Ján Turňa

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified asE. faecium (76%) andE. faecalis (23%). Several strains ofE. durans and 1 strain ofE. hirae were also present. More than 90% of strains isolated from 109 clinical enterococci wereE. faecalis, the rest belonged toE. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains andE. faecium had a higher resistance thanE. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes,cylA (coding for hemolysin),gelE (coding for gelatinase) andesp (coding for surface protein) was monitored. Differences were found in the distribution ofcylA gene between clinical and bryndza-cheeseE. faecalis strains; in contrast to clinical strains (45%),cylA gene was detected in 22% of food isolates. The distribution of 2 other virulence factors,gelE andesp, was not significantly different in the two groups ofE. faecalis strains.cylA andgelE genes were not detected inE. faecium but more than 70% of clinicalE. faecium were positive foresp, even thought none of the 79E. faecium cheese isolates contained this gene.

Salivary DNA and markers of oxidative stress in patients with chronic periodontitis

ObjectivesPrevious observational studies have shown that periodontal status is associated with salivary markers of oxidative damage. A direct comparison of periodontitis patients and controls using a wide palette of salivary markers of oxidative stress is lacking. Characteristics of salivary DNA in periodontitis are unknown. The aim of this study was to compare the salivary markers of oxidative stress and characteristics of salivary DNA between patients with chronic periodontitis and periodontitis-free controls.Materials and methodsSaliva was collected from 23 patients with chronic periodontitis and 19 periodontitis-free controls. All participants underwent a clinical periodontal examination. Markers of oxidative and carbonyl stress were measured in saliva. Human and bacterial DNA was quantified, and human DNA integrity was assessed.ResultsSalivary thiobarbituric acid-reacting substances were higher in patients than in controls; at least in men, the difference was significant (p < 0.01). In women, patients had significantly lower salivary antioxidant status (p < 0.001). No quantitative differences were found regarding salivary DNA. Tendencies towards reduced DNA integrity were found in periodontitis patients.ConclusionsThe results confirmed the association of salivary thiobarbituric acid-reacting substances with periodontitis. Lipid peroxidation in periodontitis seems to be caused by increased production of reactive oxygen species in men and by decreased antioxidant status in women. Whether lower salivary DNA integrity is involved in the pathogenesis of periodontitis remains to be elucidated.Clinical relevanceSalivary thiobarbituric acid-reacting substances are associated with periodontitis at least on a population level. Sex-specific causes of lipid peroxidation might point towards different pathogenic mechanisms.

Multiple regulatory genes in the salinomycin biosynthetic gene cluster ofStreptomyces albus CCM 4719

A DNA fragment containing part of the salinomycin biosynthetic gene cluster from industrial strainStreptomyces albus CCM 4719 was cloned. Sequence analysis of the 25.809-kbp fragment revealed the presence of 8 open reading frames (ORFs), including two large ORFs encoding three modular sets of oligoketide synthase, followed by three genes (salRI, salRII, salRIII) encoding transcriptional regulators. The first two regulators, SalRI and SalRII, belonged to the novel LAL family of large transcriptional regulators. SalRIII was highly similar to the NysRIV, AmphRIV, and FscRI transcriptional regulators from the oligoene macrolides nystatin, amphotericin, and R008/candicidin clusters, respectively.

Assessment of environmental enterococci: bacterial antagonism, pathogenic capacity and antibiotic resistance

The properties of 166 environmental strains belonging to the seven enterococcal species were studied. Enterococci originated mainly from surface- and waste-waters. They were screened for the presence of enterocins, virulence factors, and antibiotic resistance. The presence of different enterocin genes (entA, entB, entP, ent31, entL50AB) was frequently observed in our enterococcal isolates, 109 strains contained at least one enterocin gene. The distribution of enterocin genes varied according to the species, the genes were present mainly in E. hirae and E. faecium. By enterocin spot assay, 10 isolates inhibited the growth of Listeria strains. To evaluate the pathogenic ability of isolates, the distribution of selected virulence genes (cylA, gelE and esp) was investigated, eleven strains were positive in some of these genes, five of them belonged to E. faecalis. Regarding the antibiotic resistance of isolates, only two strains were multiresistant and two strains (E. hirae and E. casseliflavus) were resistant to vancomycin.

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis

Auxins and auxin transport inhibitors affect PIN-FORMED2 (PIN2) dynamics in Arabidopsis epidermal root cells through the synthetic and secretion pathway, but they do not inhibit PIN2 endocytosis. By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane.

Proteomic differences between tellurite-sensitive and tellurite-resistant E.coli.

Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level. In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins. Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control. The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.

Circulating free fetal nucleic acids in maternal plasma and preeclampsia.

Although preeclampsia represents a major threat for many pregnant women, the pathogenesis of this complication is far from being clear. Recent studies suggest that preeclampsia is an autoimmune disorder. Auto-antibodies against angiotensin receptor might explain some of the pathologic findings associated with preeclampsia. However, the origin of the autoimmune reaction is unknown. Here we hypothesize that circulating fetal RNA in maternal plasma might transfect maternal cells. Expression of fetal specific sequences could lead to an immune reaction breaking the immune tolerance against some antigens. Male fetus bearing pregnancies could be at higher risk of preeclampsia due to expression of Y-specific transcripts. This hypothesis is testable by analyzing antibodies and T-lymphocytes of pregnant women with male and female fetuses.

Association of biochemical parameters and RAGE gene polymorphisms in healthy infants and their mothers.

BACKGROUND The receptor for advanced glycation end-products (RAGEs) and its gene polymorphisms are implicated in the pathogenesis of different chronic diseases including diabetes and its complications. Infant formulas contain high amounts of advanced glycation end-products (AGEs) - the ligands of RAGE. METHODS In this cross-sectional study, we examined the impact of G82S and -374 A/T polymorphisms in the gene encoding RAGE on standard blood chemistry, soluble (s)RAGE and inflammatory markers in 244 healthy infants (3-16months of age) and in 119 healthy mothers. Children were subdivided according to age (younger and older than 8months) and for the -374 A/T polymorphism according to the feeding regimen (breast-fed vs. infant formula-fed). RESULTS Minor allele of the RAGE gene polymorphism G82S was associated with reduced plasma sRAGE in all age groups and with increased sICAM-1 in older children and mothers. Minor allele carrying mothers had decreased insulin sensitivity and HDL. The A allele of the RAGE gene promoter polymorphism -374 A/T was associated with higher indices of insulin resistance in young infant formula-fed, but not breast-fed children. In older, formerly infant formula-fed children signs of insulin resistance diminished, while formerly breast-fed children with A allele were more insulin sensitive. CONCLUSIONS The phenotype of minor allele carriers in G82S is associated with reduced levels of protective sRAGE in healthy infants. With increasing age sICAM-1 levels increased and insulin resistance developed. In early childhood the phenotype of the -374 A/T polymorphism was diet-dependently associated with changes in glucose metabolism, which diminished with increasing age.

Genetic Polymorphisms Related to Testosterone Metabolism in Intellectually Gifted Boys

Prepubertal testosterone levels are lower in intellectually gifted boys. The aim of this pilot study was to analyze potential genetic factors related to testosterone metabolism in control and gifted boys. Intellectually gifted (IQ>130; n = 95) and control (n = 67) boys were genotyped. Polymorphisms of interests were chosen in genes including androgen and estrogen receptors, 5-alpha reductase, aromatase and sex hormone binding globulin. Significant differences between control and gifted boys in genotype distributions were found for ESR2 (rs928554) and SHBG (rs1799941). A significantly lower number of CAG repeats in the AR gene were found in gifted boys. Our results support the role of genetic factors related to testosterone metabolism in intellectual giftedness. Increased androgen signaling might explain previous results of lower testosterone levels in intellectually gifted boys and add to the understanding of variability in cognitive abilities.