Ribotyping of Clostridioides difficile in the Liberec Regional Hospital: a tertiary health care facility.

The ribotyping of Clostridioides difficile is one of the basic methods of molecular epidemiology for monitoring the spread of C. difficile infections. In the Czech Republic, this procedure is mainly available in university hospitals. The introduction of ribotyping in a tertiary health care facility such as Liberec Regional Hospital not only increases safety in the facility but also supports regional professional development. In our study, 556 stool samples collected between June 2017 and June 2018 were used for C. difficile infection screening, followed by cultivation, toxinotyping, and ribotyping of positive samples. The toxinotyping of 96 samples revealed that 44.8% of typed strains could produce toxins A and B encoded by tcdA and tcdB, respectively. The ribotyping of the same samples revealed two epidemic peaks, caused by the regionally most prevalent ribotype 176 (n = 30, 31.3). C. difficile infection incidence ranged between 5.5 and 4.2 cases per 10,000 patient-bed days. Molecular diagnostics and molecular epidemiology are the two most developing parts of clinical laboratories. The correct applications of molecular methods help ensure greater safety in hospitals.

News in WHO 2021 classification of tumours of the central nervous system.

The aim of the presented communication is to clearly inform the general professional public about the newly approved modifications in this classification, including the newly approved types of tumours. A significant change is the new grading system for these tumours, including the innovative involvement of tumour profiling at the molecular level in the system for determining the degree of tumour differentiation and the application of the principle of integrated diagnostics, i. e. the synthesis of available histopathological and molecular findings in CNS tumors.

Molecular pathological profiling of selected tumors of the central nervous system using the MLPA method.

The current progress and increasing knowledge about the genetic causes of cancer opens up new possibilities for its treatment. However, it is necessary to combine the results obtained using classical pathological methods with sensitive, multiplex molecular pathological methods. The method that meets the required criteria is MLPA based on multiplex PCR reaction. This method detects both changes in gene copy number and DNA methylation and, last but not least, point mutations. The MLPA reaction is applicable to even highly fragmented DNA. At the same time, it is a robust method that can be performed on standard thermocyclers, the fluorescent tip label requires automatic sequencers. Up to 50 genetic markers can be tested in one reaction, a number that allows a diagnostic and prognostic conclusion. All these features lead to the routine use of MLPA analysis not only in diagnosis but also in cancer research. The present article aims to summarize the different types of MLPA reactions, its benefits, but also the potential pitfalls.

Genomic analysis of Acinetobacter pittii CEP14 reveals its extensive biodegradation capabilities, including cometabolic degradation of cis-1,2-dichloroethene.

Halogenated organic compounds are naturally occurring in subsurface environments; however, accumulation of the degradative intermediate cis-1,2-dichloroethene (cDCE) at soil and groundwater sites contaminated with xenobiotic chlorinated ethenes is a global environmental and public health issue. Identifying microorganisms capable of cDCE degradation in these environments is of interest because of their potential application to bioremediation techniques. In this study, we sequenced, assembled, and analyzed the complete genome of Acinetobacter pittii CEP14, a strain isolated from chloroethene-contaminated groundwater, that has demonstrated the ability for aerobic cometabolic degradation of cDCE in the presence of n-hexane, phenol, and toluene. The A. pittii CEP14 genome consists of a 3.93 Mbp-long chromosome (GenBank accession no. CP084921) with a GC content of 38.9% and three plasmids (GenBank accession no. CP084922, CP084923, and CP084924). Gene function was assigned to 83.4% of the 3,930 coding DNA sequences. Functional annotation of the genome revealed that the CEP14 strain possessed all genetic elements to mediate the degradation of a range of aliphatic and aromatic compounds, including n-hexane and phenol. In addition, it harbors gene clusters involved in cytosol detoxification and oxidative stress resistance, which could play a role in the mitigation of toxic chemical intermediates that can arise during the degradation of cDCE. Gene clusters for heavy metal and antibiotic resistance were also identified in the genome of CEP14. These results suggest that CEP14 may be a versatile degrader of xenobiotic compounds and well-adapted to polluted environments, where a combination of heavy metal and organic compound pollution is often found.

Multi-branch cooperation on SARS-CoV-2 omicron capture: a case report.

Shortly after the WHOs first notice a suspected case of omicron SARS-CoV-2 was reported in Liberec, Czech Republic. The primary goal of the following actions was to test the presence of the variant and stop the spread of the virus variant. On November 25 a sixty-year-old lady, who had recently returned from Namibia, visited a GP with flu-like symptoms and a rash on her chest. The antigen test was positive for SARS-CoV-2, a PCR test was planned. At that time, it was not known that a new variant of concern was spreading from Africa. On November 26 in the morning the GP announced a suspected omicron case to the Regional public health authority, who organized the following steps. A mobile sampling team was sent to the patient's home immediately, sample transported into the regional hospital and analyzed with the help of the national reference laboratory. The captured virus SARS-CoV-2 fitted the description of the omicron variant, was shared in the GISAID database and named hCoV-19/Czech Republic/KNL_2021-110119140/2021. Contact tracing was started immediately, eleven persons were tested and quarantined. One of them positive with no further spread. It is the first documented omicron case in the Czech Republic and one of the first cases in Europe, with an excellent systemic response to the alert. The laboratory was able to detect the omicron variant instantly after the request. This case also demonstrates how easily the virus spreads on long distances and how important it might be to increase the uptake of the booster vaccine.

Application of nanofiber carriers for sampling of microbial biomass from contaminated groundwater.

Sampling of microbial biomass is crucial for understanding and controlling remediation processes ongoing at contaminated sites in general, particularly when molecular genetic analyses are employed. In this study, fiber-based carriers with a nanofiber layer were developed and tested as a method to sample microbial biomass in groundwater for molecular genetic analysis. Nanofiber carriers, varying in the shape and the linear density of nanofibers, were examined throughout a 27-month monitoring period in groundwater contaminated with benzene, toluene, ethylbenzene and xylene isomers (BTEX), and chlorinated ethenes. The effect of carrier shape and nanofiber layer density on the microbial surface colonization and composition of the microbial biofilm was determined using real-time PCR and next-generation sequencing (NGS) analysis. Differences in microbial community composition between nanofiber carriers, groundwater, and soil samples were also analyzed to assess the applicability of carriers for biomass sampling at contaminated sites. The nanofiber carriers showed their applicability as a sampling tool, particularly because of their easy manipulation that facilitates DNA isolation. The majority of taxa (Proteobacteria, Firmicutes, and Bacteroidetes) present on the carrier surfaces were also detected in the groundwater. Moreover, the microbial community on all nanofiber carriers reflected the changes in the chemical composition of groundwater. Although the carrier characteristics (shape, nanofiber layer) did not substantially influence the microbial community on the carrier surface, the circular and planar carriers with a nanofiber layer displayed faster microbial surface colonization. However, the circular carrier was the most suitable for biomass sampling in groundwater because of its high contact area and because it does not require pre-treatment prior to DNA extraction.

Limited effect of radial oxygen loss on ammonia oxidizers in Typha angustifolia root hairs.

The benefits of plant-microbe interactions have been exploited extensively for nutrient removal. Radial oxygen loss in aquatic macrophytes potentially promotes nitrification and accelerates nitrogen removal through coupled nitrification-denitrification process. Nitrification is likely the limiting activity for an effective nitrogen removal in wetlands. In this work, we have quantified the effect of radial oxygen losses in Typha angustifolia plants in environments of contrasting salinities, including a temporary lagoon, a constructed wetland, and a river estuary. In all sites, radial oxygen diffusion occurred mainly at a narrow band, from 1 to 5 cm from the root tip, and were almost absent at the tip and basal sections of the root (> 5 cm). Root sections with active oxygen diffusion tended to show higher bacterial and archaeal densities in the rhizoplane according to 16S rRNA gene abundance data, except at higher salinities. Archaeal amoA /bacterial amoA gene ratios were highly variable among sites. Archaeal nitrifiers were only favoured over bacteria on the root surface of Typha collected from the constructed wetland. Collectively, radial oxygen loss had little effect on the nitrifying microbial community at the smaller scale (differences according to root-section), and observed differences were more likely related to prevailing physicochemical conditions of the studied environments or to long-term effects of the root microenvironment (root vs sediment comparisons).

Secondary compound hypothesis revisited: Selected plant secondary metabolites promote bacterial degradation of cis-1,2-dichloroethylene (cDCE).

Cis-1,2-dichloroethylene (cDCE), which is a common hazardous compound, often accumulates during incomplete reductive dechlorination of higher chlorinated ethenes (CEs) at contaminated sites. Simple monoaromatics, such as toluene and phenol, have been proven to induce biotransformation of cDCE in microbial communities incapable of cDCE degradation in the absence of other carbon sources. The goal of this microcosm-based laboratory study was to discover non-toxic natural monoaromatic secondary plant metabolites (SPMEs) that could enhance cDCE degradation in a similar manner to toluene and phenol. Eight SPMEs were selected on the basis of their monoaromatic molecular structure and widespread occurrence in nature. The suitability of the SPMEs chosen to support bacterial growth and to promote cDCE degradation was evaluated in aerobic microbial cultures enriched from cDCE-contaminated soil in the presence of each SPME tested and cDCE. Significant cDCE depletions were achieved in cultures enriched on acetophenone, phenethyl alcohol, p-hydroxybenzoic acid and trans-cinnamic acid. 16S rRNA gene sequence analysis of each microbial community revealed ubiquitous enrichment of bacteria affiliated with the genera Cupriavidus, Rhodococcus, Burkholderia, Acinetobacter and Pseudomonas. Our results provide further confirmation of the previously stated secondary compound hypothesis that plant metabolites released into the rhizosphere can trigger biodegradation of environmental pollutants, including cDCE.

Stratification of chlorinated ethenes natural attenuation in an alluvial aquifer assessed by hydrochemical and biomolecular tools.

Biomolecular and hydrochemical tools were used to evaluate natural attenuation of chlorinated ethenes in a Quaternary alluvial aquifer located close to a historical source of large-scale tetrachloroethylene (PCE) contamination. Distinct stratification of redox zones was observed, despite the aquifer's small thickness (2.8 m). The uppermost zone of the target aquifer was characterised by oxygen- and nitrate-reducing conditions, with mixed iron- to sulphate-reducing conditions dominant in the lower zone, along with indications of methanogenesis. Natural attenuation of PCE was strongly influenced by redox heterogeneity, while higher levels of PCE degradation coincided with iron- to sulphate reducing conditions. Next generation sequencing of the middle and/or lower zones identified anaerobic bacteria (Firmicutes, Chloroflexi, Actinobacteria and Bacteroidetes) associated with reductive dechlorination. The relative abundance of dechlorinators (Dehalococcoides mccartyi, Dehalobacter sp.) identified by real-time PCR in soil from the lower levels supports the hypothesis that there is a significant potential for reductive dechlorination of PCE. Local conditions were insufficiently reducing for rapid complete dechlorination of PCE to harmless ethene. For reliable assessment of natural attenuation, or when designing monitoring or remedial systems, vertical stratification of key biological and hydrochemical markers should be analysed as standard, even in shallow aquifers.

Microbial degradation of chloroethenes: a review.

Contamination by chloroethenes has a severe negative effect on both the environment and human health. This has prompted intensive remediation activity in recent years, along with research into the efficacy of natural microbial communities for degrading toxic chloroethenes into less harmful compounds. Microbial degradation of chloroethenes can take place either through anaerobic organohalide respiration, where chloroethenes serve as electron acceptors; anaerobic and aerobic metabolic degradation, where chloroethenes are used as electron donors; or anaerobic and aerobic co-metabolic degradation, with chloroethene degradation occurring as a by-product during microbial metabolism of other growth substrates, without energy or carbon benefit. Recent research has focused on optimising these natural processes to serve as effective bioremediation technologies, with particular emphasis on (a) the diversity and role of bacterial groups involved in dechlorination microbial processes, and (b) detection of bacterial enzymes and genes connected with dehalogenation activity. In this review, we summarise the different mechanisms of chloroethene bacterial degradation suitable for bioremediation and provide a list of dechlorinating bacteria. We also provide an up-to-date summary of primers available for detecting functional genes in anaerobic and aerobic bacteria degrading chloroethenes metabolically or co-metabolically.

Familial atypical parkinsonism with rare variant in VPS35 and FBXO7 genes: A case report.

BACKGROUND: A higher prevalence of parkinsonism was recently identified in southeastern Moravia (Czech Republic). Further research confirmed 3 large pedigrees with familial autosomal-dominant parkinsonism spanning 5 generations. METHODS: This case report concerns a patient belonging to one of these 3 pedigrees, in whom motor and oculomotor symptoms were accompanied by frontal-type dementia, who finally developed a clinical phenotype of progressive supranuclear palsy. Molecular genetic examinations were performed due to the positive family history. RESULTS: No previously described causal mutation was found. After filtering against common variants (minor allele frequency (MAF) < 0.01), 2 noncoding and 1 synonymous rare mutation potentially associable with parkinsonism were identified: GIGYF2-GRB10 Interacting GYF Protein 2, PARK11 (c.*2030G > A, rs115669549); VPS35 gene-vacuolar protein sorting 35, PARK17 (c.102 + 33G > A, rs192115886); and FBXO7-F-box only protein 7 gene, PARK15 (c.540A > G, rs41311141). CONCLUSION: As to the changes in the FBXO7 and VPS35 genes (despite phylogenetic conservation in primates), probably neither the FBXO7 nor the VPS35 variants will be direct causal mutations. Both described variants, and possibly the influence of their combination, could increase the risk of the disease.

Balancing selection and genetic drift create unusual patterns of MHCIIß variation in Galápagos mockingbirds.

The extracellular subunit of the major histocompatibility complex MHCIIß plays an important role in the recognition of pathogens and the initiation of the adaptive immune response of vertebrates. It is widely accepted that pathogen-mediated selection in combination with neutral micro-evolutionary forces (e.g. genetic drift) shape the diversity of MHCIIß, but it has proved difficult to determine the relative effects of these forces. We evaluated the effect of genetic drift and balancing selection on MHCIIß diversity in 12 small populations of Galápagos mockingbirds belonging to four different species, and one larger population of the Northern mockingbird from the continental USA. After genotyping MHCIIß loci by high-throughput sequencing, we applied a correlational approach to explore the relationships between MHCIIß diversity and population size by proxy of island size. As expected when drift predominates, we found a positive effect of population size on the number of MHCIIß alleles present in a population. However, the number of MHCIIß alleles per individual and number of supertypes were not correlated with population size. This discrepancy points to an interesting feature of MHCIIß diversity dynamics: some levels of diversity might be shaped by genetic drift while others are independent and possibly maintained by balancing selection.

Dynamics of organohalide-respiring bacteria and their genes following in-situ chemical oxidation of chlorinated ethenes and biostimulation.

Application of Fenton's reagent and enhanced reductive dechlorination are currently the most common remediation strategies resulting in removal of chlorinated ethenes. In this study, the influence of such techniques on organohalide-respiring bacteria was assessed at a site contaminated by chlorinated ethenes using a wide spectrum of molecular genetic markers, including 16S rRNA gene of the organohalide-respiring bacteria Dehaloccocoides spp., Desulfitobacterium and Dehalobacter; reductive dehalogenase genes (vcrA, bvcA) responsible for dechlorination of vinyl chloride and sulphate-reducing and denitrifying bacteria. In-situ application of hydrogen peroxide to induce a Fenton-like reaction caused an instantaneous decline in all markers below detection limit. Two weeks after application, the bvcA gene and Desulfitobacterium relative abundance increased to levels significantly higher than those prior to application. No significant decrease in the concentration of a range of chlorinated ethenes was observed due to the low hydrogen peroxide dose used. A clear increase in marker levels was also observed following in-situ application of sodium lactate, which resulted in a seven-fold increase in Desulfitobacterium and a three-fold increase in Dehaloccocoides spp. after 70 days. An increase in the vcrA gene corresponded with increase in Dehaloccocoides spp. Analysis of selected markers clearly revealed a positive response of organohalide-respiring bacteria to biostimulation and unexpectedly fast recovery after the Fenton-like reaction.

Selenium hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth promoting properties.

Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5-1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.