Noncytotoxic zinc-doped nanohydroxyapatite-based bone scaffolds with strong bactericidal, bacteriostatic, and antibiofilm activity.

Development of bone scaffolds that are nontoxic to eukaryotic cells, while revealing bactericidal activity still remains a huge challenge for the scientific community. It should be noted that only bacteriostatic (the ability of the biomaterial to inhibit the growth of bacteria) and bactericidal (the ability to kill >99.9 % bacteria) activities have clinical importance. Unfortunately, many material scientists are confused with the microbiological definition of antibacterial action and consider biomaterials causing reduction in colony-forming units (CFUs) by 50-80 % as promising antibacterial implants. The aim of this study was to synthesize three variants of Zn-doped hydroxyapatite (HA) nanopowder, which were characterized by different content of Zn2+ and served as a powder phase for the production of novel macroporous chitosan/agarose/nanoHA biomaterials with high antibacterial activity. Within this study, it was proven that the scaffold with a low zinc content (doping level 0.03 mol for 1 mol of HA; 0.2 wt%) revealed the gradual and slow release of the Zn2+ ions, preventing against accumulation of high and toxic concentration of therapeutic agents and providing prolonged antibacterial activity. Moreover, developed biomaterial was nontoxic to human osteoblasts and showed anti-biofilm properties, bactericidal activity (> 99.9 % of bacteria killed) against Staphylococcus epidermidis and Escherichia coli, significant antibacterial activity against Staphylococcus aureus (98.5 % of bacteria killed), and also bacteriostatic activity against Pseudomonas aeruginosa. Thus, the developed Zn-doped HA-based bone scaffold has excellent antibacterial properties without toxicity against eukaryotic cells, being a promising biomaterial for biomedical applications to repair bone defects and prevent post-surgery infections.

Untargetted Metabolomic Exploration of the Mycobacterium tuberculosis Stress Response to Cinnamon Essential Oil.

The antimycobacterial activity of cinnamaldehyde has already been proven for laboratory strains and for clinical isolates. What is more, cinnamaldehyde was shown to threaten the mycobacterial plasma membrane integrity and to activate the stress response system. Following promising applications of metabolomics in drug discovery and development we aimed to explore the mycobacteria response to cinnamaldehyde within cinnamon essential oil treatment by untargeted liquid chromatography-mass spectrometry. The use of predictive metabolite pathway analysis and description of produced lipids enabled the evaluation of the stress symptoms shown by bacteria. This study suggests that bacteria exposed to cinnamaldehyde could reorganize their outer membrane as a physical barrier against stress factors. They probably lowered cell wall permeability and inner membrane fluidity, and possibly redirected carbon flow to store energy in triacylglycerols. Being a reactive compound, cinnamaldehyde may also contribute to disturbances in bacteria redox homeostasis and detoxification mechanisms.

Design, synthesis and antimycobacterial activity of thiazolidine-2,4-dione-based thiosemicarbazone derivatives.

The two series of thiosemicarbazone derivatives with thiazolidine-2,4-dione (TZD) core were designed and synthesized. The antimycobacterial activity of the target compounds was tested against Mycobacterium tuberculosis H37Ra by broth microdilution method with resazurin as an indicator of the metabolic activity of mycobacteria. Conducted studies revealed antimycobacterial activity in the concentration range of 0.031-64 µg/ml for 31 synthesized derivatives with TZD core. The highest antimycobacterial activity (MIC = 0.031-0.125 µg/ml) was demonstrated for the new group of compounds: TZD-based hybrids with 4-unsubstituted thiosemicarbazone substituent. Furthermore, all the tested compounds within this group were characterized by low cytotoxicity. Among tested compounds, two compounds are the most promising potential antimycobacterial agents since they not only show very low MIC values, but also non-toxicity against Vero cells at tested concentration range. High effectiveness and safety of these synthesized compounds makes them promising candidates as antimycobacterial agents.

Synthesis and antimycobacterial activity of thiazolidine-2,4-dione based derivatives with halogenbenzohydrazones and pyridinecarbohydrazones substituents.

The two series of thiazolidine-2,4-dione (TZD) based hybrids with halogenbenzohydrazones and pyridinecarbohydrazones substituents were designed and synthesized. Target hydrazones were evaluated for their antimycobacterial activity by broth microdilution method with resazurin as an indicator of the metabolic activity of mycobacteria. Conducted studies revealed antimycobacterial activity in the concentration range of 1-512 µg/ml for 23 synthesized TZD-based derivatives. The highest antimycobacterial activity (MIC = 1 µg/ml) was demonstrated for the new group of compounds: TZD-based derivatives with pyridine-4-carbohydrazone substituent. Furthermore, all the tested compounds within this group were characterized by low cytotoxicity. On the basis of the results obtained, three compounds with the highest SI were selected. High effectiveness and safety of these synthesized derivatives makes them promising candidates as antimycobacterial agents.

Antimycobacterial Activity of Cinnamaldehyde in a Mycobacterium tuberculosis(H37Ra) Model.

The purpose of the study was to evaluate the antimycobacterial activity and the possible action mode of cinnamon bark essential oil and its main constituent-cinnamaldehyde-against the Mycobacterium tuberculosis ATCC 25177 strain. Cinnamaldehyde was proved to be the main bioactive compound responsible for mycobacterial growth inhibition and bactericidal effects. The antimycobacterial activity of cinnamaldehyde was found to be comparable with that of ethambutol, one of the first-line anti-TB antibiotics. The selectivity index determined using cell culture studies in vitro showed a high biological potential of cinnamaldehyde. In M. tuberculosis cells exposed to cinnamaldehyde the cell membrane stress sensing and envelope preserving system are activated. Overexpression of clgR gene indicates a threat to the stability of the cell membrane and suggests a possible mechanism of action. No synergism was detected with the basic set of antibiotics used in tuberculosis treatment: ethambutol, isoniazid, streptomycin, rifampicin, and ciprofloxacin.

Nigella damascena L. Essential Oil-A Valuable Source of ß-Elemene for Antimicrobial Testing.

The most commonly used plant source of ß-elemene is Curcuma wenyujin Y. H. Chen & C. Ling (syn. of Curcuma aromatic Salisb.) with its content in supercritical CO2 extract up to 27.83%. However, the other rich source of this compound is Nigella damascena L. essential oil, in which ß-elemene accounts for 47%. In this work, the effective protocol for preparative isolation of ß-elemene from a new source-N. damascena essential oil-using high performance counter-current chromatography HPCCC was elaborated. Furthermore, since sesquiterpens are known as potent antimicrobials, the need for finding new agents designed to combat multi-drug resistant strains was addressed and the purified target compound and the essential oil were tested for its activity against a panel of Gram-positive and Gram-negative bacteria, fungi, and mycobacterial strains. The application of the mixture of petroleum ether, acetonitrile, and acetone in the ratio 2:1.5:0.5 (v/v) in the reversed phase mode yielded ß-elemene with high purity in 70 min. The results obtained for antimicrobial assay clearly indicated that N. damascena essential oil and isolated ß-elemene exert action against Mycobacterium tuberculosis strain H37Ra.

The frequently occurring components of essential oils beta elemene and R-limonene alter expression of dprE1 and clgR genes of Mycobacterium tuberculosis H37Ra.

In the past few years, there has been a significant increase in detection of drug resistant strains of Mycobacterium tuberculosis. Search for new antimycobacterial drugs brought natural sources with their chemical diversity in focus. Especially essential oils, produced by plants also for toxic effect, are reservoir of potentially antitubercular compounds. In the present work, we exposed M. tuberculosis H37Ra ATCC 25177 strain to some terpenes commonly occurring in essential oils. Gene expression profiling was used to explore possible influence of these compounds on stress sensing and envelope preserving function. Expression of two genes dprE1 involved in cell wall synthesis and clgR responsible for regulation of cell membrane preservation was investigated. We report that two out of five tested compounds: ß-elemene and R-limonene alter expression of dprE1 and clgR genes. These findings indicate various mechanisms of action of essential oils compounds on M. tuberculosis. Especially the clgR expression seemed to be the perfect marker of stress sensing and envelope preserving systems status.

Quantitative analysis of biofilm formed on vascular prostheses by Staphylococcus epidermidis with different ica and aap genetic status.

OBJECTIVES: This study aims to examine biofilm formed on vascular prostheses by Staphylococcus epidermidis with different ica and aap genetic status, and to evaluate the effect of antibiotic-modified prostheses on bacterial colonization. METHODS: Biofilm formation was determined using fluorescence microscopy imaging. Quantitative analysis was conducted using the biofilm coverage ratio (BCR) calculations. RESULTS: Our investigations prove that the BCR method with fluorescent dye enabled an accurate assessment of biofilm coverage and comparison of the obtained results. The ica+ aap+ strains formed a biofilm on all of the examined vascular prostheses. Uni-Graft(®) modified with covalently immobilized amikacin was effective in preventing bacterial adherence. CONCLUSIONS: Molecular biology techniques combined with phenotype studies give a broad insight into biofilm formation mechanisms. On the other hand, fluorescence microscopy imaging along with BCR calculations are reliable and simple tools to quantitatively estimate biofilm formation, as well as the effectiveness of antimicrobial prosthesis modification.

Characterization of the developed antimicrobial urological catheters.

Antimicrobial urological catheters were developed by the mixed, covalent and non-covalent binding of sparfloxacin (SPA) to heparin (HP) film which was first deposited on the latex surface of biomaterial. The SPA-HP modified surface was characterized by SEM analysis and ATR-Fourier transform infrared spectroscopy. For the antimicrobial prevention, SPA as an antibiotic with a broad antimicrobial spectrum was chosen. Antimicrobial activity of antibiotic-modified catheter against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli strains was assessed using various procedures. On the basis of the inhibition zone and diffusion assays the efficacy around the modified catheters was demonstrated. The test samples clearly showed an antibacterial activity against all tested bacterial stains for a least one month. Inhibition of the bacterial colonization on the modified catheter surface was proved by the biofilm test. Antimicrobial activity of SPA-treated catheter surface was also quantitatively evaluated according to standard method of ISO based on JIS. The R-values were found to be higher than 3.8. The performed research indicated that the immobilization of SPA on the catheter surface by means of the mixed-type bonds resulted in stable antibacterial protection of the urological catheters for a long time.