Recreational pools filled with mineralized thermal water are potential reservoirs of pathogenic Vibrio spp.

Vibrio spp. are Gram-negative rod-shaped bacteria commonly present in marine, estuarine and natural freshwater environments. A few members of this genus are associated with human diseases. Here we present the study of Vibrio spp. isolations from 20 artificial recreational pools in Slovakia. Water samples were collected from artificial pools filled with mineralized thermal water in eight recreational areas in Slovakia in 2019 and 2020. Ninety six out of 176 samples were positive for Vibrio spp. Totally 118 different strains of Vibrio spp. were isolated, from which 77 belonged to potentially pathogenic species - V. cholerae (34 isolates), V. vulnificus (4 isolates), V. furnissii (3 isolates), V. fluvialis (25 isolates), V. alginolyticus (10 isolates) and V. mimicus (1 isolate). To our knowledge this is the first study demonstrating the presence of pathogenic or potentially pathogenic Vibrio spp. in artificial pools filled with thermal mineralized waters even disinfected with chlorine compounds.

Interfacial and structural characteristics of polyelectrolyte multilayers used as cushions for supported lipid bilayers.

The surface properties of polyelectrolyte multilayers (PEMs) obtained via sequential adsorption of oppositely charged polyions from their solutions and used as cushions for supported lipid bilayers were investigated. Five types of polyelectrolytes were used: cationic polyethyleneimine (PEI), poly(diallyldimethylammonium)chloride (PDADMAC), and poly-l-lysine hydrobromide (PLL); and anionic polysodium 4-styrenesulfonate (PSS) and poly-l-glutamic acid sodium (PGA). The wettability and surface free energy of the PEMs were determined by contact angle measurements using sessile drop analysis. Electrokinetic characterisation of the studied films was performed by streaming potential measurements of selected multilayers and the structure of the polyelectrolyte multilayer was characterized by synchrotron X-ray reflectometry. The examined physicochemical properties of the PEMs were correlated with the kinetics of the formation of supported lipid bilayers atop the PEM cushion.

Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

AIMS: The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia. METHODS AND RESULTS: The human-yeast hybrid SQS, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in Saccharomyces cerevisiae strain deleted of its own SQS gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end products, ubiquinone and dolichol, was down-regulated. CONCLUSIONS: The S. cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast strain expressing hybrid SQS with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties.

Multispecies biofilm in an artificial wound bed--A novel model for in vitro assessment of solid antimicrobial dressings.

Wound infections represent a major problem, particularly in patients with chronic wounds. Bacteria in the wound exist mainly in the form of biofilms and are thus resistant to most antibiotics and antimicrobials. A simple and cost-effective in vitro model of chronic wound biofilms applied for testing treatments and solid devices, especially wound dressings, is presented in this work. The method is based on the well-established Lubbock chronic wound biofilm transferred onto an artificial agar wound bed. The biofilm formed by four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa) was stable for up to 48h post-transplant. The applicability of the model was evaluated by testing two common iodine wound treatments. These observations indicate that this method enables assessing the effects of treatments on established resilient wound biofilms and is clinically highly relevant.

Coxsackieviral infections involved in aseptic meningitis: a study in Slovakia from 2005 to 2009.

A wide range of diseases is associated with enteroviruses.They are reported to be responsible for viral meningitis, especially in children, but also in adults.This study analysed infection with eight selected coxsackievirus serotypes as the cause of aseptic meningitis in 480 patients in Slovakia from 2005 to 2009,using a quantitative assay for the detection of intrathecal antibodies. Intrathecal production of antibodies against selected coxsackieviruses was proved in 21%of these patients. A significant decrease from 35% in 2005 to 8,5% in 2009 (p=0.004) in the proportion of patients with proven intrathecal production of virus specific antibodies was observed during the study period. We conclude that coxsackievirus B4 was the endemic serotype in Slovakia and was responsible for most cases of coxsackieviral meningitis in the study period.

[Use of PCR in diagnosis of enteroviral infections]. / Pouzitie metódy PCR v diagnostike enterovírusových infekcií.

Enteroviruses belonging to the family Picornaviridae are important human pathogens. Although most cases of infection caused by these viruses are asymptomatic, a wide range of clinical syndromes is observed in manifest cases. Conventional laboratory diagnostic methods based on virus isolation and identification, or on the detection of specific antiviral antibodies, are costly and time consuming. Therefore, they are of little benefit to treatment. We have implemented a commercially available PCR-based test for the detection of enteroviral infections. In 2008, we analyzed biological specimens from 125 patients with suspected enteroviral disease, most often involving the nervous system. The presence of enterovirus was detected in 39 patients. The results were compared with those obtained by the conventional methods. PCR appeared to be a valuable method for rapid, accurate and reliable diagnosis of enteroviral infections which is of major benefit to patient management.

[Circulation of coxsackieviruses in the Slovak population in 1985-2007]. / Cirkulácia coxsackievírusov v slovenskej populáicii v období rokov 1985 az 2007.

Enteroviral infections are of the most common infections in the human population. Circulation of these viruses is high in population and they cause a wide range of clinical syndromes in human. The aim of this study was to compare the coxsackievirus circulation intensity in the population of the Slovak Republic during the period of last 23 years and to identify changes also in relation to modification in polio vaccination scheme. Marker indicating suffered infection was the presence of virus specific antibodies, assessed by means of the virus-neutralizing test. High frequency of infections by all studied coxsackievirus serotypes was confirmed in this study. Consecutive decrease in rate of anti-coxsackievirus antibodies was identified. This may be a consequence of hygiene standard improvement. After modification of immunization scheme (2004/2005) the decrease has been stopped, a mild increase of seropositivity to all studied serotypes was observed, respectively.

Application of multivariate statistical techniques to evaluation of water quality in the Mala Welna River (Western Poland).

The paper presents the results of determinations of physico-chemical parameters of the Mala Welna waters, a river situated in Wielkopolska voivodeship (Western Poland). Samples for the physico-chemical analysis were taken in eight gauging cross-sections once a month between May and November 2006. To assess the physico-chemical composition of surface water, use was made of multivariate statistical methods of data analysis, viz. cluster analysis (CA), factor analysis (FA), principal components analysis (PCA), and discriminant analysis (DA). They made it possible to observe similarities and differences in the physico-chemical composition of water in the gauging cross-sections, to identify water quality indicators suitable for characterising its temporal and spatial variability, to uncover hidden factors accounting for the structure of the data, and to assess the impact of man-made sources of water pollution.

Antimicrobial susceptibility of Enterococcus species isolated from Slovak Bryndza cheese.

Three hundred and ten enterococcal isolates (178 Enterococcus faecium, 68 E. durans, 49 E. faecalis, 8 E. italicus, 3 E. gallinarum, 3 E. casseliflavus, and 1 E. 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 genes vanA and vanB were not detected. Resistance rates of enterococcal isolates to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin were 24, 26, 2, and 1 %, respectively. Thirty-six % of E. faecium isolates and 22 % of the E. faecalis isolates were resistant to erythromycin. Resistance to rifampicin was similar in E. faecium (31 %) and E. faecalis (29 %). Both E. faecium and E. 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 the E. faecium isolates, two (3 %) of the E. durans isolates, and six (12 %) of the E. faecalis isolates exhibited multidrug resistance. The highest frequency of resistant enterococci was observed in Bryndza produced in winter season.

Genetic diversity of Enterococcus faecium isolated from Bryndza cheese.

One hundred and seventy-six Enterococcus faecium isolates from Slovak dairy product Bryndza were tested for the presence of plasmid DNA. Eighty-two isolates were positive and their plasmid DNA was isolated and digested by EcoRI and HindIII restriction endonucleases. The patterns obtained were compared with those obtained after pulsed-field gel electrophoresis of macrorestriction fragments (PFGE), (GTG)(5)-PCR and ERIC-PCR. All these molecular approaches were applied for the study of genetic variability and determination of strain relatednesses among plasmid-positive isolates of E. faecium. In general, all methods revealed a considerable genetic diversity of E. faecium isolates. Plasmid profiling and ERIC-PCR have offered a higher resolution than PFGE and (GTG)(5)-PCR.

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. METHODS AND RESULTS: Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. CONCLUSIONS: Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.

Analysis of expression of flagella by Salmonella enterica serotype typhimurium by monoclonal antibodies recognising both phase specific and common epitopes.

Monoclonal antibodies specific for phase 1 ("i" antigen), phase 2 ("1,2" antigen) and common epitopes of the flagellins of Salmonella enterica serotype Typhimurium were raised. Having confirmed their specificity, the monoclonal antibodies were used to develop semi-quantitative ELISAs in order to assess the relative expression of the two phases by strains of Typhimurium. The majority of Typhimurium strains representative of a wide cross-section of definitive types from animal and environmental sources preferentially expressed phase 1 antigen in vitro. DT40 strains were unique in expressing phase 2 preferentially. The ratio of phase 1 to phase 2 expressed by strains tended to be constant for any one strain when strains were grown on a number of conventional laboratory media. However, the ratio of phases was shown to be modulated by incubation at 42 degrees C and buffering media at pH values, notably 4.5, other than neutral. Selenite broth and Rambach media repressed flagellation.

Characterisation of epitopes of type 1 fimbriae of Salmonella using monoclonal antibodies specific for SEF21 fimbriae of Salmonella enteritidis.

Monoclonal antibodies (mAbs) were used to identify and characterise epitopes of type 1 (SEF21) fimbriae of Salmonella enteritidis. The distribution of the epitopes among salmonellas and other enterobacteria was investigated, as well as the influence of growth media and temperatures on their expression. At least four different epitope clusters were identified on SEF21 fimbriae of S. enteritidis. Two of these clusters were associated with fimbrial haemagglutinins that were either common to all salmonellae tested, or restricted only to S. enteritidis and S. dublin. The four epitope clusters were identified on type 1 fimbriae of most Salmonella serotypes, as well as non-haemagglutinating type 2 fimbriae of S. pullorum and S. gallinarum, and on many other enterobacterial species. The expression of the epitopes was affected by growth conditions.

Salmonella enterica var Typhimurium and Salmonella enterica var Enteritidis express type 1 fimbriae in the rat in vivo.

In a series of experiments rats were dosed with purified type 1 fimbriae from Salmonella enterica var Enteritidis or with fimbriated cultures of either S. enterica var Typhimurium or S. enterica var Enteritidis. Paraffin-wax embedded histological sections of jejunal and ileal tissue were taken and stained by the streptavidin biotin complex (sABC) staining technique for the detection of salmonella and type 1 fimbriae. On oral infection with Enteritidis and Typhimurium both bacteria were shown to be closely associated with the rat ileal epithelium and expressed type 1 fimbriae, thus clearly demonstrating that type 1 fimbriae are expressed by salmonellae in vivo. Moreover, association with the ileum was also shown to occur when purified type 1 fimbriae were orally administered to rats. Our results suggest that type 1 fimbriae alone or in combination with other fimbriae may play an important role in the early stages of infection with these pathogenic bacteria.

Development and application of enzyme-linked immunosorbent assay for specific detection of Salmonella enteritidis infections in chickens based on antibodies to SEF14 fimbrial antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to the SEF14 fimbrial antigen (SEF14-DAS ELISA) and was evaluated for its use in the specific detection of chicken flocks infected with Salmonella enteritidis. The SEF14-DAS ELISA successfully discriminated between chickens experimentally infected with S. enteritidis and those infected with S. panama or S. typhimurium, although the SEF14 responses in adult birds infected with S. enteritidis were detectable but low. In contrast, ELISAs used to detect antibodies to lipopolysaccharide (LPS) and flagella were unable to discriminate between the infected groups of chicks and adult birds infected with different Salmonella serotypes. LPS and flagellar responses were low and variable in chicks, whereas in adult hens they were found to be consistently strong. When flocks naturally infected with S. enteritidis were tested by the SEF14-DAS ELISA and ELISAs to detect LPS and flagellar antibodies, it was found that they could all identify the infected flocks, although there was little correlation between individual serum samples. The study shows that the SEF14-DAS ELISA may offer advantages over existing assays with comparable sensitivities coupled with higher specificities for the serological detection of S. enteritidis-infected chicken flocks.

Characterisation of monoclonal antibodies specific to SEF 21 fimbriae of Salmonella enteritidis and their reactivity with other Salmonellae and Enterobacteria.

A panel of monoclonal antibodies (mAbs) specific to type 1 (SEF 2) fimbriae of S. enteritidis was produced using crude and HPLC purified preparations of SEF 21 fimbriae. Sixteen mAbs were selected by indirect ELISA using both purified SEF 21 antigen and whole cells of S. enteritidis. Eight mAbs were confirmed by immunoprecipitation assay to react specifically with SEF 21 fimbriae. These mAbs were further characterised for their reactivity patterns by the "whole cell" ELISA and latex agglutination test with a number of strains of Salmonella and other enterobacteria. Not all SEF 21 mAbs reacted in both ELISA and latex agglutination tests with whole bacterial cells. mAb 611 was the only one suitable for use in both tests. Unexpectedly these mAbs reacted with the type 1 fimbriae of many of the tested strains of enterobacteria. mAb 721 reacted with most strains of Salmonella (89.1%) and enterobacteria (71.4%) tested. mAb 611 reacted with 61%-75% of strains of Salmonella and with 6.9%-17.6% of enterobacteria in ELISA and latex tests respectively. These mAbs will be useful reagents for further characterisation of type 1 fimbriae expressed by members of the family Enterobacteriaceae.

3-dimensional morphometry of intact dendritic spines observed in thick sections using an electron microscope.

An experimental technique is described which allows observation of fixed neuronal dendrites at magnifications from 10-12 K. The method uses 4-7-microns-thick sections of Epon-embedded tissue with nerve cells that are first impregnated by the rapid Golgi technique and then stained with gold particles/aggregates using a modified gold-toning procedure. A relatively high acceleration voltage (200 kV) is employed to observe in fine detail the dendritic fragments of interest at different angular positions in space, by using a eucentric goniometer stage with a tilt angle of +/- 45 degrees. Image analysis methodology is proposed which permits estimation of 3-dimensional (3D) lengths and of the volume of observed intact dendritic spines. The advantages of the technique with respect to 3D reconstruction methodology are discussed.

Dendritic spines form 'collars' in hippocampal granule cells.

A quantitative study of the distribution of dendritic spines was carried out in three orders of dendritic branches of granule cells from the dentate gyrus of the rat hippocampus. Golgi-stained preparations (7-19 neurones in each of seven rats) were analysed using computerized microscopy. Identification of spines and quantification of stem-spine geometry was performed using a segmentation algorithm and a line skeleton transformation of dendritic images. Analysis of data using the statistics of point processes revealed that, in all three branch orders, the distribution of visible spines along dendrites was not evenly random, but included dense clusters of spines surrounding the dendritic stem (spine 'collars'). Three-dimensional reconstructions from serial ultrathin sections have confirmed the presence of such spine groups. We speculate the spine collars represent a functional element in which associative synaptic plasticity is fostered by the proximity of individual synapses.

Long-term increases in synaptic density in chick CNS after passive avoidance training are blocked by an inhibitor of protein synthesis.

Long-term increases in synaptic density (first recorded 24 h after training of chicks on a one-trial passive avoidance task, and still present 48 h post training), are found bilaterally in a part of the striatum, the lobus parolfactorius (LPO) [23,36], and are believed to reflect a trace of long-term memory formation. Such increases in synaptic density are most likely to occur by either de novo synthesis of new synaptic material, or via post-translational modification of pre-existing components. Several previous studies have shown that inhibitors of protein synthesis such as anisomycin injected just before, or after training, can prevent long-term memory formation in the chick. The present study therefore examined whether the long-term increases in synaptic density in the LPO that occur after passive avoidance training can be blocked by anisomycin. Our data show clearly that chicks injected with anisomycin 30 min pre-training were amnesic on testing 24 h later, and the bilateral increases in synaptic density (of spine and shaft synapses) seen in saline injected trained controls, were significantly reduced, demonstrating that protein synthesis de novo is involved in the post-training increase in synaptic density in the LPO.

The use of latex particle agglutination to specifically detect Salmonella enteritidis.

This paper reviews the development and evaluation of a latex particle agglutination test to specifically identify cultured Salmonella enteritidis organisms. The test is based on the use of two monoclonal antibody-coated latex reagents, one of which detects the recently discovered SEF14 fimbriae expressed predominantly by S. enteritidis and S. dublin organisms, while the second reagent detects the H'p' antigen of S. dublin flagella. In a series of field trials 141 out of 142 strains of S. enteritidis from eighteen phage types were correctly identified by the latex test. A further 175 salmonella isolates representing 35 serotypes were tested and only two false-positives (S. dublin) in the latex test were recorded. This is the first rapid serotype specific test for S. enteritidis to be developed, and highlights the potential advantage of fimbrial antigens as novel diagnostic antigens of the future.