Thermophilic bacteria of four genera in contrast to the commonly used production strains such as Bacillus subtilis, produce homologs other than menaquinone (MK) with seven isoprene units. The number of isoprene units and the configuration of double bonds are essential factors for their biological activity. The goal was to obtain a strain of bacteria that produces a wide range of MK homologs and only all-trans geometrical isomers, which was the strain G. kaustophilus. Using off-line two-dimensional LC-tandem MS in columns with the RP18 phase and the COSMOSIL cholester phase (separation according to the geometric configuration of double bonds) it was shown that thermophilic bacteria grown at different temperatures produce only all-trans isomers of menaquinones from MK-5 (menaquinone with five isoprenyl units) to MK-15 (fifteen isoprenyl units). Therefore, G. kaustophilus appears to be a biotechnologically important strain produces only trans isomers and additionally homologs from 5 to 15 isoprene units.
- MeSH
- Bacteria * MeSH
- butadieny * MeSH
- hmotnostní spektrometrie MeSH
- vitamin K 2 chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Vitis vinifera canes are waste material of grapevine pruning and thus represent cheap source of high-value polyphenols. In view of the fact that resistance of many pathogenic microorganisms to antibiotics is a growing problem, the antimicrobial activity of plant polyphenols is studied as one of the possible approaches. We have investigated the total phenolic content, composition, antioxidant activity, and antifungal activity against Candida biofilm of an extract from winter canes and a commercially available extract from blue grapes. Light microscopy and confocal microscopy imaging as well as crystal violet staining were used to quantify and visualize the biofilm. We found a decrease in cell adhesion to the surface depending on the concentration of resveratrol in the cane extract. The biofilm formation was observed as metabolic activity of Candida albicans, Candida parapsilosis and Candida krusei biofilm cells and the minimum biofilm inhibitory concentrations were determined. The highest inhibition of metabolic activity was observed in Candida albicans biofilm after treatment with the cane extract (30 mg/L) and blue grape extract (50 mg/L). The composition of cane extract was analyzed and found to be comparatively different from blue grape extract. In addition, the content of total phenolic groups in cane extract was three-times higher (12.75 gGA/L). The results showed that cane extract was more effective in preventing biofilm formation than blue grape extract and winter canes have proven to be a potential source of polyphenols for antimicrobial and antibiofilm treatment.
- Publikační typ
- časopisecké články MeSH
A combination of two chromatographic and two enzymatic methods was used for the analysis of molecular species of lipids from Gram-positive bacteria of the genus Kocuria. Gram-positive bacteria contain a majority of branched fatty acids (FAs), especially iso- and/or anteiso-FAs. Two strains K. rhizophila were cultivated at three different temperatures (20, 28, and 37°C) and the majority phospholipid, i.e., the mixture of molecular species of phosphatidylglycerols (PGs) was separated by means of hydrophilic interaction liquid chromatography (HILIC). After enzymatic hydrolysis of PGs by phospholipase C and derivatization of the free OH group, the sn-1,2-diacyl-3-acetyl triacylglycerols (AcTAGs) were separated by reversed phase HPLC. Molecular species such as i-15:0/i-15:0/2:0, ai-15:0/ai-15:0/2:0, and 15:0/15:0/2:0 (straight chains) were identified by liquid chromatography-positive electrospray ionization mass spectrometry. The tandem mass spectra of both standards and natural compounds containing iso, anteiso and straight chain FAs with the same carbons were identical. Therefore, for identification of the ratio of two regioisomers, i.e. i-15:0/ai-15:0/2:0 vs. ai-15:0/i-15:0/2:0, they were cleavage by pancreatic lipase. The mixture of free fatty acids (FFAs) and 2-monoacylglycerols (2-MAGs) was obtained. After their separation by TLC and esterification and/or transesterification, the fatty acid methyl esters were quantified by GC-MS and thus the ratio of regioisomers was determined. It has been shown that the ratio of PG (containing as majority i-15: 0 / i-15: 0, i-15: 0 / ai-15: 0 and / or ai-15: 0 / i-15: 0 and ai-15: 0 / ai-15: 0 molecular species) significantly affected the membrane flow of bacterial cells cultured at different temperatures.
- MeSH
- chemické techniky analytické metody MeSH
- chromatografie kapalinová * MeSH
- diglyceridy chemie izolace a purifikace MeSH
- fosfolipidy chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * MeSH
- hydrofobní a hydrofilní interakce MeSH
- mastné kyseliny chemie MeSH
- Micrococcaceae chemie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient's course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.
- Publikační typ
- kazuistiky MeSH
PURPOSE: With the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with 68Ga for imaging of bacterial infections. METHODS: In vitro characterization of [68Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [68Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens. RESULTS: DFO-B was labelled with 68Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [68Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [68Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [68Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters. CONCLUSION: DFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [68Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.
- MeSH
- deferoxamin * MeSH
- myši MeSH
- PET/CT MeSH
- počítačová rentgenová tomografie MeSH
- pozitronová emisní tomografie MeSH
- radioizotopy galia * MeSH
- Staphylococcus aureus MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plasmalogens are a group of lipids mainly found in the cell membranes. They occur in anaerobic bacteria and in some protozoa, invertebrates and vertebrates, including humans. Their occurrence in plants and fungi is controversial. They can protect cells from damage by reactive oxygen species, protect other phospholipids or lipoprotein particles against oxidative stress, and have been implicated as signaling molecules and modulators of membrane dynamics. Biosynthesis in anaerobic and aerobic organisms occurs by different pathways, and the main biosynthetic pathway in anaerobic bacteria was clarified only this year (2021). Many different analytical techniques have been used for plasmalogen analysis, some of which are detailed below. These can be divided into two groups: shotgun lipidomics, or electrospray ionization mass spectrometry in combination with high performance liquid chromatography (LC-MS). The advantages and limitations of both techniques are discussed here, using examples from anaerobic bacteria to specialized mammalian (human) organs.
Cardiolipins (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol) (CLs) are widespread in many organisms, from bacteria to higher green plants and mammals. CLs were observed in Gram-positive bacterium of the genus Kocuria, brewer's yeast Saccharomyces, the green alga Chlamydomonas, spinach and beef heart. A mixture of molecular species of CLs was obtained from total lipids by hydrophilic interaction liquid chromatography (HILIC), and these were further separated and identified by reversed phase LC/MS with negative tandem electrospray ionization. The majority of CLs molecular species from each organism were cleaved using phospholipase C from Bacillus cereus. This phospholipase cleaves CLs into 1,2-diglycerols and phosphatidylglycerol 3-phosphates, which were then separated. After CLs cleavage, diacylglycerols such as sn-1,2-diacyl-3-acetyl-glycerols (i.e., triacylglycerols) were separated and identified by chiral chromatography/MS-positive tandem ESI. Significant differences in the composition of the molecular species between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of CLs were found in all organisms tested. Molecular species of CLs that contained four different fatty acids were identified in all five samples, and CLs containing very long chain fatty acids were identified in yeast. In addition, CLs containing both enantiomers (at the sn-2 carbon) were present in the bacterium tested. These findings were further supported by data already published in GenBank where, in the same family - Micrococcaceae - both enzymes responsible for chirality in the sn-2 position, glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases, were present.
- MeSH
- chemická frakcionace MeSH
- Chlamydomonas reinhardtii chemie MeSH
- chromatografie kapalinová metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- hydrolýza MeSH
- kardiolipiny chemie MeSH
- mastné kyseliny analýza MeSH
- skot MeSH
- stereoizomerie MeSH
- triglyceridy chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Ecosystems worldwide are exposed to pollutants connected to the industrial production of pharmaceuticals. The objective of this study was to study the composition and characteristics of the soil microbial communities that had been exposed to long-term selection pressure caused by the industrial production of penicillin G. Soil samples from four sites among the penicillin G production plant were analysed using 16S rRNA profiling via Illumina MiSeq platform and were compared with the control samples from four sites outside the plant. Total metagenomic DNA from the impacted soil was also used for the preparation of E. coli T1R-based fosmid library which was consequently qualitatively tested for the presence of penicillin G acylase (PGA)-encoding genes using the method of sequence homology. Analyses of alpha diversity revealed that the long-term antibiotic presence in the soil significantly increased the microbial diversity and richness in terms of Shannon diversity index (p = 0.002) and Chao estimates (p = 0.004). Principal component analysis showed that the two types of communities (on-site and control) could be separated at the phylum, class and genus level. The on-site soil was enriched in Betaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetia, while a significant decrease in Actinobacteria was observed. Metagenomic fosmid library revealed high hit rates in identifying PGAs (14 different genes identified) and confirmed the biotechnological potential of soils impacted by anthropogenic activity. This study offers new insights into the changes in microbial communities of soils exposed to anthropogenic activity as well as indicates that those soils may represent a hotspot for biotechnologically interesting targets.
- MeSH
- antibakteriální látky biosyntéza MeSH
- Bacteria klasifikace genetika izolace a purifikace metabolismus MeSH
- biodiverzita MeSH
- DNA bakterií genetika MeSH
- Escherichia coli genetika MeSH
- fylogeneze MeSH
- látky znečišťující půdu MeSH
- metagenom MeSH
- metagenomika MeSH
- mikrobiota * genetika MeSH
- průmyslová mikrobiologie MeSH
- půda MeSH
- půdní mikrobiologie * MeSH
- RNA ribozomální 16S genetika MeSH
- Publikační typ
- časopisecké články MeSH
