Antimicrobial and Antioxidant Activity of Some Nitrogen-Containing Heterocycles and Their Acyclic Analogues.

We investigated antimicrobial and antioxidant activity of nitrogen-containing heterocycles and their acyclic analogues, some of which can be considered as promising in terms of biological activity. Based on structure, 26 tested compounds were divided into 4 groups. In the test with 2,2-diphenyl-1-picrylhydrazyl (DPPH), the compounds of the group 2 had the highest radical-binding activity (RBA) (53-78%), while those of group 3 had the lowest values (1.5-5.2%). In oxygen radical absorbance capacity assay, all compounds from groups 1, 2 and 3 showed high RBA: 44-94% at 50 µM. The highest bacteriostatic activity against Escherichia coli was found for four compounds in group 2 (MIC = 0.25-1 mM) and low bacteriostatic activity for group 3 (MIC > 4 mM). Some relationships between the structure of compounds and the values of the MIC are revealed. It was also found that four substances from different groups had the ability to inhibit the formation of colonies in E. coli from 1.3 to 5.7 times. Four compounds reduced specific biofilm formation by 40-60%. The tested substances did not induce the expression of the sulA gene controlled by the SOS system, which indicates the lack of genotoxic activity. None of the tested compounds had pro-oxidant activity. This was shown by both the absence of production hydrogen peroxide in a bacteria-free medium and inability to induce expression of the katG gene encoding HPI catalase in growing E. coli. The data obtained could be useful in the development of new drugs.

Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium.

Cysteine and its derivatives, including H2S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H2S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H2S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H2S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Phosphate starvation is accompanied by disturbance of intracellular cysteine homeostasis in Escherichia coli.

Metabolic rearrangements that occur during depletion of essential nutrients can lead to accumulation of potentially dangerous metabolites. Here we showed that depletion of phosphate (Pi), accompanied by a sharp inhibition of growth and respiration, caused a transient excess of intracellular cysteine due to a decrease in the rate of protein synthesis. High cysteine level can be dangerous due to its ability to produce ROS and reduce Fe3+ to Fenton-reactive Fe2+. To prevent these negative effects, excess cysteine was mainly incorporated into glutathione (GSH), the intracellular level of which increased by 3 times, and was also exported to the medium and partially degraded to form H2S with participation of 3-mercaptopyruvate sulfotransferase (3MST). The addition of Pi to starving cells led to a sharp recovery of respiration and growth, GSH efflux into the medium and K+ influx into the cells. A pronounced coupling of Pi, GSH, and K+ fluxes was shown upon Pi depletion and addition, which may be necessary to maintain the ionic balance in the cytoplasm. We suggest that processes aimed at restoring cysteine homeostasis may be an integral part of the universal response to stress under different types of stress and for different types of bacteria.

Influence of Growth Medium Composition on Physiological Responses of Escherichia coli to the Action of Chloramphenicol and Ciprofloxacin.

The ability of hydrogen sulfide (H2S) to protect bacteria from bactericidal antibiotics has previously been described. The main source of H2S is the desulfurization of cysteine, which is either synthesized by cells from sulfate or transported from the medium, depending on its composition. Applying electrochemical sensors and a complex of biochemical and microbiological methods, changes in growth, respiration, membrane potential, SOS response, H2S production and bacterial survival under the action of bactericidal ciprofloxacin and bacteriostatic chloramphenicol in commonly used media were studied. Chloramphenicol caused a sharp inhibition of metabolism in all studied media. The physiological response of bacteria to ciprofloxacin strongly depended on its dose. In rich LB medium, cells retained metabolic activity at higher concentrations of ciprofloxacin than in minimal M9 medium. This decreased number of surviving cells (CFU) by 2-3 orders of magnitude in LB compared to M9 medium, and shifted optimal bactericidal concentration (OBC) from 0.3 µg/mL in M9 to 3 µg/mL in LB. Both drugs induced transient production of H2S in M9 medium. In media containing cystine, H2S was produced independently of antibiotics. Thus, medium composition significantly modifies physiological response of E. coli to bactericidal antibiotic, which should be taken into account when interpreting data and developing drugs.

Study of the early response of Escherichia coli lpcA and ompF mutants to ciprofloxacin.

In most previous studies the sensitivity of Escherichia coli outer membrane mutants to ciprofloxacin (CF) was studied by MIC method. In the present work, the early response of these mutants to CF was studied using physiological and biochemical methods and electrochemical sensors. The use of sensors made it possible to monitor dissolved oxygen, potassium and extracellular sulfide continuously directly in growing cultures in real time. In the absence of CF, no significant differences were found between the mutants deficient in porin OmpF and lipopolysaccharide (LPS) and the parent. The only exception was 5-6 times higher extracellular glutathione and 1.5-3 times lower intracellular glutathione in the lpcA compared to the parent and the ompF. Ciprofloxacin inhibited growth, respiration, membrane potential and K+ consumption, which was less pronounced in both mutants compared to the parent. Changes in these parameters correlated with each other, but not with survival. A reversible increase in sulfide level was observed at 3 µg ml-1 CF in the parent, at 20 µg ml-1 CF in ompF and was absent in lpcA at all concentrations. The data obtained show that the use of electrochemical sensors can provide a more complete understanding of the early response of bacteria to CF.

Study of the contribution of active defense mechanisms to ciprofloxacin tolerance in Escherichia coli growing at different rates.

Using rpoS, tolC, ompF, and recA knockouts, we investigated their effect on the physiological response and lethality of ciprofloxacin in E. coli growing at different rates on glucose, succinate or acetate. We have shown that, regardless of the strain, the degree of changes in respiration, membrane potential, NAD+/NADH ratio, ATP and glutathione (GSH) strongly depends on the initial growth rate and the degree of its inhibition. The deletion of the regulator of the general stress response RpoS, although it influenced the expression of antioxidant genes, did not significantly affect the tolerance to ciprofloxacin at all growth rates. The mutant lacking TolC, which is a component of many E. coli efflux pumps, showed the same sensitivity to ciprofloxacin as the parent. The absence of porin OmpF slowed down the entry of ciprofloxacin into cells, prolonged growth and shifted the optimal bactericidal concentration towards higher values. Deficiency of RecA, a regulator of the SOS response, dramatically altered the late phase of the SOS response (SOS-dependent cell death), preventing respiratory inhibition and a drop in membrane potential. The recA mutation inverted GSH fluxes across the membrane and abolished ciprofloxacin-induced H2S production. All studied mutants showed an inverse linear relationship between logCFU ml-1 and the specific growth rate. Mutations shifted the plot of this dependence relative to the parental strain according to their significance for ciprofloxacin tolerance. The crucial role of the SOS system is confirmed by dramatic shift down of this plot in the recA mutant.

Study of antioxidant activity of fodder grasses using microbial test systems.

AIM: To measure the biological activities of extracts of fodder grasses Onobrýchis arenária, Galéga orientális and Rhaponticum carthamoides that are commonly planted in Europe, Middle East and eastern Africa. METHODS AND RESULTS: Microbial test-systems based on Escherichia coli BW25113 that allow measurement of gene expression, growth and survival, biofilm formation (BF) in combination with the standard chemical procedures were used. The extracts studied had radical scavenging and metal-chelating activities and induced expression of antioxidant genes via generation of hydrogen peroxide. However, the extracts did not affect bacterial growth in planktonic cultures but dose-dependently inhibited BF. CONCLUSIONS: The most remarkable effects were observed in G. orientalis, a high-yielding crop, rich in crude protein and fibres. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account the antibiofilm activities of the extracts, a perspective for decreasing colonization of ruminants' gut with pathogenic bacteria might be suggested in case of feeding with all the grasses studied.

Effect of resveratrol and quercetin on the susceptibility of Escherichia coli to antibiotics.

Activities of plant polyphenols (PPs), resveratrol and quercetin, alone or in combination with four conventional antibiotics against Escherichia coli have been investigated. In medium without antibiotics, both polyphenols caused a dose-dependent growth inhibition. However, pretreatment with resveratrol (40 and 100 µg ml-1) and quercetin (40 µg ml-1) reduced the bacteriostatic effect of kanamycin, streptomycin, cefotaxime and partially of ciprofloxacin. With few exceptions, both PPs also reduced the bactericidal effect of tested antibiotics. Paradoxically, low doses of PPs enhanced the bactericidal effect of kanamycin and partially ciprofloxacin. Compared to quercetin, resveratrol showed a weaker effect on the induction of antioxidant genes and the resistance of E. coli to the oxidative stress generated by hydrogen peroxide treatment. Both polyphenols at high doses reduced membrane potential. Altogether, these findings suggest that the decrease in the bactericidal effect of antibiotics by high doses of polyphenols is mostly due to bacteriostatic action of the latter. In the case of quercetin, the contribution of its antioxidant activity for antibiotic protection may be significant. There is a growing interest in the use of plant-derived compounds to enhance the toxicity of traditional antibiotics. This and other studies show that, under certain conditions, the use of polyphenols as adjuvants may not exert the expected therapeutic effect, but rather to decrease antimicrobial activity of antibiotics.

Study of the relationship between extracellular superoxide and glutathione production in batch cultures of Escherichia coli.

Aerobically growing Escherichia coli generates superoxide flux into the periplasm via the oxidation of dihydromenaquinone and simultaneously carries out continuous transmembrane cycling of glutathione (GSH). Here we have shown that, under the conditions of a gradual decrease in dissolved oxygen (dO2), characteristic of batch culture, the global regulatory system ArcB/ArcA can play an important role in the coordinated control of extracellular superoxide and GSH fluxes and their interaction with intracellular antioxidant systems. The lowest superoxide production was observed in the menA and arcB mutants, while the atpA, atpC and atpE mutants generated superoxide 1.3-1.5 times faster than the parent. The share of exported glutathione in the ubiC, atpA, atpC, and atpE mutants was 2-3 times higher compared to the parent. A high direct correlation (r = 0.87, p = 0.01) between extracellular superoxide and GSH was revealed. The menA and arcB mutants, as well as the cydD mutant lacking the GSH export system CydDC, were not capable of GSH excretion with a decrease in dO2, which indicates a positive control of GSH export by ArcB. In contrast, ArcB downregulates sodA, therefore, an inverse correlation (r = -0.86, p = 0.013) between superoxide production and sodA expression was observed.

Cysteine homeostasis under inhibition of protein synthesis in Escherichia coli cells.

Increased intracellular cysteine poses a potential danger to cells due to the high ability of cysteine to reduce free iron and promote the Fenton reaction. Here, we studied ways to maintain cysteine homeostasis in E. coli cells while inhibiting protein synthesis with valine or chloramphenicol. When growing wild-type bacteria on minimal medium with sulfate, an excess of cysteine resulting from the inhibition of protein synthesis is mainly incorporated into glutathione (up to 90%), which, therefore, can be considered as cysteine buffer. The share of hydrogen sulfide, which is the product of cysteine degradation by cysteine synthase B (CysM), does not exceed 1-3%, the rest falls on free cysteine, exported from cells. As a result, intracellular free cysteine is maintained at a low level (about 0.1 mM). The lack of glutathione in the gshA mutant increases H2S production and excretion of cysteine and leads to a threefold increase in the level of intracellular cysteine in response to valine and chloramphenicol. The relA mutants, exposed to valine, produce more H2S, dramatically accelerate the export of glutathione and accumulate more cysteine in the cytoplasm than their parent, which indicates that the regulatory nucleotide (p)ppGpp is involved in maintaining cysteine homeostasis. Disruption of cysteine homeostasis in gshA and relA mutants increases their sensitivity to peroxide stress.

Tannic and gallic acids alter redox-parameters of the medium and modulate biofilm formation.

Tannic (TA) and gallic (GA) acids are known to have both anti- and prooxidant properties however recently they have been described as potential anti-biofilm agents although their mechanisms of action on bacterial cells remain obscure. The aim of our research was to elucidate the role of prooxidant actions of these plant phenolic compounds in bactericidal effects and biofilm formation. In our experiments, both compounds demonstrated strong oxidative properties that altered activity of stress regulons and contributed to decrease of CFU and ability of cells to maintain membrane potential. Stimulation of biofilm formation was observed in all the strains with the exception of the strains deficient in flagella synthesis. Both compounds demonstrated bactericidal effect which was weakened in biofilms. TA efficiently killed bacteria in the bioflms of pgaA mutant which pointed out an important role of poly-beta-1,6-N-acetyl-D-glucosamine (PGA) polysaccharide in matrix formation. Similar effects of TA in recA mutant indicate involvement of SOS-response into reaction towards exposure with TA. Gallic acid-induced killing was more pronounced in the biofilms of csgA mutant revealing role of curli in protection against GA toxicity.

The sharp phase of respiratory inhibition during amino acid starvation in Escherichia coli is RelA-dependent and associated with regulation of ATP synthase activity.

Amino acid starvation causes an RelA-dependent increase in the regulatory nucleotide (p)ppGpp that leads to pleiotropic changes in Escherichia coli metabolism, but the role of (p)ppGpp in regulation of respiration remains unclear. Here we demonstrate that amino acid starvation is accompanied by sharp RelA-dependent inhibition of respiration. The sharp phase of inhibition is absent in relA mutants, and can be prevented by translation inhibitors chloramphenicol and tetracycline, which abolish accumulation of (p)ppGpp. Single knockouts of any components of the respiratory chain do not affect inhibition of respiration. Studies of dO2 changes in various atp mutants indicate that ATP synthase is probably the primary target of (p)ppGpp-mediated respiratory control. Inhibition of respiration induced by amino acid starvation is followed by transient perturbations in the membrane potential (Δψ) and K+ fluxes and leads to transient acceleration of superoxide production and H2O2 accumulation in the medium. High levels of H2O2 and superoxide formation and induced activity of antioxidant systems in the atpC mutant indicate the important role of ATP synthase in controlling the production of reactive oxygen species. The new function of (p)ppGpp, discovered here, expands the understanding of its role in metabolic reprogramming during the adaptive response to stresses.

The role of sulfides in stress-induced changes of Eh in Escherichia coli cultures.

Real-time monitoring of the state of bacterial cultures is important in both experiment and biotechnology. Using Eh and sulfide sensors, we demonstrated that the abrupt reversible reduction in Eh (Eh jump), occurring during transition of E. coli from exponential growth to starvation and antibiotic-induced stresses, is the result of sulfide excretion from the cells. Changes in the potential of sensors had a two-phase mode. The potential reduced within 10-15min and returned within 10-30min. In the parental strain, maximum amplitudes of Eh jumps (ΔEh) were 25±2mV, 57±6mV and 36±7mV under isoleucine starvation, glucose depletion and ciprofloxacin exposure that corresponded to 43±3nM, 96±5nM and 140±1nM of sulfide, respectively. In the glutathione-deficient mutant (ΔgshA), ΔEh values and sulfide concentration increased 1.5-4 times compared to the parent. Stress-induced sulfide excretion occurred in the background of inhibition of growth and respiration and a decrease in the membrane potential. The formation of sulfide caused by cysteine desulfurization may be related with maintaining of cysteine homeostasis under conditions of slow metabolism. There was a close relationship between transmembrane fluxes of sulfide, cysteine and glutathione.

Relationship between Escherichia coli growth rate and bacterial susceptibility to ciprofloxacin.

The effect of Escherichia coli growth rate on its susceptibility to ciprofloxacin was investigated using bacteria grown on different carbon sources and harboring mutations in genes encoding tricarboxylic acid cycle enzymes. A 1-h treatment of the wild type (wt) grown on glucose, succinate, malate, α-ketoglutarate or acetate with 0.3 µg ml-1 ciprofloxacin decreased the number of surviving cells (CFU ml-1), 560, 110, 74, 62 and 5 times, respectively. Among the mutants tested, sucB strain, which grew 1.75 times slower than wt, was 7.4-fold more tolerant to 0.3 µg ml-1 of ciprofloxacin than wt. Strong inverse correlations between log(CFU ml-1) after 1-h exposure to 0.3 and 3.0 µg ml-1 ciprofloxacin and the specific growth rate prior to antibiotic treatment (r = - 0.93 and -0.96, respectively) were observed. Data from the current and previous studies on the inhibitory effect of ciprofloxacin on cultures exhibiting a wide range of growth rates (0.01-1.3 h-1) were collated. Statistical analysis revealed a significant inverse correlation between log(CFU ml-1) after exposure to 3.0 µg ml-1 of ciprofloxacin and the specific bacterial growth rate prior to antibiotic exposure (r = -0.92). These data may be used in a design of antibiotic treatment protocols.

Ciprofloxacin provokes SOS-dependent changes in respiration and membrane potential and causes alterations in the redox status of Escherichia coli.

An in-depth understanding of the physiological response of bacteria to antibiotic-induced stress is needed for development of new approaches to combatting microbial infections. Fluoroquinolone ciprofloxacin causes phase alterations in Escherichia coli respiration and membrane potential that strongly depend on its concentration. Concentrations lower than the optimal bactericidal concentration (OBC) do not inhibit respiration during the first phase. A dose higher than the OBC provokes immediate SOS-independent inhibition of respiration and growth that can contribute to a decreased SOS response and lowered susceptibility to high concentrations of ciprofloxacin. Cells retain their metabolic activity, membrane potential and accelerated K+ uptake and produce low levels of superoxide and H2O2 during the first phase. The time before initiation of the second phase is inversely correlated with the ciprofloxacin concentration. The second phase is SOS-dependent and characterized by respiratory inhibition, membrane depolarization, K+ and glutathione leakage and cessation of glucose consumption and may be considered as cell death. atpA, gshA and kefBkefC knockouts, which perturb fluxes of protons and K+, can modify the degree and duration of respiratory inhibition and potassium retention. Loss of K+ efflux channels KefB and KefC enhances the susceptibility of E. coli to ciprofloxacin.

Roles of the glutathione- and thioredoxin-dependent systems in the Escherichia coli responses to ciprofloxacin and ampicillin.

Recently, it was proposed that some antibiotics stimulate the production of reactive oxygen species (ROS), which contribute to cell death. Later, other research groups have provided arguments against ROS-mediated killing of bacteria by antibiotics. At present, there remain a number of unanswered questions in understanding of the role of ROS in killing by antibiotics. Mutants of Escherichia coli in components of the thioredoxin and glutaredoxin redox pathways used in this study possess a great variability in antioxidant activity, and they therefore are a useful model for the investigation of the role of oxidative stress in bactericidal effect of antibiotics. Statistical analysis did not reveal a significant correlation between the susceptibility of the mutants to ciprofloxacin and ampicillin and their resistance to peroxide stress. However, we found strong reverse correlations between the bactericidal activity of antibiotics and the specific growth rate of these mutants at the moment of drug addition. Supplements changing the level of intra- and extracellular glutathione considerably affected E. coli susceptibility to ciprofloxacin and ampicillin. The effect of GSH precursors on bactericidal activity of antibiotics was also observed in gshA mutants.

Extracellular superoxide provokes glutathione efflux from Escherichia coli cells.

The aim of the study was to elucidate a possible relationship between transmembrane cycling of glutathione and changes in levels of external superoxide. Exposure of growing Escherichia coli to exogenous reactive oxygen species (ROS) generated by xanthine and xanthine oxidase (XO) stimulates reversible glutathione (GSH) efflux from the cells that is considerably lowered under phosphate starvation. This GSH efflux is prevented by exogenous SOD, partially inhibited by catalase, and is not dependent on the GSH exporter CydDC. The γ-glutamyl transpeptidase (GGT) deficiency completely prevents a return of GSH to the cytoplasm. In contrast to wild-type E. coli, mutants devoid of GGT and glutathione reductase (GOR) show enhanced accumulation of oxidized glutathione in the medium after exposure to xanthine and XO. Under these conditions, sodC, ggt and especially gshA mutants reveal more intensive and prolonged inhibition of growth than wild-type cells. Treatment with XO does not influence E. coli viability, but somewhat increases the number of cells with lost membrane potential. In summary, data obtained here indicate that transmembrane cycling of GSH may be involved in E. coli protection against extracellular ROS and may promote rapid growth recovery.

Medicinal plant extracts can variously modify biofilm formation in Escherichia coli.

Low concentrations of black tea and water extracts from medicinal plants Arctostaphylos uva-ursi, Vaccinium vitis-idaea, Tilia cordata, Betula pendula and Zea mays stimulated biofilm formation in Escherichia coli BW25113 up to three times. Similar effect was observed for tannic acid and low concentrations of quercetin. In contrast, the extract from Urtica dioica reduced biofilm production. Pretreatment with plant extracts variously modified antibiotic effects on specific biofilm formation (SBF). Extract from V. vitis-idaea increased SBF, while the extracts from Achillea millefolium, Laminaria japonica and U. dioica considerably decreased SBF in the presence of ciprofloxacin, streptomycin and cefotaxime. Stimulatory effect of the extracts and pure polyphenols on biofilm formation was probably related to their prooxidant properties. The rpoS deletion did not affect SBF significantly, but stimulation of biofilm formation by the compounds tested was accompanied by inhibition of rpoS expression, suggesting that a RpoS-independent signal transduction pathway was apparently used.

Medicinal plant extracts variously modulate susceptibility of Escherichia coli to different antibiotics.

Antioxidant activity of green and black tea and extracts of medicinal plants and their ability to modulate antibiotic susceptibility in Escherichia coli were studied. Among a number of extracts tested the maximal capacity to scavenge DPPH radicals and chelate iron in chemical tests was found in green and black tea, Arctostaphylos uva-ursi and Vaccinium vitis-idaea. These extracts contained high level of polyphenols and in aerobic conditions exhibited prooxidant features, producing H2O2 and inducing expression of the katG gene encoding catalase HPI in E. coli cells. A good correlation between the polyphenol content and the ability of extracts to protect bacteria against peroxide stress was observed (r = 0.88). Polyphenol-rich extracts and iron chelators demonstrated the highest modulating effect on the antibiotic susceptibility by changing the time period before lysis started and by influencing the colony-forming ability of bacteria. The direction of the modulating effect was dependent on nature of antibiotic applied: under treatment with ciprofloxacin and ampicillin the extracts predominantly provided protective effects, while under treatment with kanamycin a bactericidal action was enhanced. Mechanism of modulating action of extracts on bacterial antibiotic susceptibility probably involves antioxidant, preferentially iron-chelating, or prooxidant properties of polyphenols.

Transmembrane glutathione cycling in growing Escherichia coli cells.

Glutathione (GSH) plays an important role in bacterial cells, participating in maintenance of redox balance in the cytoplasm and in defense against many toxic compounds and stresses. In this study we demonstrate that in aerobic, exponentially growing Escherichia coli culture endogenous reduced glutathione undergoes continuous transmembrane cycling between the cells and medium. As a result of an establishment of a dynamic balance between GSH efflux and uptake, a constant extracellular concentration of GSH counting per biomass unit is maintained. The magnitude of this concentration strictly depends on external pH. GSH cycling is carried out in respiring cells and disturbed by influences, which change the level of ΔµH(+) and ATP. Export of GSH is modified by phosphate deficiency in the medium.