We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert®C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes' detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.

• Klíčová slova
• MLVA, Slovakia, Thr82Ile, binary toxin, ribotyping, tcdC, Δ117,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Clostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon. The spread of certain strains represents a significant economic burden for health-care. The epidemic successful strains are also associated with severe clinical features of CDI. Therefore, a proteomic study has been conducted that comprises proteomes released from in vitro cultured panel of eight different PCR ribotypes (RTs) and employs the combination of shotgun proteomics and label-free quantification (LFQ) approach. RESULTS: The comparative semi-quantitative analyses enabled investigation of a total of 662 proteins. Both hierarchical clustering and principal component analysis (PCA) created eight distinctive groups. From these quantifiable proteins, 27 were significantly increased in functional annotations. Among them, several known factors connected with virulence were identified, such as toxin A, B, binary toxin, flagellar proteins, and proteins associated with Pro-Pro endopeptidase (PPEP-1) functional complex. Comparative analysis of protein expression showed a higher expression or unique expression of proteins linked to pathogenicity or iron metabolism in RTs 027 and 176 supporting their genetic relatedness and clinical importance at the proteomic level. Moreover, the absence of putative nitroreductase and the abundance of the Abc-type fe3+ transport system protein were observed as biomarkers for the RTs possessing binary toxin genes (027, 176 and 078). Higher expression of selected flagellar proteins clearly distinguished RTs 027, 176, 005 and 012, confirming the pathogenic role of the assembly in CDI. Finally, the histidine synthesis pathway regulating protein complex HisG/HisZ was observed only in isolates possessing the genes for toxin A and B. CONCLUSIONS: This study showed the applicability of the LFQ approach and provided the first semi-quantitative insight into the proteomes released from in vitro cultured panel of eight RTs. The observed differences pointed to a new direction for studies focused on the elucidation of the mechanisms underlining the CDI nature.

• Klíčová slova
• Binary toxin, Clostridium difficile, Flagellins, Label-free quantification, PCR ribotype 027, PCR ribotype 176, Proteome, Toxins A/B,
• Publikační typ
• časopisecké články MeSH

Fungal Immunomodulatory Proteins from Ganoderma species (gFIPs) have garnered significant interest due to their potential therapeutic applications in modulating immune responses. This study investigates the sequence, structural, and functional relationships of gFIPs with other proteins involved in immune modulation. Utilizing molecular modelling, multiple sequence alignments, and structural superimposition, we analysed two FIP crystallized structures (PDB IDs: 3F3H and 3KCW) alongside homologous sequences from various taxonomic groups. Our results reveal conserved motifs across fungal, bacterial, and human sequences, indicating potential functional similarities. Comparative structural analysis highlights significant conservation in FIP architecture, with variations primarily in the N-terminal regions. Notably, structural alignment with bacterial toxins, such as ADP-ribosylating binary toxin from Clostridium difficile or protective antigen of Anthrax toxin from Bacillus anthracis suggests mechanistic insights into FIP's immunomodulatory actions. Structural similarities between gFIPs and immune-related proteins, such as bacterial toxin-binding domains, antibody fragments, T-cell receptor components, and immune checkpoint regulators (PD-1) suggest their potential involvement in immune response/inflammation signalling pathways. This comprehensive analysis elucidates the structural basis for the diverse biological activities of gFIPs and underscores their potential as therapeutic agents in immune-related diseases.

• Klíčová slova
• 3D molecular modeling, Comparative structural analysis, Fungal immunomodulatory proteins (FIPs), Ganoderma, Immune modulation, Immune response, Natural products, Protein-protein interaction energy,
• MeSH
• bakteriální toxiny chemie   MeSH
• fungální proteiny * chemie   imunologie   genetika   metabolismus   MeSH
• Ganoderma * imunologie   chemie   genetika   metabolismus   MeSH
• imunologické faktory * chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální toxiny MeSH
• fungální proteiny * MeSH
• imunologické faktory * MeSH

Clostridium difficile is a leading nosocomial pathogen and molecular typing is a crucial part of monitoring its occurrence and spread. Over a three-year period (2013-2015), clinical C. difficile isolates from 32 Czech hospitals were collected for molecular characterisation. Of 2201 C. difficile isolates, 177 (8%) were non-toxigenic, 2024 (92%) were toxigenic (tcdA and tcdB) and of these, 677 (33.5%) carried genes for binary toxin production (cdtA, cdtB). Capillary-electrophoresis (CE) ribotyping of the 2201 isolates yielded 166 different CE-ribotyping profiles, of which 53 were represented by at least two isolates for each profile. Of these, 29 CE-ribotyping patterns were common to the Leeds-Leiden C. difficile reference strain library and the WEBRIBO database (83.7% isolates), and 24 patterns were recognized only by the WEBRIBO database (11.2% isolates). Isolates belonging to these 53 CE-ribotyping profiles comprised 94.9% of all isolates. The ten most frequent CE-ribotyping profiles were 176 (n=588, 26.7%), 001 (n=456, 20.7%), 014 (n=176, 8%), 012 (n=127, 5.8%), 017 (n=85, 3.9%), 020 (n=68, 3.1%), 596 (n=55, 2.5%), 002-like (n=45, 2.1%), 010 (n=35, 1.6%) and 078 (n=34, 1.6%). Multi-locus sequence typing (MLST) of seven housekeeping genes performed in one isolate of each of 53 different CE-ribotyping profiles revealed 40 different sequence types (STs). We conclude that molecular characterisation of Czech C. difficile isolates revealed a high diversity of CE-ribotyping profiles; the prevailing RTs were 001 (20.7%) and 176 (027-like, 26.7%).

• Klíčová slova
• Capillary electrophoresis ribotyping, Clostridium difficile, MLST, Molecular typing, Toxin genes, tcdC,
• MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• dítě MeSH
• dospělí MeSH
• genetická variace * MeSH
• genotyp MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• molekulární epidemiologie MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ribotypizace MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• bakteriální toxiny MeSH

BACKGROUND: Clostridium difficile is currently a significant cause of nosocomial diarrhea. For several years, the number of infectious cases in the community has also been increasing. Since the beginning of 2010, quite a large increase in the number of Clostridium difficile infections (CDIs) has been noted in Pardubice Regional Hospital (PRH). The objectives of this study were to describe and evaluate the methods used in the laboratory diagnosis of CDIs in PRH, and to describe the laboratory diagnostic algorithm used here. MATERIAL AND METHODS: Samples of stools were taken from symptomatic patients hospitalized or examined in the outpatient departments of PRH from 1 July 2010 to 31 December 2012. For the detection of glutamate dehydrogenase (GDH) and toxin A/B, the dual test based upon the principle enzyme immunoassays C. Diff Quik Chek Complete, Techlabo (D-EIA) was used. The system GeneXpert PCR Cepheid (PCR) was used for confirmation of laboratory findings. Since the beginning of 2011, all the GDH-positive samples were cultured. RESULTS: A total of 2,040 samples were examined. The D-EIA test was used for examination of 2,014 samples. Of those, 1,373 (68.2 %) samples were GDH- and toxin A/B-negative. In 359 (17.8 %) samples, both GDH and toxin A/B were detected. The D-EIA sensitivity and specificity for detecting toxigenic strains in stool samples were 21.8% and 97.2%, respectively. The PPV and NPV rates calculated for the populations with prevalence rates of disorders of 5%, 10%, 20% and 50 % were 0.29, 0.46, 0.66, 0.88 and 0.96, 0.92, 0.83, 0.55, respectively. The sensitivity and specificity of GDH for the detection of Clostridium difficile in stools were 100.0% and 96.2%, respectively. PCR examination was carried out in 140 samples. Of those, 82 samples were PCR-positive. The gene for the production of toxin B was detected in 47%, the finding suspected for ribotype 027 (gene for toxin B, binary toxin and deletion of tcdC) in 48%. In 5% of the samples, the gene for toxin B and the gene for the binary toxin were detected. CONCLUSION: Considering the low sensitivity of the D-EIA test for detecting the toxigenic strain of Clostridium difficile, if used as the only one, a two-step algorithm was introduced for routine laboratory examination of infections with Clostridium difficile in the Clinical Microbiology Department of PRH. In the first step, the D-EIA test diagnosed 86 % of examined samples in 30 minutes as positive (GDH +; toxin A/B +) or negative (GDH -; toxin A/B -). The examination with PCR in the second step increased the number of patients diagnosed with CDI. The test results are available within two hours. This enables quick introduction of isolation measures in the departments of PRH and appropriate antibiotic treatment of the patients.

• MeSH
• algoritmy MeSH
• azurová mořidla MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bakteriální toxiny genetika   metabolismus   MeSH
• Clostridioides difficile enzymologie   genetika   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• glutamátdehydrogenasa genetika   metabolismus   MeSH
• imunoenzymatické techniky metody   MeSH
• lidé MeSH
• methylenová modř MeSH
• polymerázová řetězová reakce metody   MeSH
• pseudomembranózní enterokolitida diagnóza   mikrobiologie   MeSH
• senzitivita a specificita MeSH
• xantheny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• azurová mořidla MeSH
• bakteriální proteiny MeSH
• bakteriální toxiny MeSH
• Diff Quik MeSH Prohlížeč
• glutamátdehydrogenasa MeSH
• methylenová modř MeSH
• xantheny MeSH

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální toxiny analýza   genetika   MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• diskové difúzní antimikrobiální testy MeSH
• incidence MeSH
• klostridiové infekce epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• minisatelitní repetice MeSH
• molekulární typizace * MeSH
• nemocnice MeSH
• ribotypizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální toxiny MeSH

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30 min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6-24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

• MeSH
• alkaloidy MeSH
• Aphanizomenon metabolismus   MeSH
• bakteriální toxiny MeSH
• buněčné extrakty toxicita   MeSH
• buněčné linie MeSH
• Cylindrospermopsis metabolismus   MeSH
• epitelové buňky účinky léků   metabolismus   MeSH
• fluoreny toxicita   MeSH
• karcinogeny toxicita   MeSH
• krysa rodu Rattus MeSH
• látky znečišťující životní prostředí toxicita   MeSH
• mezerový spoj účinky léků   metabolismus   MeSH
• mezibuněčná komunikace účinky léků   fyziologie   MeSH
• mikrocystiny toxicita   MeSH
• mořské toxiny MeSH
• polychlorované bifenyly toxicita   MeSH
• synergismus léků MeSH
• toxiny kmene Cyanobacteria MeSH
• uracil analogy a deriváty   toxicita   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2,4,5,2',4',5'-hexachlorobiphenyl MeSH Prohlížeč
• alkaloidy MeSH
• bakteriální toxiny MeSH
• buněčné extrakty MeSH
• cyanoginosin LR MeSH Prohlížeč
• cylindrospermopsin MeSH Prohlížeč
• fluoranthene MeSH Prohlížeč
• fluoreny MeSH
• karcinogeny MeSH
• látky znečišťující životní prostředí MeSH
• mikrocystiny MeSH
• mořské toxiny MeSH
• polychlorované bifenyly MeSH
• toxiny kmene Cyanobacteria MeSH
• uracil MeSH

As human co-exposure to natural toxins through food and water is inevitable, risk assessments to safeguard health are necessary. Aflatoxin B1 and fumonisin B1, frequent co-contaminants of maize and microcystin-LR, produced in freshwater by cyanobacteria are all naturally occurring potent toxins that threaten human health. Populations in the poorest regions of the world may suffer repeated simultaneous exposure to these contaminants. Using High Content Analysis, multiple cytotoxicity endpoints were measured for the individual toxins and mixtures in various cell lines. Results highlighted that significant cytotoxic effects were observed for aflatoxin B1 in all cell lines while no cytotoxic effects were observed for fumonisin B1 or microcystin-LR. Aflatoxin B1/microcystin-LR was cytotoxic in the order HepG2 > Caco-2 > MDBK. Fumonisin B1/microcystin-LR affected MDBK cells. The ternary mixture was cytotoxic to all cell lines. Most combinations were additive, however antagonism was observed for binary and ternary mixtures in HepG2 and MDBK cell lines at low and high concentrations. Synergy was observed in all cell lines, including at low concentrations. The combination of these natural toxins may pose a significant risk to populations in less developed countries. Furthermore, the study highlights the complexity around trying to regulate for human exposure to multiple contaminants.

• Klíčová slova
• Aflatoxin B(1), Combined exposure, Cytotoxicity, Fumonisin B(1), High content analysis, Microcystin-LR,
• MeSH
• aflatoxin B1 aplikace a dávkování   chemie   toxicita   MeSH
• biologické markery moč   MeSH
• biologické toxiny MeSH
• buněčné linie MeSH
• fumonisiny aplikace a dávkování   chemie   toxicita   MeSH
• kontaminace potravin MeSH
• lidé MeSH
• mikrocystiny aplikace a dávkování   chemie   toxicita   MeSH
• mořské toxiny MeSH
• skot MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aflatoxin B1 MeSH
• biologické markery MeSH
• biologické toxiny MeSH
• cyanoginosin LR MeSH Prohlížeč
• fumonisin B1 MeSH Prohlížeč
• fumonisiny MeSH
• mikrocystiny MeSH
• mořské toxiny MeSH

In 2014, 18 hospitals in the Czech Republic participated in a survey of the incidence of Clostridium difficile infections (CDI) in the country. The mean CDI incidence was 6.1 (standard deviation (SD):7.2) cases per 10,000 patient bed-days and 37.8 cases (SD: 41.4) per 10,000 admissions. The mean CDI testing frequency was 39.5 tests (SD: 25.4) per 10,000 patient bed-days and 255.8 tests (SD: 164.0) per 10,000 admissions. A total of 774 C. difficile isolates were investigated, of which 225 (29%) belonged to PCR ribotype 176, and 184 isolates (24%) belonged to PCR ribotype 001. Multilocus variable-number tandem repeat analysis (MLVA) revealed 27 clonal complexes formed by 84% (190/225) of PCR ribotype 176 isolates, and 14 clonal complexes formed by 77% (141/184) of PCR ribotype 001 isolates. Clonal clusters of PCR ribotypes 176 and 001 were observed in 11 and 7 hospitals, respectively. Our data demonstrate the spread of two C. difficile PCR ribotypes within 18 hospitals in the Czech Republic, stressing the importance of standardising CDI testing protocols and implementing mandatory CDI surveillance in the country.

• Klíčová slova
• C. difficile infection, CDI surveillance, MLVA, PCR ribotype 001, PCR ribotype 176, binary toxin, ribotyping,
• MeSH
• Clostridioides difficile klasifikace   genetika   izolace a purifikace   MeSH
• elektroforéza kapilární MeSH
• feces mikrobiologie   MeSH
• incidence MeSH
• klostridiové infekce diagnóza   epidemiologie   MeSH
• lidé MeSH
• minisatelitní repetice MeSH
• nemocnice statistika a číselné údaje   MeSH
• oznamovací povinnost MeSH
• polymerázová řetězová reakce MeSH
• průjem epidemiologie   mikrobiologie   MeSH
• ribotypizace metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH

BACKGROUND: Clostridium difficile infection (CDI) is becoming a serious problem predominantly in geriatric patients, who are a significant risk group. The goal of this study was to evaluate the risk factors for mortality in CDI patients and to construct a binary logistic regression model that describes the probability of mortality in geriatric patients suffering from CDI. METHODS: In this retrospective study, we evaluated a group of 235 patients over 65 years of age with confirmed diagnoses of CDI, hospitalized at the Department of Internal Medicine, Geriatrics and General Practice, Brno, from January 2008 to December 2013. The examined group comprised 148 women (63 %) and 87 men (37 %). For the diagnosis of CDI, confirmation of A and B toxins in the patients' stool or an autopsy confirmation was crucial. RESULTS: The impact of antibiotic therapy on the increased incidence of CDI was clearly confirmed in our study group when examining patients' histories. Other risk factors included cerebrovascular disease, dementia, the presence of pressure ulcers, and immobility. Our new model consisted of a combination of the following parameters: the number of antibiotics used (from patients' history), nutritional status (Mini Nutritional Assessment short-form test), presence of pressure ulcers, and occurrence of fever. CONCLUSION: Our logistic regression model may predict mortality in geriatric patients suffering from CDI. This could help improve the therapeutic process.

• Klíčová slova
• Antibiotic therapy, Binary logistic regression model, Clostridium difficile, Geriatric patient, Pseudomebranous colitis,
• MeSH
• antibakteriální látky terapeutické užití   MeSH
• Clostridioides difficile * MeSH
• geriatrické hodnocení statistika a číselné údaje   MeSH
• incidence MeSH
• lidé MeSH
• míra přežití MeSH
• prognóza MeSH
• pseudomembranózní enterokolitida mikrobiologie   mortalita   terapie   MeSH
• rizikové faktory MeSH
• rozložení podle pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• věkové rozložení MeSH
• zlepšení kvality MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH