of oxidized molecules. Aldo-keto reductase, using NADPH as an electron donor, detoxifies carbonyl compounds resulting from the oxidation of lipids and proteins. Thus, many antioxidative and redox enzyme genes are expressed and aggressively protect gametes and embryos in reproductive systems.     

The origin of reactive oxygen species in mouse embryos cultured in vitro.

The increase in production of reactive oxygen species such as H2O2 at the G2/M phase of the second cell cycle may be related to the in vitro block to development of mouse 2-cell embryos. The occurrence of the H2O2 rise is independent of the activation of the embryonic genome and of passage through the S, G2 and M phases of the first cell cycle and G1 and M phases of the second cell cycle, but does require the activation of the unfertilized oocyte. The H2O2 is produced via dismutation of superoxide by the enzyme superoxide dismutase. Production of superoxide via mitochondrial, NADPH-oxidase and xanthine/xanthine oxidase systems has been investigated. The evidence suggests that superoxide, and thereby H2O2, is produced by the xanthine/xanthine oxidase system, but an involvement of the other superoxide generating systems has not been excluded. The relation between H2O2 and development in vitro is discussed.     

Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II

Photosystem II (PSII) is a multisubunit protein complex in cyanobacteria, algae and plants that use light energy for oxidation of water and reduction of plastoquinone. The conversion of excitation energy absorbed by chlorophylls into the energy of separated charges and subsequent water-plastoquinone oxidoreductase activity are inadvertently coupled with the formation of reactive oxygen species (ROS). Singlet oxygen is generated by the excitation energy transfer from triplet chlorophyll formed by the intersystem crossing from singlet chlorophyll and the charge recombination of separated charges in the PSII antenna complex and reaction center of PSII, respectively. Apart to the energy transfer, the electron transport associated with the reduction of plastoquinone and the oxidation of water is linked to the formation of superoxide anion radical, hydrogen peroxide and hydroxyl radical. To protect PSII pigments, proteins and lipids against the oxidative damage, PSII evolved a highly efficient antioxidant defense system comprising either a non-enzymatic (prenyllipids such as carotenoids and prenylquinols) or an enzymatic (superoxide dismutase and catalase) scavengers. It is pointed out here that both the formation and the scavenging of ROS are controlled by the energy level and the redox potential of the excitation energy transfer and the electron transport carries, respectively. The review is focused on the mechanistic aspects of ROS production and scavenging by PSII. This article is part of a Special Issue entitled: Photosystem II.     

喜温嗜酸硫杆菌SM-1的ROS防护机制

【目的】从基因组水平探讨生物冶金微生物——喜温嗜酸硫杆菌(Acidithiobacillus caldus)的活性氧类物质(Reactive oxygen species,ROS)防护机制。【方法】采用罗氏454 GS FLX测序平台对喜温嗜酸硫杆菌SM-1进行全基因组测序,利用NCBI非冗余蛋白数据库、Uniport蛋白数据库对全基因组序列进行功能注释,并采用基因组百科全书数据库(KEGG)进行基因组代谢途径重构,通过比较基因组学方法分析SM-1基因组中参与ROS防护相关的基因及可能的分子机制。【结果】SM-1细胞内的酶促抗氧化系统可用于清除细胞内产生的ROS物质,而非酶促抗氧化系统可用于维持细胞内的还原性内环境;细胞内的DNA损伤修复系统可用于修复DNA的氧化损伤从而保持个体遗传物质的稳定性。此外,SM-1基因组中大量的转座元件可能会增加基因组的可塑性以适应极端冶金环境。【结论】SM-1基因组序列的获得为从整体水平揭示喜温嗜酸硫杆菌适应生物冶金环境ROS氧化损伤的防护机制提供了帮助,对于SM-1的ROS防护机制的认知也为进一步通过遗传改造、提升其在高浓度重金属离子冶金环境中的抗性、提高冶金效率提供了理论指导。     

粪肠球菌和屎肠球菌耐药性分析

目的 监测我院肠球菌中粪肠球菌株和屎肠球菌株的耐药性,为临床合理应用抗菌药物提供依据。方法 采用法国生物梅里埃公司的GPI板进行细菌鉴定及药敏试验,应用whonet5软件统计粪肠球菌和屎肠球菌的耐药率。结果 粪肠球菌和屎肠球菌对氯霉素、呋喃妥因、万古霉素有较好体外抗菌活性,耐药率都在50%以下,对万古霉素的耐药率在1%以下。粪肠球菌对青霉素、高水平庆大霉素、环丙沙星、利福平、红霉素等大部分抗菌素的耐药率有逐年下降趋势,而屎肠球菌对环丙沙星、利福平、呋喃妥因等抗菌素的耐药率则有上升趋势,屎肠球菌对大多数抗菌素耐药率都高于粪肠球菌。结论 粪肠球菌和屎肠球菌呈多重耐药,临床用药应结合药敏试验结果合理选择抗菌药物。     

Antioxidants and reactive oxygen species in plants

Reactive oxygen species, antioxidants and oxygen stress arethe subject of investigation by many research laboratories worldwide.The use of molecular biology techniques is propelling the subjectalong at a fast pace and the number of research papers on thetopic is growing fast. Antioxidants and Reactive Oxygen Speciesin Plants summarizes much recent     

Triticale and barley microspore embryogenesis induction requires both reactive oxygen species generation and efficient system of antioxidative defence

Plant Cell, Tissue and Organ Culture (PCTOC) - The effectiveness of microspore embryogenesis (ME) is determined by a complex network of internal and environmental factors. In the present study on...     

Imaging reactive oxygen species in arthritis

Reactive oxygen species (ROS) have been shown to play a role in the pathogenesis of arthritides. Luminol was used as the primary reporter of ROS and photons resulting from the chemiluminescence reaction were detected using a super-cooled CCD photon counting system. Luminol was injected intravenously into groups of animals with different models of arthritis. Imaging signal correlated well with the severity of arthritis in focal and pan-arthritis as determined by histological measurement of ROS by formazan. Measurements were highly reproducible, sensitive, and repeatable. In vivo chemiluminescence imaging is expected to become a useful modality to elucidate the role of ROS in the pathogenesis of arthritides and in determining therapeutic efficacy of protective therapies.     

Imaging reactive oxygen species in asthma

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.     

Phagocytic leukocytes and reactive oxygen species

Phagocytic leukocytes, when appropriately stimulated, display a respiratory burst in which they consume oxygen and produce superoxide anions. Superoxide is produced by the phagocyte NADPH-oxidase system which is a multiprotein complex that is dissociated in quiescent cells and is assembled into the functional oxidase following stimulation of these cells. Also associated with the respiratory burst is the generation of other reactive oxygen species. The identity of components of the NADPH-oxidase system and their interactions are known in considerable molecular detail. Understanding of the regulation of superoxide production is less well known. This review also points out the important role of microscopy in complementing biochemical studies to understand better the cell biology of the phagocyte respiratory burst. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Mammalian peroxisomes and reactive oxygen species

The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor- on peroxisome function and peroxisome proliferator activated receptor-. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5^–/– knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.     

Mitochondrial reactive oxygen species and inflammation: Molecular mechanisms,diseases and promising therapies

Over the last few decades, many different groups have been engaged in studies of new roles for mitochondria, particularly the coupling of alterations in the redox pathway with the inflammatory responses involved in different diseases, including Alzheimer’s disease, Parkinson's disease, atherosclerosis, cerebral cavernous malformations, cystic fibrosis and cancer. Mitochondrial dysfunction is important in these pathological conditions, suggesting a pivotal role for mitochondria in the coordination of pro-inflammatory signaling from the cytosol and signaling from other subcellular organelles. In this regard, mitochondrial reactive oxygen species are emerging as perfect liaisons that can trigger the assembly and successive activation of large caspase-1- activating complexes known as inflammasomes. This review offers a glimpse into the mechanisms by which inflammasomes are activated by mitochondrial mechanisms, including reactive oxygen species production and mitochondrial Ca²⁺ uptake, and the roles they can play in several inflammatory pathologies.     

Therapeutic efficacy of Kalpaamruthaa on reactive oxygen/nitrogen species levels and antioxidative system in mammary carcinoma bearing rats

Reactive oxygen species play an important role in cancer and metastasis. Kalpaamruthaa is a modified Siddha preparation, which has been formulated in our laboratory. The preparation is an amalgamation of Semecarpus anacardium (SA), Emblica officinalis (EO) and honey, which gives an extra protectiveness to mammary carcinoma bearing animals (Sprague-Dawley stains were used for this study). The aim of our research is to determine the therapeutic efficiency of the drug with respect to lipid peroxidation and antioxidant status. The levels of lipid peroxides and antioxidant levels were measured in blood, and vital organs (liver, kidney and breast tissue) of control and experimental animals. In cancer condition, the LPO was increased and antioxidant levels were decreased. On drug (SA and KA) administration, decreased LPO and increased antioxidant levels were seen in control and experimental animals. This may be due to additive property of the drugs (SA, Emblica and honey), which possesses anticancer effect. The present study shows the good therapeutic efficacy of Kalpaamruthaa against mammary carcinoma.     

Catabolite repression in Enterococcus faecalis

Metabolism of citrate, pyruvate and sugars by Enterococcus faecalis E-239 and JH2-2 and an isogenic, catabolite derepressed mutant of JH2-2, strain CL4, was investigated. The growth rates of E. faecalis E-239 on citrate and pyruvate were 0.58 and 0.63 h(-1), respectively, indicating that both acids were used as energy sources. Fructose and glucose prevented the metabolism of citrate until all the glucose or fructose had been metabolised. Diauxie growth was not observed but growth on glucose and fructose was much faster than on citrate. In contrast, citrate was co-metabolized with galactose or sucrose and pyruvate with glucose. When glucose was added to cells growing on citrate, glucose metabolism began immediately but inhibition of citrate utilisation did not begin for approximately 1.5 h. Growth rates of E. faecalis JH2-2 and its isogenic, catabolite derepressed mutant, strain CL4, on citrate, were 0.41 and 0.36 h(-1), respectively. The catabolite derepressed mutant was able to co-metabolise citrate and glucose at all concentrations of glucose tested (3-25 mM), while its parent, could only metabolise citrate once all the glucose had been consumed. In strains JH2-2 and E-239, the growth rate on citrate decreased as the glucose concentration increased and, in 25 mM glucose, consumption of citrate was inhibited for several hours after glucose had been consumed. These results indicate that catabolite repression by glucose and fructose occurs in enterococci.     

Measuring mitochondrial reactive oxygen species

This article examines recent methods for measuring reactive oxygen species produced in isolated mitochondria and within live cells, with particular emphasis on the detection of hydrogen peroxide. Protocols for reliable measurements of mitochondrial hydrogen peroxide are presented, while the advantages and pitfalls of these and other methods are discussed. New developments in the detection of lipid peroxidation are outlined. Advice is also provided to aid the interpretation of cellular data with respect to the contribution of oxygen radical production by different components of the mitochondrial respiratory chain.