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
- Anti-Bacterial Agents pharmacology MeSH
- Bacterial Toxins analysis genetics MeSH
- Clostridioides difficile classification genetics isolation & purification MeSH
- Disk Diffusion Antimicrobial Tests MeSH
- Incidence MeSH
- Clostridium Infections epidemiology microbiology MeSH
- Humans MeSH
- Minisatellite Repeats MeSH
- Molecular Typing * MeSH
- Hospitals MeSH
- Ribotyping MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic epidemiology 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%).
- MeSH
- Bacterial Toxins analysis genetics MeSH
- Clostridioides difficile classification genetics isolation & purification MeSH
- Child MeSH
- Adult MeSH
- Genetic Variation * MeSH
- Genotype MeSH
- Clostridium Infections epidemiology microbiology MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Molecular Epidemiology MeSH
- Multilocus Sequence Typing MeSH
- Hospitals MeSH
- Infant, Newborn MeSH
- Child, Preschool MeSH
- Ribotyping MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Infant, Newborn MeSH
- Child, Preschool MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic epidemiology 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.
- MeSH
- Aflatoxin B1 administration & dosage chemistry toxicity MeSH
- Biomarkers urine MeSH
- Toxins, Biological MeSH
- Cell Line MeSH
- Fumonisins administration & dosage chemistry toxicity MeSH
- Food Contamination MeSH
- Humans MeSH
- Microcystins administration & dosage chemistry toxicity MeSH
- Cattle MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cíl studie: Clostridium difficile je v současnosti významným původcem nozokomiálních průjmů, v posledních letech narůstá i počet případů infekcí vzniklých v komunitě. Od začátku roku 2010 jsme v Pardubické krajské nemocnici, a. s. (PKN) zaznamenali výrazný nárůst počtu případů infekcí Clostridium diffícile. Cílem práce bylo popsat a vyhodnotit metody používané v laboratorní diagnostice infekcí Clostridium diffícile v PKN a popsat používaný laboratorní diagnostický algoritmus. Materiál a metody: Vzorky stolic byly odebrány od symptomatických pacientů hospitahzovaných nebo vyšetřených na ambulancích PKN v období od 1. 7. 2010 do 31. 12. 2012. K detekci glutamát dehydrogenázy (GDH) a toxinu A/B byl použit duální test na principu enzymoimunoeseje C. Diff Quik Chek Complete, Techlab® (D-ELA). Ke konfirmaci byl použit systém GeneXpert PCR Cepheid® (PCR). Od začátku roku 2011 byla prováděna kultivace u všech GDH pozitivních vzorků. Výsledky: Celkem bylo vyšetřeno 2 040 vzorků. D-EIA test byl použit k vyšetření 2 014 vzorků. I 373 (68,2 %) vzorků bylo GDH a toxin A/B negativní. Ve 359 (17,8 %) vzorcích byla prokázána GDH i toxin A/B. Zjištěná senzitivita a specificita D-ElA testu pro průkaz toxigenního kmene ve vzorku stolice byla 21,8 a 97,2 %. PPV a NPV kalkulovaná pro populaci s prevalencí onemocnění 5,10,20,50 % byla 0,29, 0,46, 0,66, 0,88 a 0,96, 0,92, 0,83, 0,55. Senzitivita a specificita GDH pro detekci Clostridium diffícile ve stolici byla 100,0 a96,2%. PCR vyšetření bylo celkem provedeno u 140 vzorků. 82 vzorků bylo PCR pozitivních. Gen pro produkci toxinu B byl prokázán ve 47 %, nález suspektní pro ribotyp 027 (gen pro toxin B, binární toxin a delece tcdC) ve 48 %, u 5 % vzorků byl detekován současně gen pro toxin B a gen pro binární toxin. Závěr: Vzhledem k nízké senzitivitě EIA testu pro průkaz toxigenního kmene Clostridium diffícile, pokud je užit jako jediný, byl pro rutinní laboratorní vyšetřování infekcí Clostridium difficile na Oddělení klinické mikrobiologie PKN zaveden dvoustupňový testovaci algoritmus. Použití D-EIA testu v prvním kroku umožnilo diagnostikovat 86 % vyšetřovaných vzorků během 30 minut jako pozitivní (GDH +; toxin A/B +) nebo negativní (GDH -; toxin A/B -). Vyšetření pomocí PCR ve druhém kroku zvyšuje počet diagnostikovaných případů CDI. Výsledek testu je k dispozici do dvou hodin, což umožňuje rychlé zavedení izolačních opatření na odděleních PKN a adekvátní antibiotickou léčbu pacientů.
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 (GDIs) 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 GDIs 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, Techlab® (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 difricile, if used as the only one, a two-step algorithm was introduced for routine laboratory examination of infections with Clostridium difRcile 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 -i-) or negative (GDH -; toxin A/B -). The examination with PCR in the second step increased the number of patients diagnosed with GDI. 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.
Man-made shallow fishponds in the Czech Republic have been facing high eutrophication since the 1950s. Anthropogenic eutrophication and feeding of fish have strongly affected the physicochemical properties of water and its aquatic community composition, leading to harmful algal bloom formation. In our current study, we characterized the phytoplankton community across three eutrophic ponds to assess the phytoplankton dynamics during the vegetation season. We microscopically identified and quantified 29 cyanobacterial taxa comprising non-toxigenic and toxigenic species. Further, a detailed cyanopeptides (CNPs) profiling was performed using molecular networking analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data coupled with a dereplication strategy. This MS networking approach, coupled with dereplication, on the online global natural product social networking (GNPS) web platform led us to putatively identify forty CNPs: fourteen anabaenopeptins, ten microcystins, five cyanopeptolins, six microginins, two cyanobactins, a dipeptide radiosumin, a cyclooctapeptide planktocyclin, and epidolastatin 12. We applied the binary logistic regression to estimate the CNPs producers by correlating the GNPS data with the species abundance. The usage of the GNPS web platform proved a valuable approach for the rapid and simultaneous detection of a large number of peptides and rapid risk assessments for harmful blooms.
- MeSH
- Bacterial Toxins analysis toxicity MeSH
- Spectrometry, Mass, Electrospray Ionization * MeSH
- Risk Assessment MeSH
- Metabolomics MeSH
- Water Microbiology MeSH
- Microbiota MeSH
- Environmental Monitoring * MeSH
- Marine Toxins analysis toxicity MeSH
- Online Social Networking * MeSH
- Population Dynamics MeSH
- Seasons MeSH
- Ponds microbiology MeSH
- Cyanobacteria classification growth & development metabolism MeSH
- Harmful Algal Bloom * MeSH
- Tandem Mass Spectrometry * MeSH
- Chromatography, High Pressure Liquid * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Mycotoxins found in randomly selected commercial milk thistle dietary supplement were evaluated for their toxicity in silico and in vitro. Using in silico methods, the basic physicochemical, pharmacological, and toxicological properties of the mycotoxins were predicted using ACD/Percepta. The in vitro cytotoxicity of individual mycotoxins was determined in mouse macrophage (RAW 264.7), human hepatoblastoma (HepG2), and human embryonic kidney (HEK 293T) cells. In addition, we studied the bioavailability potential of mycotoxins and silibinin utilizing an in vitro transwell system with differentiated human colon adenocarcinoma cells (Caco-2) simulating mycotoxin transfer through the intestinal epithelial barrier. The IC50 values for individual mycotoxins in studied cells were in the biologically relevant ranges as follows: 3.57-13.37 nM (T-2 toxin), 5.07-47.44 nM (HT-2 toxin), 3.66-17.74 nM (diacetoxyscirpenol). Furthermore, no acute toxicity was obtained for deoxynivalenol, beauvericin, zearalenone, enniatinENN-A, enniatin-A1, enniatin-B, enniatin-B1, alternariol, alternariol-9-methyl ether, tentoxin, and mycophenolic acid up to the 50 nM concentration. The acute toxicity of these mycotoxins in binary combinations exhibited antagonistic effects in the combinations of T-2 with DON, ENN-A1, or ENN-B, while the rest showed synergistic or additive effects. Silibinin had a significant protective effect against both the cytotoxicity of three mycotoxins (T-2 toxin, HT-2 toxin, DAS) and genotoxicity of AME, AOH, DON, and ENNs on HEK 293T. The bioavailability results confirmed that AME, DAS, ENN-B, TEN, T-2, and silibinin are transported through the epithelial cell layer and further metabolized. The bioavailability of silibinin is very similar to mycotoxins poor penetration.
- MeSH
- Cell Line MeSH
- Coculture Techniques MeSH
- Comet Assay MeSH
- Drug Interactions MeSH
- Humans MeSH
- Mycotoxins toxicity MeSH
- Mice MeSH
- Protective Agents pharmacology MeSH
- Silybum marianum chemistry MeSH
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism MeSH
- Computer Simulation MeSH
- Dietary Supplements MeSH
- Silybin pharmacology MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PURPOSE: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. METHODS: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. RESULTS: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. CONCLUSION: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.
- MeSH
- ADP Ribose Transferases genetics MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Drug Resistance, Bacterial MeSH
- Bacterial Proteins genetics MeSH
- Bacterial Toxins analysis genetics MeSH
- Clostridioides difficile classification drug effects genetics isolation & purification MeSH
- DNA, Bacterial analysis MeSH
- DNA Gyrase genetics MeSH
- Adult MeSH
- Enterotoxins genetics MeSH
- Fluoroquinolones pharmacology MeSH
- Genotype MeSH
- Clostridium Infections epidemiology microbiology MeSH
- Middle Aged MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Minisatellite Repeats genetics MeSH
- Young Adult MeSH
- Molecular Epidemiology MeSH
- Moxifloxacin MeSH
- Multilocus Sequence Typing methods MeSH
- Polymerase Chain Reaction methods MeSH
- Repressor Proteins genetics MeSH
- Ribotyping methods MeSH
- Base Sequence MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Bacterial Typing Techniques * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Finland 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.
- Publication type
- Journal Article MeSH
This article describes a brief history of chemical warfare, which culminated in the signing of the Chemical Weapons Convention. It describes the current level of chemical weapons and the risk of using them. Furthermore, some traditional technology for the development of chemical weapons, such as increasing toxicity, methods of overcoming chemical protection, research on natural toxins or the introduction of binary technology, has been described. In accordance with many parameters, chemical weapons based on traditional technologies have achieved the limit of their development. There is, however, a big potential of their further development based on the most recent knowledge of modern scientific and technical disciplines, particularly at the boundary of chemistry and biology. The risk is even higher due to the fact that already, today, there is a general acceptance of the development of non-lethal chemical weapons at a technologically higher level. In the future, the chemical arsenal will be based on the accumulation of important information from the fields of chemical, biological and toxin weapons. Data banks obtained in this way will be hardly accessible and the risk of their materialization will persist.
- MeSH
- Biological Warfare Agents history MeSH
- Chemical Warfare history trends MeSH
- Chemical Warfare Agents chemistry history toxicity MeSH
- Riot Control Agents, Chemical chemistry history toxicity MeSH
- History, 20th Century MeSH
- History, 21st Century MeSH
- History, Medieval MeSH
- Humans MeSH
- International Cooperation MeSH
- Nanotechnology trends MeSH
- Toxicity Tests MeSH
- Public Policy MeSH
- Military Science history MeSH
- Animals MeSH
- Check Tag
- History, 20th Century MeSH
- History, 21st Century MeSH
- History, Medieval MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Historical Article MeSH
- Review MeSH
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.
- Publication type
- Journal Article MeSH
