Enterotoxigenic Staphylococcus aureus in raw milk poses a potential health hazard to consumers, and the identification of such strains should be used as part of a risk analysis of milk and milk products. The primary purpose of this study was to investigate the occurrence of enterotoxigenic S. aureus strains in raw milk supplied for dairy processing in the Czech Republic. A further aim was to compare the production of staphylococcal enterotoxins (SEs) with the presence of the corresponding genes. This was undertaken using multiplex polymerase chain reaction (PCR) and reversed passive latex agglutination (RPLA). Out of 440 bulk tank milk samples from 298 dairy herds, 70 proved positive for S. aureus (15.9%). Staphylococcal enterotoxin genes (ses) were detected in 39 (55.7%) isolates. The genes most commonly detected were sei (38.6%), seg (31.4%) and sea (27.1%). Genes seb, seh, sed, sej and sec were observed in 10%, 4.3%, 2.9%, 2.9% and 1.4% of strains respectively. Genes see and sel did not occur. The most frequently detected genotypes were seg, sei at 11.4%; sea at 10.0%; and sea, seg, sei at 8.6%. Toxin production was observed in nine (12.9%) S. aureus isolates. Seven strains were detected as SEB- (10%) and two as SED- (2.9%) producing. A relatively high number (32%) of discrepancies between the results with multiplex PCR and RPLA assays was obtained, particularly on account of SEA. Nineteen strains were sea positive by PCR but SEA negative by RPLA, and one strain was sec positive and SEC negative. The results of both methods were identical concerning SEB and SED. It was concluded that detection of ses by PCR was a useful additional tool to support identification of enterotoxigenic strains.

The objective was to determine the proportions and bacterial counts of major mastitis pathogens in samples of bulk tank milk (BTM), as well as to clarify the relationship between these bacteria counts and bulk tank somatic cell count (BTSCC). The purpose was to judge the importance of the counts of mammary pathogens for BTSCC at the population level. Samples of BTM were collected from 268 randomly selected anonymous dairy herds (with approximately 29 000 dairy cows). Staphylococcus aureus, other coagulase-positive staphylococci, Streptococcus agalactiae and Streptococcus dysgalactiae were grouped as contagious pathogens, and Streptococcus uberis, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Enterococcus faecium as environmental pathogens. Based on assessment of the dominant pathogen in each herd, environmental pathogens were found to dominate. Counts of specific mammary pathogens in BTM samples did not exceed a geometric mean of 1000 cfu/ml. Significant differences were documented in BTSCC between BTM samples containing Str. agalactiae, Staph. aureus, and Str. dysgalactiae and BTM samples that were pathogen-free. Geometric means of BTSCC associated with these pathogens were noticeably higher than the overall BTSCC (Str. agalactiae 243 628; Staph. aureus 205 610; Str. dysgalactiae 203 978; overall 173 000 cells/ml). It follows that the somatic cell count (SCC) associated with these pathogens contributed substantially to the overall BTSCC. Environmental pathogens predominated in samples of BTM with SCC <300x103/ml and contagious mastitis pathogens predominated in BTM samples with SCC >300x103/ml. No correlation was detected between bacterial counts of specific pathogens and BTSCC. This study revealed that the assessment of bacterial counts of mammary pathogens in samples of BTM in relation to BTSCC is applicable for the monitoring of changes in the occurrence of major mastitis pathogens in dairy herds at a national level.

The objective of this study was to probe the relationship between prevalence of selected principal mastitis pathogens and somatic cell counts in bulk tank milk samples. The sources of milk contamination were evaluated. The samples were collected from 298 dairy herds (with approximately 32 000 dairy cows). Only 48.3% of the bulk tank milk samples were free of contamination of pathogens of interest. Approximately 38.9% of the milk samples were contaminated with only one, 12.4% with two and 0.3% with three pathogens. The arithmetic mean of logarithmically transformed data of bulk tank milk somatic cell count rise in order: pathogen free, <i>Pseudomonas aeruginosa</i>, <i>Streptococcus uberis</i>, <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> (5.381; 5.413; 5.495; 5.518; 5.563, respectively). The arithmetic mean differences between bulk tank milk somatic cell counts in pathogen-free and single-pathogen contaminated samples have revealed a significance for the <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> groups (<i>P</i> &lt; 0.01). Using binary logistic regression, a statistically highly significant relationship (<i>P</i> &lt; 0.001) has been found between the number of contaminations of bulk tank milk samples with mastitis pathogens and bulk tank milk somatic cell counts. The relationship allows the determination of the probability of finding relevant mastitis pathogens in bulk tank milk samples with different levels of bulk tank milk SCC. A 63% probability can be defined at a cell count level of 400 000/ml and 20% at a cell count level of 100 000/ml. Analysis may reveal the potential sources of the bulk tank milk sample contamination, i.e. infected mammary glands or the environment. The presence of high levels of contamination along with a low bulk tank SCC may suggest an environmental source of contamination. The study clarified that a potential source of bulk tank milk contamination by relevant pathogens (the environment or the mammary gland) may be elucidated and the probability of the contamination of bulk tank milk samples with mastitis pathogens predicted by the analysis of relationship between the bulk tank milk somatic cell counts and the number of mastitis pathogen contaminations.

Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions—N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member—the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

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Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel—the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN—its oligomerization ability—is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.