Anna Belicová

Identification and characterization of enterococci from bryndza cheese

Aims:  To identify enterococci isolated from sheep milk cheese – bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants.

Probiotic Potential and Safety Properties of Lactobacillus plantarum from Slovak Bryndza Cheese

One hundred and twenty-five acid-resistant presumptive lactobacilli were isolated from Slovak Bryndza cheese and screened for their antimicrobial activity against eight bacterial pathogens using spot agar assay. Out of twenty-six Lactobacillus strains with strong inhibition activity, twenty were identified as Lactobacillus plantarum and six as Lactobacillus fermentum. The most active eleven L. plantarum isolates were further characterized in vitro for some probiotic and safety properties. Only three isolates K10, K21, and ZS07 showed the ability to grow over 50% in the presence of 0.3% bile. Strong deconjugation efficiency was determined for CK06 and K21. The highest β-galactosidase activity was shown in isolates ZS11, B01, CK06, and ZS07. Only three of the strains had the ability to produce tyramine: CK06, LM1, and ZS11. Strains K09, K21, ZS11, and ZS15 were susceptible to all tested antibiotics. Analysis of the results confirmed the L. plantarum isolates ZS07 and K21 as the most suitable for probiotic use, due to their desirable probiotic and safety characteristics.

Phenolic acids inhibit chloroplast mutagenesis in Euglena gracilis

The mutagenicity (bleaching activity) of ofloxacin (43 microM) and acridine orange (AO) (13.5 microM) in Euglena gracilis is inhibited by plant phenolics. Caffeic acid (CA), p-coumaric acid (PCA), ferulic acid (FA) and gentisic acid (GA) (25, 50, 100 and 250 microM) exhibited a significant concentration-dependent inhibitory effect against ofloxacin-induced mutagenicity, which was very effectively eliminated by the highest concentration of all four of those phenolic acids. The mutagenicity of AO was also significantly reduced in the presence of CA, PCA and FA (25, 50, 100 and 250 microM). However, GA exhibited no significant activity, even at the concentration of 250 microM. Based on the UV spectrophotometric measurements, we suggest that the antimutagenic effect of CA, PCA, FA and GA resulted from the scavenging of reactive oxygen species (ROS) produced by ofloxacin. On the other hand, the reduction of AO-induced mutagenicity correlates with the binding capabilities of CA, PCA and FA, with the exception of GA.

Antimicrobial susceptibility ofEnterococcus species isolated from slovak bryndza cheese

Three hundred and ten enterococcal isolates (178Enterococcus faecium, 68E. durans, 49E. faecalis, 8E. italicus, 3E. gallinarum, 3E. casseliflavus, and 1E. hirae) from Slovak Bryndza cheese were evaluated for susceptibility to nine antimicrobial agents (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, erythromycin, rifampicin, nitrofurantoin, and ciprofloxacin). All enterococcal isolates from Bryndza cheese were susceptible to ampicillin, streptomycin, gentamicin, vancomycin, and teicoplanin as determined by the disk diffusion method. Vancomycin resistance genesvanA andvanB were not detected. Resistance rates of enterococcal isolates to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin were 24, 26, 2, and 1 %, respectively. Thirty-six % ofE. faecium isolates and 22 % of theE. faecalis isolates were resistant to erythromycin. Resistance to rifampicin was similar inE. faecium (31 %) andE. faecalis (29 %). BothE. faecium andE. faecalis strains showed the same resistance to ciprofloxacin (2 %).E. durans isolates showed low levels of resistance to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin (1–4 %). Forty-eight (30 %) of theE. faecium isolates, two (3 %) of theE. durans isolates, and six (12 %) of theE. faecalis isolates exhibited multidrug resistance. The highest frequency of resistant enterococci was observed in Bryndza produced in winter season.

Phenolic acids reduce the genotoxicity of acridine orange and ofloxacin inSalmonella typhimurium

Naturally occurring plant phenolics,p-coumaric acid (PA), caffeic acid (CA), ferulic acid (FA) and gentisic acid (GA) (25–100 nmol/L) had protective effects on acridine orange (AO; 216 μmol/L)- and ofloxacin (3 μmol/L)-induced genotoxicity inSalmonella typhimurium. FA, GA and CA exhibited a significant concentration-dependent protective effect against the genotoxicity of AO and ofloxacin, with the exception of PA, which at all concentrations tested abolished the AO and ofloxacin genotoxicity. UV spectrophotometric measurements showed the interaction of PA, FA, GA and CA with AO but not with ofloxacin; this interaction is obviously responsible for the reduction of AO-inducedS. typhimurium mutagenicity. In the case of ofloxacin the antimutagenic effect of PA, FA, GA and CA is assumed to be a result of their ability to scavenge reactive oxygen species (ROS) produced by ofloxacin.

Antimutagenicity of milk fermented byEnterococcus fœcium

The diethyl ether extracts isolated from unfermented milk and milk fermented byEnterococcus fœcium exhibited dose-dependent inhibition of mutagenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), nitrovin (NIT), 5-nitro-2-furylacrylic acid (NFA) and UV-irradiation on the Ames bacterial test (Salmonella typhimurium strains TA97 and TA100) and the unicellular flagellateEuglena gracilis. Overall, the fermented milk extract was the most active against UV-irradiation, less active against NIT and MNNG, and the least active against NFA on bacteria. The highest antibleaching effects were observed against MNNG. The differences between antimutagenic effects from fermented and unfermented milk extracts were determined to be statistically significant at the 0.95 CI level.

Synergic activity of selenium and probiotic bacteriumEnterococcus faecium M-74 against selected mutagens inSalmonella assay

Concentrated extracts of MRS (De Man-Rogosa-Sharpe) media in which probiotic bacteriumEnterococcus faecium strain M-74 was grown exerted different antimutagenic activity against ofloxacin-,N-methyl,N′-nitro-N-nitrosoguanidine- and sodium 5-nitro-2-furylacrylate-induced mutagenicity inSalmonella typhimurium assay depending on the presence (+Se) or absence of disodium selenite pentahydrate (−Se). The antimutagenicity of MRS(+Se) extract was higher than that of MRS(−Se) extract. Selenium enhanced also the antimutagenic effect of both live and killed cells ofE. faecium M-74, respectively. The live bacteria decreased the mutagenicity of selected substances more than killed cells. Synergic activity of selenium with the bacterium was also manifested.

Antimutagenicity of a suberin extract from Quercus suber cork.

The possible protective effect of a suberin extract from Quercus suber cork on acridine orange (AO)-, ofloxacin- and UV radiation-induced mutagenicity (bleaching activity) in Euglena gracilis was examined. To our knowledge, the present results are the first attempt to analyse suberin in relation to mutagenicity of some chemicals. Suberin exhibits a significant dose-dependent protective effect against AO-induced mutagenicity and the concentration of 500 micrograms/ml completely eliminates the Euglena-bleaching activity of AO. The mutagenicity of ofloxacin is also significantly reduced in the presence of suberin (125, 250 and 500 micrograms/ml). However, the moderate protective effect of suberin on UV radiation-induced mutagenicity was observed only at concentrations 500 and 1000 micrograms/ml. Our data shows that suberin extract from Q. suber cork possess antimutagenic properties and can be included in the group of natural antimutagens acting in a desmutagenic manner.

Transcriptome analysis of the Euglena gracilis plastid chromosome

The characterisation of transcript levels of chloroplast genes and their changes under different conditions is an initial step towards understanding chloroplast gene expression and the functional integration of the plastid chromosome into the entire integrated compartmentalised genome of the plant cell. Using RNA from cells of 12 different developmental stages and stress treatments, we have studied the transcript patterns of all 96 genes of the circular plastid chromosome of Euglena gracilis, Pringsheim strain Z, by a macroarray-based approach and Northern analysis of selected genes representing approximately half a dozen operons. The unicellular alga possesses complex, triple-envelope chloroplasts that were acquired by secondary endosymbiosis. (1) Transcripts were detected from all genes, although stationary concentrations varied substantially between individual loci. No obvious economy in the expression pattern with respect to transcription units and genes for complex structures was noted. (2) The chromosome appears to be constitutively expressed under all chosen conditions including stresses such as UV light, temperature, antiplastidial agents, herbicide and heavy metal exposure. (3) The euglenoid organelle transcriptome is qualitatively relatively insensitive to the environment, but exhibited marked overall quantitative changes. The more or less global changes demonstrate that primarily RNA turnover, translational, proteolytic and/or metabolic control regulate organelle gene expression in the alga.

Molecular Effects of Some Stress Factors on the Chloroplast Genetic Apparatus of the Flagellate Euglena gracilis

The chloroplast system of the flagellate Euglena gracilis is particularly sensitive to various chemical and physical factors. Ofloxacin, a quinolone antibacterial chemotherapeutical, the mutagen nitrosoguanidine, as well as the hyperthermic cultivation at 33°C for 7 days, induce complete bleaching of Euglena cultures. Treatment with all those factors results in a mass degradation of E. gracilis chloroplast DNA. Significant decrease in the hybridisation signal intensity is connected with prolongation of cell exposure to the stress factors. No evidence for chloroplast genome rearrangement was obtained for chloroplasts of chemically stressed cells. On the contrary, heat induced loss of chloroplast functions is associated with partial restructurations of the chloroplast genome. Electrophoretograms of E. gracilis total RNA show distinct difference between wild type cells and bleached mutants. Bands for 23S and 16S rRNA are absent in all mutants. Subsequent hybridisation with DNA probes for nuclear encoded chloroplast proteins as Rubisco SSU and LHCPII provided identical signals for wild type cells and all mutants. However, in the case of the petJ gene for cytochrome c6 variable hybridisation signals were obtained with northern blots from an ofloxacin-bleached mutant depending on the light regime.