Sublethal Photodynamic Treatment Does Not Lead to Development of Resistance

A promising new alternative approach for eradication of antibiotic-resistant strains is to expose microbes to photosensitizers, which upon illumination generate reactive oxygen species. Among the requirements for a potent, medically applicable photosensitizer, are high efficacy in killing microbes and low toxicity to the host. Since photodynamic treatment is based on production of reactive species which are potentially DNA damaging and mutagenic, it might be expected that under selective pressure, microbes would develop resistance. The aim of this study was to determine if antibacterial photodynamic treatment with a highly photoefficient photosensitizer, Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin would lead to development of resistance. To answer that question, bacterial cultures were subjected to multiple cycles of sublethal photodynamic stress and regrowth, and to continuous growth under photodynamic exposure. Antibiotic-resistant Staphylococcus aureus and Escherichia coli clinical isolates were also tested for susceptibility to photodynamic inactivation and for development of resistance. Results demonstrated that multiple photodynamic exposures and regrowth of surviving cells or continuous growth under sublethal photodynamic conditions, did not lead to development of resistance to photosensitizers or to antibiotics. Antibiotic-resistant E. coli and S. aureus were as sensitive to photodynamic killing as were their antibiotic-sensitive counterparts and no changes in their sensitivity to antibiotics or to photodynamic inactivation after multiple cycles of photodynamic treatment and regrowth were observed. In conclusion, photosensitizers with high photodynamic antimicrobial efficiency can be used successfully for eradication of antibiotic-resistant bacterial strains without causing development of resistance

Effect of growth media on the MTT colorimetric assay in bacteria.

Reduction of tetrazolium salts to colored formazan products by metabolically active cells is widely used for assessment of cell viability. Among the tetrazolium compounds most commonly used is MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Numerous studies about sites and mechanisms of cellular reduction of MTT, performed in mammalian cell cultures, have identified various parameters that affect formazan production and can lead to overestimation/underestimation of viable cells or effects of treatment. Irrespective of lack of such data for prokaryotic cells, the MTT assay is commonly used for microbiological studies, which often leads to contradictory results or misinterpretation of data. The aim of this study was to investigate how components of growth media and conditions of growth, affect formazan formation by microbial cells. Results showed that MTT reduction depended on the amino acid composition of the medium. Several amino acids potentiated formazan production by Gram-positive and Gram-negative bacteria, with histidine having the strongest effect. Results of this study demonstrate that data obtained with the MTT test should be interpreted with caution, particularly when different growth media are used or treatments affect metabolic pathways, and that evaluation of the reliability of the MTT assay under specific conditions should be performed, to avoid erroneous results. Performing the assay with cells suspend in glucose-supplemented buffer would eliminate the effects of metabolites and will limit cell division during incubation with MTT. Another critical element to be considered is the choice of a proper solvent for dissolution of formazan crystals

Role of rpoS in the regulation of glyoxalase III in Escherichia coli.

Methylglyoxal is an endogenous electrophile produced in Escherichia coli by the enzyme methylglyoxal synthase to limit the accumulation of phosphorylated sugars. In enteric bacteria methylglyoxal is detoxified by the glutathione-dependent glyoxalase I/II system, by glyoxalase III, and by aldehyde reductase and alcohol dehydrogenase. Here we demonstrate that glyoxalase III is a stationary-phase enzyme. Its activity reached a maximum at the entry into the stationary phase and remained high for at least 20 h. An rpoS- mutant displayed normal glyoxalase I and II activities but was unable to induce glyoxalase III in stationary phase. It thus appears that glyoxalase III is regulated by rpoS and might be important for survival of non-growing E. coli cultures

Simple biological systems for assessing the activity of superoxide dismutase mimics

Significance: Half a century of research provided unambiguous proof that superoxide and species derived from it - reactive oxygen species (ROS) - play a central role in many diseases and degenerative processes. This stimulated the search for pharmaceutical agents that are capable of preventing oxidative damage, and methods of assessing their therapeutic potential. Recent Advances: The limitations of superoxide dismutase (SOD) as a therapeutic tool directed attention to small molecules, SOD mimics, that are capable of catalytically scavenging superoxide. Several groups of compounds, based on either metal complexes, including metalloporphyrins, metallocorroles, Mn(II) cyclic polyamines, and Mn(III) salen derivatives, or non-metal based compounds, such as fullerenes, nitrones, and nitroxides, have been developed and studied in vitro and in vivo. Very few entered clinical trials. Critical Issues and Future Directions: Development of SOD mimics requires in-depth understanding of their mechanisms of biological action. Elucidation of both molecular features, essential for efficient ROS-scavenging in vivo, and factors limiting the potential side effects requires biologically relevant and, at the same time, relatively simple testing systems. This review discuses the advantages and limitations of genetically engineered SOD-deficient unicellular organisms, Escherichia coli and Saccharomyces cerevisiae as tools for investigating the efficacy and mechanisms of biological actions of SOD mimics. These simple systems allow the scrutiny of the minimal requirements for a functional SOD mimic: the association of a high catalytic activity for superoxide dismutation, low toxicity, and an efficient cellular uptake/biodistribution. Antioxid. © Copyright 2014, Mary Ann Liebert, Inc. 2014

Antibacterial Activity of Synthetic Cationic Iron Porphyrins

Widespread antibiotic resistance demands new strategies for fighting infections. Porphyrin-based compounds were long ago introduced as photosensitizers for photodynamic therapy, but light-independent antimicrobial activity of such compounds has not been systematically explored. The results of this study demonstrate that synthetic cationic amphiphilic iron -alkylpyridylporphyrins exert strong bactericidal action at concentrations as low as 5 μM. Iron porphyrin, FeTnHex-2-PyP, which is well tolerated by laboratory animals, efficiently killed Gram-negative and Gram-positive microorganisms. Its bactericidal activity was oxygen-independent and was controlled by the lipophilicity and accumulation of the compound in bacterial cells. Such behavior is in contrast with the anionic gallium protoporphyrin IX, whose efficacy depends on cellular heme uptake systems. Under aerobic conditions, however, the activity of FeTnHex-2-PyP was limited by its destruction due to redox-cycling. Neither iron released from the Fe-porphyrin nor other decomposition products were the cause of the bactericidal activity. FeTnHex-2-PyP was as efficient against antibiotic-sensitive and as against their antibiotic-resistant counterparts. Our data demonstrate that development of amphiphilic, positively charged metalloporphyrins might be a promising approach in the introduction of new weapons against antibiotic-resistant strains