Renata Los

A comparative analysis of phenotypic and genotypic methods for the determination of the biofilm-forming abilities of Staphylococcus epidermidis

The collection of 146 Staphylococcus epidermidis strains isolated from the nasopharynx of lung cancer patients has been studied for the ability of slime secretion and biofilm formation using the Congo red agar (CRA) test and the microtiter plate (MtP) method, respectively. The prevalence of the icaAD and the aap genes was also analyzed. Some isolates (35.6%) were biofilm positive by the MtP method, while 58.9% of isolates exhibited a slime-positive phenotype by the CRA test. The sensitivities of the CRA test evaluated using the MtP method as a gold standard of biofilm production were 73.1%, 97.3% and 13.3% for all the strains screened, ica-positive and ica-negative strains, respectively. The genotype ica(+)aap(+) was correlated with a strong biofilm-producer phenotype. Interestingly, some of the ica(-)aap(-) isolates could also form a biofilm. The correlation between the presence of icaAD genes and the biofilm-positive phenotype by the MtP method as well as slime production by the CRA test was statistically significant (P<0.0001). However, some S. epidermidis strains possess the potential ability of ica-independent biofilm formation; thus, further studies are needed to determine reliable, short-time criteria for an in vitro assessment of biofilm production by staphylococci.

Energy conservation in aerobically grown Staphylococcus aureus.

The present studies provide new data on the involvement of menaquinol oxidases in substrate oxidation and energy conservation in aerobically grown, resting cells of Staphylococcus aureus 17810R, starved of endogenous energy reserves and supplemented with glutamate or L-lactate. These cells were energetically competent, since they oxidized both substrates, generated an electrochemical proton gradient (deltamuH+) and synthesized ATP via oxidative phosphorylation. Studies with KCN showed that: (i) L-lactate oxidation occurred via two terminal menaquinol oxidases - the ba3-type sensitive to low KCN and the bo-type insensitive to cyanide, (ii) glutamate oxidation proceeded via the bo-type oxidase, and (iii) ATP synthesis with glutamate or L-lactate was coupled only to the bo-type oxidase. Also in glucose-grown cells oxidizing L-lactate, ATP synthesis was coupled to the highly repressed bo-type oxidase. It is suggested that in the respiratory chain of strain 17810R two energy coupling sites may be present: in the complex of NADH-menaquinone oxidoreductase and in the complex of the bo-type menaquinol oxidase. The rate of ATP synthesis was similar with both substrates, but the rate of their oxidation differed significantly: the P/O ratios were 1.5 and 0.03 with glutamate and L-lactate, respectively. CCCP accelerated glutamate oxidation by 50% but was without effect on L-lactate oxidation. In cell lysates, the rates of NADH and L-lactate oxidation were equal. It is concluded that in whole cells of S. aureus 17810R oxidation of NADH derived from glutamate breakdown is tightly coupled to phosphorylation, while L-lactate oxidation seems to be rather loosely coupled.

Cytotoxic, antioxidant, antimicrobial properties and chemical composition of rose petals

BACKGROUND Rosa rugosa petals are used for production of teas, jams, wines and juices. Despite the wide availability of rose cultivars, comprehensive information on petal chemical composition and healthful properties is still lacking. Therefore, the aim of this study was analysis of cytotoxic, antioxidant and antimicrobial activity and chemical composition of rugosa rose petals. RESULTS Petals of R. rugosa were evaluated for their cytotoxic effect against cervical (HeLa) and breast cancer (T47D) cell lines and for antiradical activity (with DPPH•). As a result, significant cytotoxic (up to 100% of dead cells) and antiradical properties (IC₅₀ 1.33-0.08 mg mg⁻¹ DPPH•) were demonstrated. Moreover, notable antimicrobial activity against eight bacterial (i.e. Staphylococcus. epidermidis, S. aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis) and two yeast strains (Candida. albicans, C. parapsilosis) was shown. Total phenolic, flavonoid, phenolic acid, tannin, carotenoid and polysaccharide content in petals was determined using spectrophotometric methods. Liquid chromatography-electrospray ionization-tandem mass spectrometry was used to thoroughly analyze phenolic acids and flavonoid glycosides in the methanolic extract and fractions obtained after its separation. Five phenolic acids and six flavonoids previously not reported in the plant material were identified. CONCLUSION This is the first such detailed report on chemical composition and biological activity of R. rugosa petals.

Biological activity and composition of teas and tinctures prepared from Rosa rugosa Thunb.

The study was designed to determine the total phenolic, flavonoid, o-dihydroxyphenol, tannin, and carotenoid content as well as the antiradical, antitumor and antimicrobial properties of two types of galenic preparations from Rosa rugosa Thunb. Such extracts obtained from various plant parts have not been studied to date. Our findings have revealed high antiradical activity of the examined galenic preparations, with root, leaf and flower extracts (IC50 ranging from 0.27 to 0.19 mg of dry extract per mg DPPH·) showing the greatest potential. MIC and MBC values against 8 reference bacterial strains (i.e. Staphylococcus epidermidis, Staphylococcus aureus, Bacillussubtilis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis) were determined. Generally, tinctures were found to be more active than teas with MIC ranging from 0.08 to 2.5 mg mL−1 and 0.31 to 1.25 mg mL−1, respectively. Anticancer activities against ovarian (TOV-112D), cervical (HeLa), breast (T47D) and lung cancer (A549) cell lines were evaluated using the BrdU test. The data obtained demonstrate considerable impact of polyphenols on the anticancer activity of extracts (ethanolic, in particular).

Phenolic acids content, antioxidant and antimicrobial activity of Ligusticum mutellina L.

A simple HPLC method has been used for separation and quantitative analysis of the phenolic acids in the methanolic extracts of Ligusticum mutellina aerial parts. Chlorogenic acid was the predominant phenolic acid. Additionally, gallic, p-OH-benzoic, caffeic, p-coumaric and ferulic acids were identified. Moderate antibacterial and antifungal activity (MIC = 1.25–2.5 mg mL−1) was observed for the methanol extract of L. mutellina herb received from plants in flowering stage against a broad spectrum of bacteria. Micrococcus luteus, Pseudomonas aeruginosa and Candida spp. were the most sensitive to this plant material. Total phenolic content for the methanol extract of L. mutellina herb received from plants in flowering stage was 1.56 g of chlorogenic acid equivalents/100 g dry weight. The methanol extract of L. mutellina herb received from plants in flowering stage showed antioxidant activity with DPPH (IC50 value of 0.40 mg mL−1) and with ABTS (IC50 value of 8.65 mg mL−1).

Antibacterial, Antiradical Potential and Phenolic Compounds of Thirty-One Polish Mushrooms

Background Among many sources of natural bioactive substances, mushrooms constitute a huge and almost unexplored group. Fungal compounds have been repeatedly reported to exert biological effects which have prompted their use in pharmaceutical and cosmetic industry. Therefore, the aim of this study was analysis of chemical composition and biological activity of 31 wild growing mushroom species (including saprophytic and parasitic) from Poland. Methods Qualitative and quantitative LC-ESI-MS/MS analysis of fourteen phenolic acids in the mushrooms analysed was performed. Moreover, total phenolic content was determined by the modified Folin-Ciocalteau method. Antioxidative activity of ethanolic extracts towards DPPH• free radical was examined. Antibacterial activity against Gram-positive (S. epidermidis, S. aureus, B. subtilis, M. luteus) and Gram-negative (E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis) microbial strains was analyzed. Results As a result, the first such broad report on polyphenolic composition, antiradical and antimicrobial potential of wild growing Polish mushrooms was developed. Mushroom extracts were found to contain both benzoic (protocatechuic, 4-OH-benzoic, vanillic, syringic) and cinnamic acid derivatives (caffeic, p-coumaric, ferulic). Total phenolic content in mushrooms ranged between 2.79 and 53.13 mg gallic acid equivalent /g of dried extract in Trichaptum fuscoviolaceum and Fomes fomentarius, respectively. Fungi showed much differentiated antiradical activity, from highly active F. fomentarius to poorly effective Russula fragilis (IC50 1.39 to 120.54 mg per mg DPPH•, respectively). A quite considerable relationship between phenolic content and antiradical activity has been demonstrated. Mushrooms varied widely in antimicrobial potential (MIC from 0.156 to 5 mg/ml). Generally, a slightly higher activity against Gram-positive than Gram-negative strains was observed. This is the first study concerning the chemical composition and biological activity of the majority of investigated species.

Anaerobic bacteria colonizing the lower airways in lung cancer patients.

Anaerobes comprise most of the endogenous oropharyngeal microflora, and can cause infections of airways in lung cancer patients who are at high risk for respiratory tract infections. The aim of this study was to determine the frequency and species diversity of anaerobes in specimens from the lower airways of lung cancer patients. Sensitivity of the isolates to conventional antimicrobial agents used in anaerobe therapy was assessed. Respiratory secretions obtained by bronchoscopy from 30 lung cancer patients were cultured onto Wilkins-Chalgren agar in anaerobic conditions at 37°C for 72-96 hours. The isolates were identified using microtest Api 20A. The minimal inhibitory concentrations for penicillin G, amoxicillin/clavulanate, piperacillin/tazobactam, cefoxitin, imipenem, clindamycin, and metronidazole were determined by E-test. A total of 47 isolates of anaerobic bacteria were detected in 22 (73.3%) specimens. More than one species of anaerobe was found in 16 (53.3%) samples. The most frequently isolated were Actinomyces spp. and Peptostreptococcus spp., followed by Eubacterium lentum, Veillonella parvula, Prevotella spp., Bacteroides spp., Lactobacillus jensenii. Among antibiotics used in the study amoxicillin/clavulanate and imipenem were the most active in vitro (0% and 2% resistant strains, respectively). The highest resistance rate was found for penicillin G and metronidazole (36% and 38% resistant strains, respectively). The results obtained confirm the need to conduct analyses of anaerobic microflora colonizing the lower respiratory tract in patients with lung cancer to monitor potential etiologic factors of airways infections, as well as to propose efficient, empirical therapy.

Antimicrobial Activity of Fatty Acids from Fruits of Peucedanum cervaria and P. alsaticum

Plants of the genus Peucedanum have been used in traditional medicine for a long time to treat different diseases including infectious diseases. The hexane fruits extracts of Peucedanum cervaria and P. alsaticum were examined for antimicrobial activity and analyzed for their fatty acid content. Fatty acid composition of oils were analyzed by GC/FID in methyl ester form. Minimal inhibitory concentrations (MICs) of fatty acid fractions against twelve reference bacterial and yeast strains were performed by the twofold serial microdilution broth method. Fourteen fatty acids were identified. Oleic and linoleic acids were found to be dominant. The extracts from both plants examined exhibited inhibitory effects against Gram‐positive strains tested with different MIC values (0.25–2 mg/ml); however, extract from P. alsaticum possessed stronger antibacterial properties and a broader spectrum. The growth of Gram‐negative bacteria and Candida spp. strains was not inhibited even at the highest extract concentration used (MIC>4 mg/ml). Standard fatty acids exhibited inhibitory effects towards all bacterial and yeast strains used in this study; however, the majority of bacteria were more sensitive to linoleic than to oleic acid. These results revealed, for the first time, that hexane extracts obtained from fruits of P. alsaticum and P. cervaria possess moderate in vitro antibacterial activity against Gram‐positive bacteria including staphylococci. Linoleic and oleic acids appear to be the compounds responsible for this effect, and a synergistic antimicrobial effect between these two fatty acids was indicated.

Volatile compounds in fruits of Peucedanum cervaria (Lap.) L.

The volatile compounds from Peucedanum cervaria (Lap.) L. were obtained by hydrodistillation (HD) and headspace solid‐phase microextraction techniques (HS‐SPME), and then analyzed by GC/MS methods. The composition of samples from a botanical garden was compared with plants collected in the wild. The main compounds of the essential oils of P. cervaria were identified as α‐pinene, sabinene, and β‐pinene (more than 80% of oil). The content of β‐myrcene, limonene+β‐phellandrene, and germacrene D was higher than 1%. The in vitro antibacterial activity of the essential oil was evaluated by the agar dilution method against ten reference strains of Gram‐positive and Gram‐negative bacteria.

Synthesis and Antibacterial Activity of Novel Fused 1,3‐Thiazoles and 1,3‐Thiazines Incorporating a 2,4‐Dihydroxyphenyl Residue

In an attempt to find a new class of antimicrobial agents, a series of benzothiazoles, 1,3‐thiazolo[5,4‐b]pyridines, 4H‐3,1‐benzothiazines, naphtho[2,3‐d][1,3]thiazole‐4,9‐diones and other related compounds containing a 2,4‐dihydroxyphenyl moiety were prepared. They were obtained via the reaction of aryl‐modified sulfinyl[bis(2,4‐dihydroxyphenylmethanethione)]s with appropriate commercial chemical reagents in the endocyclization processes. The MIC values of the compounds towards eight reference bacterial strains were assessed by the two‐fold serial micro‐dilution broth method. They exhibited inhibitory effects against the Gram‐positive strains tested opposite to Gram‐negative ones. Some compounds were more effective than the reference drug. 4‐(6‐Chloro‐4H‐3,1‐benzothiazin‐2‐yl)‐6‐methylbenzene‐1,3‐diol (5b) due to its very good activity (MIC from 1.56 to 3.13 µg/mL) and low cytotoxicity (IC50 > 50 µg/mL) may be regarded as a promising precursor for the development of novel antibacterial agents.