Comparison of quantitative and qualitative methods of detecting hydrogen peroxide produced by human vaginal strains of lactobacilli

A quantitative method was developed for the measurement of micromolar quantities of H₂O₂ produced in Rogosa broth and peptonized milk broth by vaginal strains of lactobacilli isolated from women. The production of substantial amounts reproducibly was dependent on the growth of the organisms in acid media (pH ≤6.0) under anaerobic or micro-aerophilic conditions with continuous agitation. The addition to the media of the enzyme inhibitor, 3-amino-l,2,4-triazole, with or without catalase sometimes induced the production of H₂O₂ especially in non-agitated cultures. However, other agents such as concanavalin and o -dianisidine had no enhancing effect, and catalase or peroxidase alone completely inhibited H₂O₂ production.
The H₂O₂ produced in the acid media was stable for more than a month at 5°C but not in media at pH ≥ 7.0. Of five strains of lactobacilli tested by the quantitative method and by a chromogenic qualitative method (Rogosa-catalase or -peroxidase agar), three consistently produced H₂O₂ measurable by the former method, but none did so after growth of the organisms on Rogosa-catalase/peroxidase agar which suggested that the qualitative method was unreliable. The fact that H₂O₂ was produced in substantial quantities by some strains and not at all by others enabled H₂O₂-producers and non-producers to be distinguished easily.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

Aerobic and anaerobic microbial consumption of hydrogen in geothermal spring water

Abstract Hydrogen consumption was measured in hot geothermal water from two ponds of the San Federigo solfatara, Tuscany, Italy, where emanation gases contained approx. 4% H₂. H₂ consumption was completely inhibited by NaOH and partially by HgCl₂ indicating microbial utilization. Aerobic and anaerobic H₂ consumption activities coexisted in the same water with aerobic activity being higher in one pond and anaerobic activity in the other. The kinetics of H₂ consumption were consistent with those of 'Knallgas', methanogenic or sulfidogenic bacteria.     

Distribution of transition metal ions in multiple forms of Methanosarcina hydrogenase

There were significant levels of in vitro hydrogenase activity in Methanosarcina strains. The multiple forms of hydrogenase were observed in cell free extracts of cells grown on methanol. Strains having poor growth on H₂ : CO₂ had four forms while strains having normal growth on all substrates contained two forms of hydrogenase. These multiple forms differ in their charges as well as in their composition of transition metal ions. The strain having normal growth showed higher incorporation of ⁶³Ni²⁺ and ⁶⁵Zn²⁺. Both hydrogenases, A and D, of strain P₃ had methylviologen and F₄₂₀-reducing activity and contained Zn²⁺ and Co²⁺ respectively. Hydrogenases A and D of strains P₁ and P₄ also had similar characteristics whereas hydrogenases B and C had only methylviologen reducing activity.     

Menaquinone composition of some micrococci determined by high performance liquid chromatography

The menaquinone composition of some species of the genus Micrococcus was analysed by high performance liquid chromatography. Both unsaturated and hydrogenated menaquinones were detected. Micrococcus kristinae, M. lylae and M. nishinomiyaensis contained hydrogenated menaquinones while M. sedentarius contained unsaturated menaquinone. The predominant menaquinone isoprenologues of these species are MK- 7 (H₂), MK- 8 (H₂), MK- 8 (H₂) and MK- 8 , respectively. These observations confirm further the heterogeneity of the genus.     

Hydrogen Peroxide Production by Rat Brain In Vivo

Abstract: H₂ O₂ production by rat brain in vivo was observed with a method based on the measurement of brain catalase. The administration to the rat of 3-amino-1, 2, 4-triazole, an H₂ O_2- dependent inhibitor of catalase, caused progressive inhibition of brain catalase activity in both the supernatant and pellet fractions of homogenates of the striatum and prefrontal cortex. The prevention of catalase inhibition by prior administration of ethanol confirmed that catalase inhibition in vivo was dependent upon H₂ O₂. A significant portion of the catalase (30-33%) appeared in the supernatant fraction from a slow-speed homogenization procedure and was not significantly contaminated by either erythrocytes or capillaries. In the whole homogenate, less than 6% of the catalase activity was attributed to erythrocytes. Modification of intracellular monoamine oxidase activity by either pargyline or reserpine did not change the rate of inhibition of catalase by aminotriazole. A probable interpretation of these data is that H₂ O₂ generated by mitochondrial monoamine oxidase does not reach the catalase compartment; the catalase is contained in particles described by other investigators as the microperoxisomes of brain. In studies in vitro , the production of H₂ O₂ by rat brain mitochondria with either dopamine or serotonin as substrate was confirmed.     

Association mapping of cadmium, copper and hydrogen peroxide tolerance of roots and translocation capacities of cadmium and copper in Arabidopsis thaliana

Association mapping analysis of Cd, Cu and H ₂O ₂ tolerance, judged by relative root length (RRL: % of root length in stress condition relative to that in control condition), and Cd and Cu translocation ratios (amount of metal in the shoot to the total) were performed using 90 accessions of Arabidopsis thaliana . Using 140 SNPs that were distributed across the genome, association mapping analysis was performed with a haploid setting by the Q + K method, which minimizes detection of false associations by combining the Q-matrix of the structured association (Q) with kinship (K) to control for the population structure. Six, five and five significant (−log ₁₀ P -value is 1.3 ≥) linkages were detected between the SNPs and Cd, Cu and H₂O₂ resistant RRLs, respectively. In addition, six significant linkages were identified with translocation capacities of Cd and Cu. Among those detected loci, two each of Cu and Cd tolerance RRLs were collocated with those of H₂O₂ tolerance RRL, while one locus each was detected by Cu and Cd tolerance RRLs that collocated with their translocation ratios. These results suggested that these factors might partly explain the phenotypic variation of tolerance RRLs to Cd and Cu of Arabidopsis thaliana . Finally, using a different approach to analyze interactions between individual phenotypes, namely clustering analysis, we found an expected segregation of resistant SNPs (single-nucleotide polymorphisms) of the multiple RRLs in the typical accession groups carrying multiple traits. Almost none of the loci detected by association mapping analysis were linked to the loci of previously identified critical genes regulating the traits, suggesting that this could be useful to identify complex architecture of genetic factors determining variation among multiple accessions.     

Uptake hydrogenase system in urd bean (Vigna mungo) Rhizobium in relation to nitrogen fixation

Twenty-five isolates of urd bean ( Vigna mungo ) Rhizobium were tested for the presence of an H₂-uptake system using triphenyl tetrazolium chloride reduction as the screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle H₂ both in culture as well as in nodules. H₂-uptake positive, H₂-uptake negative strains and H₂-uptake negative mutants were compared on the basis of their effect on dry matter accumulation and N₂-fixation. Greenhouse experiments have shown the beneficial effect of the H₂-uptake positive system in nodules on N₂-fixation. Plants inoculated with H₂-uptake positive strains produced higher N content and dry matter over the plants inoculated with H₂-uptake negative strains.     

Isolation and structural elucidation of a dihydroubiquinone-9 from the fungus Aureobasidium pullulans

Abstract A naturally occurring member of ubiquinone (Q) group, a dihydroubiquinone-9 (Q-9 (H₂)), has been isolated as a minor ubiquinone component from the fungus Aureobasidium pullulans . By ultraviolet absorption, mass and nuclear magnetic resonance spectrometric studies, the structure of Q-9 (H₂) was found to be 2,3-dimethoxy-5-methyl-6-IX-dihydromultiprenyl⁹-1,4-benzoquinone (I).     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

H2 and CO2 production from methanol or formaldehyde by the methanogenic bacterium strain Gö1 treated with 2-bromoethanesulfonic acid

Abstract In cell suspensions of the methanogenic bacterium strain Gö1 or Methanosarcina barkeri H₂ formation from methanol in the presence of 2-bromoethanesulfonic acid (BES) was strictly dependent on sodium ions; apparent K _S for Na⁺, 1.3±0.3 mM.H₂ formation was inhibited by the uncoupler tetrachlorosalicylanilide (TCS), but this inhibition could be temporarily overcome, when a sodium pulse (100 mM) was given to the cell suspension. On the other hand, H₂ formation from formaldehyde in the presence of BES (rate: 300 nmol H₂/h·mg protein as compared to 25 nmol H₂/h·mg protein from methanol) was not sodium-dependent, not TCS-sensitive and not inhibited by addition of monensin. H₂ formation was accompanied by CO₂ formation in stoichiometric amounts, 3 H₂:1 CO₂ for methanol and 2 H₂:1 CO₂ for formaldehyde oxidation.     

HISTAMINE-SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5-HT) and drugs such as isoprenaline (ISP), 2-(2-pyridyl)ethylamine (H₂ stimulant—H_ls), 4-methyl-histamine (H₂ stimulant H_2s), mepyramine (H₁ antagonistp H_la), cimetidine (H₂ antagonist—H_2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.
The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H_2s, induced a greater stimulation of adenylate cyclase than H_ls. On the other hand, the H_2a inhibited HA stimulation to a greater extent than the H_la, H_la and H_2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans-mitter role in the hypothalamus, and in this area there appears to be a mixed population of H₁ and H₂ receptors, with a majority of H₂ receptors.     

H2 oxidation in the presence of thiosulfate, by a Thermoanaerobacter strain isolated from an oil-producing well

Abstract A thermophilic rod (strain SEBR 5268), isolated from an oil-producing well, was identified as a Thermoanaerobacter strain that was phenotypically related to T. finnii . Both SEBR 5268 and T. finnii oxidized H₂ by reducing thiosulfate to sulfide using yeast extract as growth substrate. H₂ oxidation in the presence of thiosulfate was significant at the end of the exponential growth of SEBR 5268 and was maintained during the lysis phase. In the absence of thiosulfate, H₂ was inhibitory for both strains. The role of H₂ consumption by these bacteria is discussed with regard to their metabolism on organic compounds.     

The C Terminal Tail of the Histamine H2 Receptor Contains Positive and Negative Signals Important for Signal Transduction and Receptor Down-Regulation

Abstract: To examine the role of the C terminal tail in H₂ receptor regulation, three cDNAs, encoding truncated histamine H₂ receptor mutants (H₂T295, H₂T307, and H₂T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H₂ receptor binding of the H₂T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H₂T307) did not affect the binding properties of H₂ antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H₂T341), which was able to form a ternary complex but was unable to fully activate the G_s protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H₂ receptor down-regulation was more profound for the H₂T307 receptor, indicating that different structural elements of the H₂ receptor protein are involved in the cyclic AMP-dependent and independent pathways of H₂ receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H₂ receptor that act as positive and/or negative signals in H₂ receptor signaling and down-regulation.     

The presence of oxygen in rumen liquor and its effects on methanogenesis

In situ measurement of O₂ in the rumen liquor of cows, sheep and goats using a membrane-covered O₂ electrode revealed the presence of up to 1630 nmol/l O₂; O₂ became undetectable immediately after feeding of animals. The effects of O₂ on H₂ production and methanogenesis in samples of rumen liquor were investigated using a mass spectrometer fitted with a membrane inlet system. Methanogenesis was totally and irreversibly inhibited after short term exposure (about 10 min) to 5 KPa (0·05 atm) O₂; H₂ production was unaffected. Glucose additions produced rapid transient increases in H₂ levels and increased O₂ uptake.     

RAT BRAIN FOLATE IDENTIFICATION

Abstract— Endogenous rat brain folates were identified using column chromatography for folate separation, authentic folate standards, and microbiological assay. The total folate content of rat brain was 360 ng per gram brain (wet wt) and consisted of tetrahydropteroylpentaglutamate (H₄ PteGlu₅, 29%), H₄ PteGlu₆ (14%), H₄PteGlu₇ (7%), 5-CH₃-H₄PteGlu₄ (7%), 5-CH₃-H₄PteGlu₅ (13%), and small amounts of formyl-, methyl-, and H₄PteGlu_1-6 (~25%) and non-methylated folates having a glutamate chain over seven glutamates long (4%). Overall folate oxidation state and one carbon forms were H₄PteGlu_n (58%), 5-CH₃-H₄PteGlu_n (18%), 5- and 10-CHO-H₄PteGlu_n (16%), and H₂ PteGlu_n (8%). No PteGlu_n was detected.     

Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides

Aims:  Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H₂O₂) and stabilized H₂O₂ combination products.
Methods and Results:  The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H₂O₂ combined with silver possessed a higher bactericidal and fungicidal activity compared to native H₂O₂ with and without glycerol.
Conclusions:  Validation showed that the RMM may replace the PCCT. When applying the RMM, H₂O₂ combined with silver was clearly a more potent disinfectant compared to H₂O₂ in killing bacteria and fungi.
Significance and Impact of the Study:  The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H₂O₂ with silver may replace native H₂O₂ to increase overall disinfection efficiency.     

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.