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.

Synthesis and Preclinical Evaluation of Small-Molecule Prostate-Specific Membrane Antigen-Targeted Abiraterone Conjugate.

Prostate cancer is the second most common type of cancer among men. The main method of its treatment is androgen deprivation therapy, which has a wide range of side effects. One of the solutions to this challenge is the targeted delivery of drugs to prostate cancer cells. In this study, we performed the synthesis of a novel small-molecule PSMA-targeted conjugate based on abiraterone. Cytotoxicity, the induction of intracellular reactive oxygen species, and P450-cytochrome species inhibition were investigated for this conjugate PSMA-abiraterone. The conjugate demonstrated a preferential effect on prostate tumor cells, remaining inactive at up to 100 µM in human fibroblast cells. In addition, it revealed preferential efficacy, specifically on PSMA-expressing lines with a 65% tumor growth inhibition level on 22Rv1 (PSMA+) xenografts after 14-fold oral administration of PSMA-Abi at a single dose of 500 mg/kg (7.0 g/kg total dose) was observed. This compound showed significantly reduced acute toxicity with comparable efficacy compared to AbiAc.

A novel glycosylated indolocarbazole derivative LCS1269 effectively inhibits growth of human cancer cells in vitro and in vivo through driving of both apoptosis and senescence by inducing of DNA damage and modulating of AKT/mTOR/S6K and ERK pathways.

In recent decades, indolocarbazole glycosides containing sugar moieties have attracted attention due to their diverse anti-tumor activities. In the present study, a series of new indolo [2,3-a]pyrrolo [3,4-c]carbazole derivatives were synthesized for the first time. First of all, we have shown that compound 6e (LCS1269) had the most pronounced effect on inhibiting tumor growth in the transferable solid and non-solid murine tumors as compared with other synthesized indolocarbazole derivatives. The results of the in vivo nude mice xenoraft study also confirmed that LCS1269 treatment strongly suppressed the growth of human colon cancer SW620 xenografts. It is important to note that the antiproliferative activity of LCS1269 against three human cancer cell lines (MCF-7, HCT-116 and A549) was considerably higher than that against the non-tumor cell lines (immortalized breast cells and normal embryonic fibroblasts). Furthermore, the treatment of MCF-7, HCT-116 and A549 cells with LCS1269 caused the statistically significant inhibition of anchorage-dependent and anchorage-independent colony formation. We further revealed that LCS1269 treatment of investigated human cancer cells resulted in the DNA damage and G2/M cell cycle arrest followed by the decrease of mitochondrial membrane potential with subsequent initiation of intrinsic apoptosis and the triggering of senescence via p53-dependent mechanisms. In addition, our western blotting findings and molecular docking data suppose that LCS1269 could at least partially attenuate cancer cells growth by modulation of AKT/mTOR/S6K and ERK signaling pathways. Therefore, we concluded that LCS1269 might be the promising compound for implementation and probable use in the clinical practice.

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.

PSMA-targeted small-molecule docetaxel conjugate: Synthesis and preclinical evaluation.

Prostate cancer is one of the most commonly diagnosed men's cancers and remains one of the leading causes of cancer death. The development of approaches to the treatment of this oncological disease is an ongoing process. In this work, we have carried out the selection of ligands for the creation of conjugates based on the drug docetaxel and synthesized a series of three docetaxel conjugates. In vitro cytotoxicity of these molecules was evaluated using the MTT assay. Based on the assay results, we selected the conjugate which showed cytotoxic potential close to unmodified docetaxel. At the same time, the molar solubility of the resulting compound increased up to 20 times in comparison with the drug itself. In vivo evaluation on 22Rv1 (PSMA+) xenograft model demonstrated a good potency of the synthesized conjugate to inhibit tumor growth: the inhibition turned out to be more than 80% at a dose of 30 mg/kg. Pharmacokinetic parameters of conjugate distribution were analyzed. Also, it was found that PSMA-targeted docetaxel conjugate is less toxic than docetaxel itself, the decrease of molar acute toxicity in comparison with free docetaxel was up to 20%. Obtained conjugate PSMA-DOC is a good candidate for further expanded preclinical trials because of high antitumor activity, fewer side toxic effects and better solubility.

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.

Synthesis, Characterization, and Preclinical Evaluation of a Small-Molecule Prostate-Specific Membrane Antigen-Targeted Monomethyl Auristatin E Conjugate.

Prostate cancer is the second most common type of cancer among men. Its main method of treatment is chemotherapy, which has a wide range of side effects. One of the solutions to this challenge is targeted delivery to prostate cancer cells. Here we synthesized a novel small-molecule PSMA-targeted conjugate based on the monomethyl auristatin E. Its structure and conformational properties were investigated by NMR spectroscopy. Cytotoxicity, intracellular reactive oxygen species induction, and stability under liver microsomes and P450-cytochrome species were investigated for this conjugate. The conjugate demonstrated 77-85% tumor growth inhibition levels on 22Rv1 (PSMA (+)) xenografts, compared with a 37% inhibition level on PC-3 (PSMA (-)) xenografts, in a single dose of 0.3 mg/kg and a sufficiently high therapeutic index of 21. Acute, chronic, and subchronic toxicities and pharmacokinetics have shown that the synthesized conjugate is a promising potential agent for the chemotherapy of prostate cancer.

Synthesis and Biological Evaluation of PSMA Ligands with Aromatic Residues and Fluorescent Conjugates Based on Them.

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is a suitable target for specific delivery of antitumor drugs and diagnostic agents due to its overexpression in prostate cancer cells. In the current work, we describe the design, synthesis, and biological evaluation of novel low-molecular PSMA ligands and conjugates with fluorescent dyes FAM-5, SulfoCy5, and SulfoCy7. In vitro evaluation of synthesized PSMA ligands on the activity of PSMA shows that the addition of aromatic amino acids into a linker structure leads to a significant increase in inhibition. The conjugates of the most potent ligand with FAM-5 as well as SulfoCy5 demonstrated high affinities to PSMA-expressing tumor cells in vitro. In vivo biodistribution in 22Rv1 xenografts in Balb/c nude mice of PSMA-SulfoCy5 and PSMA-SulfoCy7 conjugates with a novel PSMA ligand demonstrated good visualization of PSMA-expressing tumors. Also, the conjugate PSMA-SulfoCy7 demonstrated the absence of any explicit toxicity up to 87.9 mg/kg.

Pharmacophore hybridization approach to discover novel pyrazoline-based hydantoin analogs with anti-tumor efficacy.

In search for new and safer anti-cancer agents, a structurally guided pharmacophore hybridization strategy of two privileged scaffolds, namely diaryl pyrazolines and imidazolidine-2,4-dione (hydantoin), was adopted resulting in a newfangled series of compounds (H1-H22). Herein, a bio-isosteric replacement of "pyrrolidine-2,5-dione" moiety of our recently reported antitumor hybrid incorporating diaryl pyrazoline and pyrrolidine-2,5-dione scaffolds with "imidazoline-2,4-dione" moiety has been incorporated. Complete biological studies revealed the most potent analog among all i.e. compound H13, which was at-least 10-fold more potent compared to the corresponding pyrrolidine-2,5-dione, in colon and breast cancer cells. In-vitro studies showed activation of caspases, arrest of G0/G1 phase of cell cycle, decrease in the expression of anti-apoptotic protein (Bcl-2) and increased DNA damage. In-vivo assay on HT-29 (human colorectal adenocarcinoma) animal xenograft model unveiled the significant anti-tumor efficacy along with oral bioavailability with maximum TGI 36% (i.p.) and 44% (per os) at 50 mg/kg dose. These findings confirm the suitability of hybridized pyrazoline and imidazolidine-2,4-dione analog H13 for its anti-cancer potential and starting-point for the development of more efficacious analogs.

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.

Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents.

In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, in vitro and in vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC50 =0.78±0.01 µM), HT29 (IC50 =0.92±0.15 µM) and K562 (IC50 =47.25±1.24 µM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G1 /G0 phase and decreased cell population in G2 /M phase. The excellent in vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50 mg kg-1 dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents.

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.

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.

Binding characterization of the targeting drug AIMPILA to AFP receptors in human tumor xenografts.

The main objective of this study was the characterization of preclinical tumor models based on their expression of alpha-fetoprotein receptor (RECAF) for targeting cancer cells with a new non-covalent complex (AIMPILA) containing alpha-fetoprotein as the carrier and Atractyloside as an apoptosis-inducing agent. For that purpose, we measured the amount of RECAF in the homogenates of the grafted tumors T47D and SW620 and in HepG2 cell extracts. We also determined the alpha-fetoprotein binding specificity of the targeting drug AIMPILA using a solid-phase chemiluminescent assay with AIMPILA-Acrdidinium. We found that RECAF is practically absent from healthy mice tissues (100 Units/mg) where in malignant cells, the amount of alpha-fetoprotein receptors follows this order: T47D (9152 Units/mg) > HepG2 (4865 Units/mg) > SW620 (2839 Units/mg). This agrees with our findings regarding AIMPILA-induced tumor growth inhibition (T47D (T/C = 22%) > HepG2 (T/C = 51%) > SW620 (T/C = 70%), where T/C is the ratio of tumor volume in treated vs control animals). Our results demonstrate that the therapeutic response to the targeting drug AIMPILA strongly depends on the RECAF expression by human tumors and confirms the choice of the tumor models used for an AIMPILA preclinical study.

New Poly(3-hydroxybutyrate) Microparticles with Paclitaxel Sustained Release for Intraperitoneal Administration.

BACKGROUND: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations. OBJECTIVE: This study was conducted to develop new prolong paclitaxel (PTX) formulation based on poly(3- hydroxybutyrate) (PHB) microparticles. METHOD: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The PTX release kinetics in vitro from PHB microparticles and their cytotoxity on murine hepatoma cell line MH-22a were studied. Microparticles antitumor activity in vivo was studied using intraperitoneally (i.p.) transplanted tumor models: murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. RESULTS: Uniform PTX release from PHB-microparticles during 2 months was observed. PTX-loaded PHB microparticles have demonstrated a significant antitumor activity versus pure drug both in vitro in murine hepatoma cells and in vivo when administered i.p. to mice with murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. CONCLUSION: The developed technique of PTX sustained delivery from PHB-microparticles has therapeutic potential as prolong anticancer drug formulation.

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.