Increasing Incidence of Geomyces destructans Fungus in Bats from the Czech Republic and Slovakia

BACKGROUND: White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval. CONCLUSIONS/SIGNIFICANCE: G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme

Effect of knock out of yxkO gene on environmental stress adaptation in genus Bacillus

We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to..

Bacillus subtilis motility

Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Barn Owl Productivity Response to Variability of Vole Populations.

We studied the response of the barn owl annual productivity to the common vole population numbers and variability to test the effects of environmental stochasticity on their life histories. Current theory predicts that temporal environmental variability can affect long-term nonlinear responses (e.g., production of young) both positively and negatively, depending on the shape of the relationship between the response and environmental variables. At the level of the Czech Republic, we examined the shape of the relationship between the annual sum of fledglings (annual productivity) and vole numbers in both non-detrended and detrended data. At the districts' level, we explored whether the degree of synchrony (measured by the correlation coefficient) and the strength of the productivity response increase (measured by the regression coefficient) in areas with higher vole population variability measured by the s-index. We found that the owls' annual productivity increased linearly with vole numbers in the Czech Republic. Furthermore, based on district data, we also found that synchrony between dynamics in owls' reproductive output and vole numbers increased with vole population variability. However, the strength of the response was not affected by the vole population variability. Additionally, we have shown that detrending remarkably increases the Taylor's exponent b relating variance to mean in vole time series, thereby reversing the relationship between the coefficient of variation and the mean. This shift was not responsible for the increased synchrony with vole population variability. Instead, we suggest that higher synchrony could result from high food specialization of owls on the common vole in areas with highly fluctuating vole populations

Epidemiology of Methicillin-Resistant Staphylococcus aureus in Slovakia, 2020 – Emergence of an Epidemic USA300 Clone in Community and Hospitals

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of health care-associated infections. Additionally, over the decades, the spread of community-associated (CA-MRSA) clones has become a serious problem. The aim of this study was to gain data on the current epidemiology of MRSA in Slovakia. Between January 2020 and March 2020, single-patient MRSA isolates (invasive and/or colonizing) were collected in Slovakia from hospitalized inpatients (16 hospitals) or outpatients (77 cities). Isolates were characterized via antimicrobial susceptibility testing, spa typing, SCCmec typing, the detection of mecA/mecC, genes coding for Panton-Valentine leukocidin (PVL), and the arcA gene (part of the arginine catabolic mobile element [ACME]). Out of 412 isolates, 167 and 245 originated from hospitalized patients and outpatients, respectively. Inpatients were most likely older (P < 0.001) and carried a strain exhibiting multiple resistance (P = 0.015). Isolates were frequently resistant to erythromycin (n = 320), clindamycin (n = 268), and ciprofloxacin/norfloxacin (n = 261). 55 isolates were resistant to oxacillin/cefoxitin only. By clonal structure, CC5-MRSA-II (n = 106; spa types t003, t014), CC22-MRSA-IV (n = 75; t032), and CC8-MRSA-IV (n = 65; t008) were the most frequent. We identified PVL in 72 isolates (17.48%; 17/412), with the majority belonging to CC8-MRSA-IV (n = 55; arcA+; t008, t622; the USA300 CA-MRSA clone) and CC5-MRSA-IV (n = 13; t311, t323). To the best of our knowledge, this is the first study on the epidemiology of MRSA in Slovakia. The presence of the epidemic HA-MRSA clones CC5-MRSA-II and CC22-MRSA-IV was found, as was, importantly, the emergence of the global epidemic USA300 CA-MRSA clone. The extensive spread of USA300 among inpatients and outpatients across the Slovakian regions warrants further investigation. IMPORTANCE The epidemiology of MRSA is characterized by the rise and fall of epidemic clones. Understanding the spread, as well as the evolution of successful MRSA clones, depends on the knowledge of global MRSA epidemiology. However, basic knowledge about MRSA epidemiology is still fragmented or completely missing in some parts of the world. This is the first study of MRSA epidemiology in Slovakia to identify the presence of the epidemic HA-MRSA clones CC5-MRSA-II and CC22-MRSA-IV and, importantly and unexpectedly, the emergence of the global epidemic USA300 CA-MRSA clone in the Slovakian community and hospitals. So far, USA300 has failed to spread in Europe, and this study documents an extensive spread of this epidemic clone in a European country for the first time

NAD(P)H-Hydrate Dehydratase- A Metabolic Repair Enzyme and Its Role in <i>Bacillus subtilis</i> Stress Adaptation

<div><p>Background</p><p>One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in <i>Bacillus</i> sp. The GSR is one of the largest regulon in <i>Bacillus</i> sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the <i>Bacillus subtilis</i>168 osmosensitive mutant, defective in the <i>yxkO</i> gene (encoding a putative ribokinase), which was recently assigned <i>in vitro</i> as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon.</p><p>Methods and Results</p><p>We show the impact of YxkO on the activity of SigB-dependent P<i>ctc</i> promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress.</p><p>Conclusion</p><p>We performed the first characterization of a <i>Bacillus subtilis</i>168 knock-out mutant in the <i>yxkO</i> gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.</p></div

Transcription level of P<i>ctc</i> on genetic background of WT and MP2 mutant.

<p>P<i>ctc</i> activation measurements of WT and MP2 were performed under ethanol stress in LB medium (A), osmotic stress in MM medium with 10 mM K⁺ concentration (B), osmotic stress in MM medium with 0.5 mM K⁺ concentration (C), shift from MM medium with 10 mM K⁺ concentration to MM medium with 0.5 mM K⁺ concentration (D). Time 0 indicates application of stress. Details of transcription activity measurements, growth, and stress conditions are described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a>.</i></p

Comparison of microleakage under orthodontic brackets bonded with five different adhesive systems: in vitro study

Abstract Background Orthodontic treatment is associated with numerous adverse side effects, such as enamel discoloration, demineralization or even caries. The presence of microleakage between the enamel and the adhesive and between the adhesive and the base of the orthodontic bracket allows penetration of the bacteria, molecules, and liquids into the enamel and can lead to unpleasant “white spot lesions” or secondary caries beneath and around the brackets. The aim of this in vitro study was to evaluate microleakage in five adhesive systems commonly used in orthodontic practice for bonding brackets. Methods One hundred extracted premolars were divided into five groups of twenty teeth. Stainless steel Legend medium metal brackets were bonded to teeth using five adhesive systems: resin-reinforced glass ionomer cement GC Fuji Ortho LC (GCF) and composite materials Light Bond (LB), Transbond XT (TB), Trulock™ Light Activated Adhesive (TL), and GC Ortho Connect (GCO). The specimens were subjected to thermal cycling, stained with 2% methylene blue, sectioned with low–speed diamond saw Isomet and evaluated under a digital microscope. Microleakage was detected at the enamel-adhesive and adhesive-bracket interfaces from occlusal and gingival margins. Statistical analysis was performed using generalized linear mixed models with beta error distribution. Results Microleakage was observed in all materials, with GCF showing the highest amount of microleakage. Composite materials GCO, TB, and LB exhibited the lowest amount of microleakage with no statistical difference between them, while TL showed a statistically significantly higher amount of microleakage (p < 0.001). The enamel–adhesive interface had more microleakage in all composite materials (GCO, LB, TB, and TL) than the adhesive bracket–interface (p < 0.001). The highest amount of microleakage occurred in the gingival region in all materials. Conclusion Composite materials showed better adhesive properties than a resin-reinforced glass ionomer cement. The presence of microleakage at the enamel-adhesive interface facilitates the penetration of various substances into enamel surfaces, causing enamel demineralization and the development of dental caries

Comparative 2DE analysis of WT versus MP2 mutant exposed to osmotic and ethanol stress – metabolic enzymes.

<p>Proteins with protein level profiles that are similar for both stresses. For experimental conditions and data evaluation, see <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a></i>. Separate columns of the bar charts show the protein level of respective proteins, as calculated from the quantification of the spot volume by PDQuest 8.0 software; y-axes are scaled in intensity for each particular protein. Bars represent each strains and conditions, and there are in the same order as the protein level profiles are presented.</p