The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.
Methods and Results:  The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO₂) or hydrogen peroxide/peracetic acid (H₂O₂/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO₂ or H₂O₂/PA inactivated B. anthracis spores at levels ranging from 2·2 to ≥7·6 log reductions.
Conclusions:  Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface.
Significance and Impact of the Study:  This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.     

A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste

Aims:  The aim of this study was to enrich, characterize and identify strict anaerobic extreme thermophilic hydrogen (H₂) producers from digested household solid wastes.
Methods and Results:  A strict anaerobic extreme thermophilic H₂ producing bacterial culture was enriched from a lab-scale digester treating household wastes at 70°C. The enriched mixed culture consisted of two rod-shaped bacterial members growing at an optimal temperature of 80°C and an optimal pH 8·1. The culture was able to utilize glucose, galactose, mannose, xylose, arabinose, maltose, sucrose, pyruvate and glycerol as carbon sources. Growth on glucose produced acetate, H₂ and carbon dioxide. Maximal H₂ production rate on glucose was 1·1 mmol l⁻¹ h⁻¹ with a maximum H₂ yield of 1·9 mole H₂ per mole glucose. 16S ribosomal DNA clone library analyses showed that the culture members were phylogenetically affiliated to the genera Bacillus and Clostridium. Relative abundance of the culture members, assessed by fluorescence in situ hybridization, were 87 ± 5% and 13 ± 5% for Bacillus and Clostridium , respectively.
Conclusions:  An extreme thermophilic, strict anaerobic, mixed microbial culture with H₂-producing potential was enriched from digested household wastes.
Significance and Impact of the Study:  This study provided a culture with a potential to be applied in reactor systems for extreme thermophilic H₂ production from complex organic wastes.     

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.     

Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.     

Resistance of Escherichia coli grown at different temperatures to various environmental stresses

Aims:  To study the influence of growth temperature on the resistance of Escherichia coli to three agents of different nature: heat, pulsed electric field (PEF) and hydrogen peroxide.
Methods and Results:  Escherichia coli cells were grown to stationary phase at 10°C, 20°C, 30°C, 37°C and 42°C. Survival curves to a heat treatment at 57·5°C, to a PEF treatment at 22 kV cm⁻¹ and to 40 mmol l⁻¹ hydrogen peroxide were obtained and fitted to a model based on the Weibull distribution to describe and compare the inactivation. Time to inactivate the first log cycle of the population at 57·5°C of cells grown at 42°C was sixfold higher than that corresponding to cells grown at 10°C. On the contrary, cells grown at 10°C and 20°C were more resistant to PEF and hydrogen peroxide treatments.
Conclusions:  The influence of growth temperature on bacterial resistance depends on the stress applied. Cells grown at higher temperatures were more heat resistant, but more sensitive to PEF and hydrogen peroxide.
Significance and Impact of the Study:  Results obtained in this investigation help in understanding the physiology of bacterial resistance and the inactivation mechanisms of different technologies.     

Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase

Aims:  Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H₂O₂ concentration that is low compared to current nonenzymatic formulations.
Methods and Results:  Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus ) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l⁻¹ H₂O₂ and 100 mmol l⁻¹ Br⁻ at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme.
Conclusions:  The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions.
Significance and Impact of the Study:  For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.     

Fusarium response to oxidative stress by H2O2 is trichothecene chemotype-dependent

The present study aims at clarifying the impact of oxidative stress on type B trichothecene production. The responses to hydrogen peroxide (H₂O₂) of an array of Fusarium graminearum and Fusarium culmorum strains were compared, both species carrying either the chemotype deoxynivalenol (DON) or nivalenol (NIV). In both cases, levels of in vitro toxin production are greatly influenced by the oxidative parameters of the medium. A 0.5 mM H₂O₂ stress induces a two- to 50-fold enhancement of DON and acetyldeoxynivalenol production, whereas the same treatment results in a 2.4- to sevenfold decrease in NIV and fusarenone X accumulation. Different effects of oxidative stress on toxin production are the result of a variation in Fusarium 's antioxidant defence responses according to the chemotype of the isolate. Compared with DON strains, NIV isolates have a higher H₂O₂-destroying capacity, which partially results from a significant enhancement of catalase activity induced by peroxide stress. A 0.5 mM H₂O₂ treatment leads to a 1.3- to 1.7-fold increase in the catalase activity of NIV isolates. Our data, which show the higher adaptation to oxidative stress developed by NIV isolates, are consistent with the higher virulence of these Fusarium strains on maize compared with DON isolates.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos

Aim:  To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos.
Methods and Results:  A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0·01 g l⁻¹). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0·2 g l⁻¹, was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0·01 g l⁻¹) by ChlD strain. The best degradation efficiency was observed at 0·1 g l⁻¹ supplement of biosurfactant, as validated by GC and HPLC studies.
Conclusion:  The addition of biosurfactant at 0·1 g l⁻¹ resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation.
Significance and Impact of the Study:  This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

Sodium acetate enhances hydrogen peroxide production in Weissella cibaria

Aims:  To investigate hydrogen peroxide production by lactic acid bacteria (LAB) and to determine the key factors involved.
Methods and Results:  Six strains of Weissella cibaria produced large amounts (2·2–3·2 mmol l⁻¹) of hydrogen peroxide in GYP broth supplemented with sodium acetate, but very low accumulations in glucose yeast peptone broth without sodium acetate. Increased production of hydrogen peroxide was also recorded when strains of W. cibaria were cultured in the presence of potassium acetate, sodium isocitrate and sodium citrate. Oxidases and peroxidases were not detected, or were present at low levels in W. cibaria . However, strong nicotinamide adenine dinucleotide (NADH) oxidase activity was recorded, suggesting that the enzyme plays a key role in production of hydrogen peroxide by W. cibaria .
Conclusions:  Weissella cibaria produces large quantities of hydrogen peroxide in aerated cultures, in a process that is dependent on the presence of acetate in the culture medium. NADH oxidase is likely the key enzyme in this process.
Significance and Impact of the Study:  This is the first study showing that sodium acetate, normally present in culture media of LAB, is a key factor for hydrogen peroxide production by W. cibaria . The exact mechanisms involved are not known.     

Effects of carotenoids from Deinococcus radiodurans on protein oxidation

Aims:  To evaluate the antioxidant effect of carotenoids from Deinococcus radiodurans on protein.
Methods and Results:  Deinococcus radiodurans strain R1 (ATCC 13939) and its mutant strain R1ΔcrtB were used for this study. The total carotenoids (R1ex) from D. radiodurans were obtained by extraction with acetone/methanol (7 : 2, by vol), and their antioxidant activity was measured using the DPPH˙ (2,2-diphenyl-1-picrylhydrazyl) system. The protein oxidation level, in vitro and in the cell, was measured using the DNPH (2,4-dinitrophenyl hydrazine) method. The carotenoid extract R1ex scavenged 40·2% DPPH˙ radicals compared to β-carotene (31·7%) at a concentration of 0·5 mg ml⁻¹. The intracellular level of protein oxidation in mutant R1ΔcrtB, which does not contain carotenoid, was 0·0212 mmol mg⁻¹ protein which is significantly greater than that in the wild type (0·0169 mmol mg⁻¹ protein) following the treatment with H₂O₂. The purified major carotenoid product (deinoxanthin) from the wild type showed a greater inhibition of oxidative damage in bovine serum albumin than lycopene or lutein.
Conclusions:  Carotenoids prevent protein oxidation and contribute to the resistance to cell damage in D. radiodurans .
Significance and Impact of the Study:  Our results provide the evidence that carotenoids can protect proteins in D. radiodurans against oxidative stress.     

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p -nitrophenyl butyrate was 0·3 mmol l⁻¹ while on p -nitrophenyl caprylate, it was 0·04 mmol l⁻¹. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l⁻¹ phenylmethylsulphonyl fluoride, β-mercaptoethanol and N -ethylmaleimide, and was stimulated by Zn²⁺ at 1 mmol l⁻¹.     

Petite mutation in aged and oxidatively stressed ale and lager brewing yeast

Aims:  To determine the role of oxidative stress and chronological ageing on the propensity of brewing yeast strains to form respiratory deficient 'petites'.
Methods and Results:  Four industrial yeast strains (two ale and two lager strains) were exposed to oxidative stress in the form of H₂O₂ (5 mmol l⁻¹) for two hours. Cell viability and occurrence of petites were determined by the slide culture and TTC-overlay techniques, respectively. Increases in petite frequency were observed but only in those strains sensitive to oxidative stress. Chronological ageing under aerobic conditions led to an increase in petites in strains sensitive to oxidative stress. No such increase was observed under anaerobic conditions.
Conclusion:  Ageing may contribute to mitochondrial DNA damage and increase the propensity of brewing yeast cells to become respiratory deficient. Tolerant strains may be less likely to generate petites as a result of serial re-pitching.
Significance and Impact of the Study:  Continuous re-use of brewing yeast is associated with an increase in the frequency of petites within brewery yeast slurries, a phenomenon resulting in reduced fermentative capacity. The cause of petite generation during brewery handling is unknown. We show that endogenous oxidative stress has the potential to generate petites within brewing yeast populations.