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.

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 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.

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.

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.