Outer membrane vesicles (OMVs), nanoparticles released by Shiga toxin-producing Escherichia coli (STEC), have been identified as novel efficient virulence tools of these pathogens. STEC O157 OMVs carry a cocktail of virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, flagellin, and lipopolysaccharide. OMVs are taken up by human intestinal epithelial and microvascular endothelial cells, the major targets during STEC infection, and deliver the virulence factors into host cells. There the toxins separate from OMVs and are trafficked via different pathways to their target compartments, i.e., the cytosol (Stx2a-A subunit), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). This leads to a toxin-specific host cell injury and ultimately apoptotic cell death. Besides their cytotoxic effects, STEC OMVs trigger an inflammatory response via their lipopolysaccharide and flagellin components. In this chapter, we describe methods for the isolation and purification of STEC OMVs, for the detection of OMV-associated virulence factors, and for the analysis of OMV interactions with host cells including OMV cellular uptake and intracellular trafficking of OMVs and OMV-delivered toxins.

• MeSH
• bakteriální toxiny metabolismus   MeSH
• endoteliální buňky metabolismus   mikrobiologie   patologie   MeSH
• Escherichia coli O157 * metabolismus   patogenita   MeSH
• faktory virulence metabolismus   MeSH
• lidé MeSH
• mikropartikule metabolismus   MeSH
• shiga toxin 2 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell's plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions.IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

• MeSH
• bakteriální adheze * MeSH
• buněčná membrána chemie   MeSH
• buněčné linie MeSH
• buňky HT-29 MeSH
• epitelové buňky mikrobiologie   MeSH
• Escherichia coli O157 fyziologie   MeSH
• flagella metabolismus   MeSH
• fosfolipidy analýza   MeSH
• interakce hostitele a patogenu * MeSH
• kyselina alfa-linolenová analýza   MeSH
• kyselina palmitová analýza   MeSH
• lidé MeSH
• membránové mikrodomény chemie   MeSH
• unilamelární lipozómy chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super-shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super-shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal-to-animal cross-contaminations and reinfections are reduced. We hypothesized that unlike super-shedders, low-shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic-treated chicks are more prone to become super-shedders; (ii) super or low-shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low- and super-shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.

• MeSH
• Escherichia coli O157 * MeSH
• fenotyp MeSH
• kur domácí MeSH
• Salmonella MeSH
• střevní mikroflóra * MeSH
• vylučování bakterií z těla MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted from the cell envelope of all Gram-negative bacteria. Originally considered as an artifact of the cell wall, OMVs are now recognized as a general secretion system, which serves to improve the fitness of bacteria and facilitate bacterial interactions in polymicrobial communities as well as interactions between the microbe and the host. In general, OMVs are released in increased amounts from pathogenic bacteria and have been found to harbor much of the contents of the parental bacterium. They mainly encompass components of the outer membrane and the periplasm including various virulence factors such as toxins, adhesins, and immunomodulatory molecules. Numerous studies have clearly shown that the delivery of toxins and other virulence factors via OMVs essentially influences their interactions with host cells. Here, we review the OMV-mediated intracellular deployment of toxins and other virulence factors with a special focus on intestinal pathogenic Escherichia coli. Especially, OMVs ubiquitously produced and secreted by enterohemorrhagic E. coli (EHEC) appear as a highly advanced mechanism for secretion and simultaneous, coordinated and direct delivery of bacterial virulence factors into host cells. OMV-associated virulence factors are not only stabilized by the association with OMVs, but can also often target previously unknown target structures and perform novel activities. The toxins are released by OMVs in their active forms and are transported via cell sorting processes to their specific cell compartments, where they can develop their detrimental effects. OMVs can be considered as bacterial "long distance weapons" that attack host tissues and help bacterial pathogens to establish the colonization of their biological niche(s), impair host cell function, and modulate the defense of the host. Thus, OMVs contribute significantly to the virulence of the pathogenic bacteria.

• MeSH
• bakteriální sekreční systémy metabolismus   MeSH
• bakteriální toxiny metabolismus   MeSH
• enterohemoragická Escherichia coli metabolismus   patogenita   MeSH
• enterotoxigenní Escherichia coli metabolismus   patogenita   MeSH
• enterotoxiny metabolismus   MeSH
• faktory virulence metabolismus   MeSH
• fyziologický stres MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• lidé MeSH
• proteolipidy metabolismus   ultrastruktura   MeSH
• střeva mikrobiologie   MeSH
• transport proteinů MeSH
• virulence MeSH
• vnější bakteriální membrána metabolismus   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

Druhá kazuistika se zabývá případem akutní gastroenteritidy u batolete vyvolané třemi patogeny najednou – duální virovou infekcí (rotaviry a noroviry typ I) současně s Escherichia coli O 157:H7 a komplikované rozvojem hemolyticko-uremického syndromu s nutností dialýzy a krevních transfuzí.

The second case report deals with a case of acute gastroenteritis in a toddler caused simultaneously by three pathogens – dual viral infection (rotaviruses and noroviruses type I) together with Escherichia coli O157:H7 and complicated by the development of haemolytic-uremic syndrome with the necessity of dialysis and blood transfusions.

• MeSH
• dehydratace terapie   MeSH
• dialýza ledvin metody   MeSH
• dítě MeSH
• enterohemoragická Escherichia coli patogenita   MeSH
• Escherichia coli O157 izolace a purifikace   patogenita   MeSH
• gastroenteritida * terapie   virologie   MeSH
• gastrointestinální látky terapeutické užití   MeSH
• hemolyticko-uremický syndrom komplikace   terapie   MeSH
• lidé MeSH
• předškolní dítě MeSH
• probiotika terapeutické užití   MeSH
• rehydratační roztoky terapeutické užití   MeSH
• rotavirové infekce * komplikace   terapie   MeSH
• shiga toxiny izolace a purifikace   škodlivé účinky   MeSH
• vakcína proti rotavirům terapeutické užití   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• kazuistiky MeSH

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H-, the most common non-O157 serotype causing hemolytic uremic syndrome worldwide, are evolutionarily highly dynamic with new pathogenic clones emerging rapidly. Here, we investigated the population structure of EHEC O26 isolated from patients in several European countries using whole genome sequencing, with emphasis on a detailed analysis of strains of the highly virulent new European clone (nEC) which has spread since 1990s. RESULTS: Genome-wide single nucleotide polymorphism (SNP)-based analysis of 32 EHEC O26 isolated in the Czech Republic, Germany, Austria and Italy demonstrated a split of the nEC (ST29C2 clonal group) into two distinct lineages, which we termed, based on their temporal emergence, as "early" nEC and "late" nEC. The evolutionary divergence of the early nEC and late nEC is marked by the presence of 59 and 70 lineage-specific SNPs (synapomorphic mutations) in the genomes of the respective lineages. In silico analyses of publicly available E. coli O26 genomic sequences identified the late nEC lineage worldwide. Using a PCR designed to target the late nEC synapomorphic mutation in the sen/ent gene, we identified the early nEC decline accompanied by the late nEC rise in Germany and the Czech Republic since 2004 and 2013, respectively. Most of the late nEC strains harbor one of two major types of Shiga toxin 2a (Stx2a)-encoding prophages. The type I stx2a-phage is virtually identical to stx2a-phage of EHEC O104:H4 outbreak strain, whereas the type II stx2a-phage is a hybrid of EHEC O104:H4 and EHEC O157:H7 stx2a-phages and carries a novel mutation in Stx2a. Strains harboring these two phage types do not differ by the amounts and biological activities of Stx2a produced. CONCLUSIONS: Using SNP-level analyses, we provide the evidence of the evolutionary split of EHEC O26:H11/H- nEC into two distinct lineages, and a recent replacement of the early nEC by the late nEC in Germany and the Czech Republic. PCR targeting the late nEC synapomorphic mutation in ent/sen enables the discrimination of early nEC strains and late nEC strains in clinical and environmental samples, thereby facilitating further investigations of their geographic distribution, prevalence, clinical significance and epidemiology.

• MeSH
• biologická evoluce * MeSH
• DNA bakterií MeSH
• enterohemoragická Escherichia coli klasifikace   genetika   izolace a purifikace   MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• genom bakteriální * MeSH
• genomika MeSH
• infekce vyvolané Escherichia coli diagnóza   epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• molekulární epidemiologie MeSH
• sekvenování celého genomu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H- induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines. H7 flagellin was the key IL-8-inducing component of EHEC O157:H7 OMVs, whereas cytolethal distending toxin V and O157 lipopolysaccharide (LPS) largely contributed to IL-8 production elicited by flagellin-lacking OMVs from SF EHEC O157:H-. The H7 flagellin-mediated signaling via Toll-like receptor (TLR) 5, and O157 LPS-mediated signaling via TLR4/MD-2 complex, which were followed by activation of the nuclear factor NF-κB were major pathways underlying IL-8 production induced by EHEC O157 OMVs. The proinflammatory and immunomodulatory capacities of EHEC O157 OMVs have pathogenetic implications and support the OMVs as suitable vaccine candidates.

• MeSH
• buněčná membrána metabolismus   MeSH
• buňky HT-29 MeSH
• Caco-2 buňky MeSH
• epitelové buňky metabolismus   MeSH
• Escherichia coli O157 patogenita   MeSH
• faktory virulence metabolismus   MeSH
• flagelin metabolismus   MeSH
• infekce vyvolané Escherichia coli mikrobiologie   patologie   MeSH
• interleukin-8 biosyntéza   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• NF-kappa B metabolismus   MeSH
• proteiny vnější bakteriální membrány metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   mikrobiologie   patologie   MeSH
• toll-like receptor 4 metabolismus   MeSH
• toll-like receptor 5 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

• MeSH
• Escherichia coli O157 klasifikace   genetika   MeSH
• hybridizace nukleových kyselin MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• Listeria monocytogenes klasifikace   genetika   MeSH
• multiplexová polymerázová řetězová reakce MeSH
• Salmonella klasifikace   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Food of non-animal origin is a major component of the human diet and has been considered to pose a low risk from the point of view of bacteriological safety. However, an increase in the number of outbreaks of illness caused by such pathogens and linked to the consumption of fresh fruit and vegetables have been reported from around the world recently. Salmonella spp., STEC (Shiga toxin producing Escherichia coli) and Listeria monocytogenes are among the most frequently identified agents. Additionally, the transmission of antibiotic resistant strains including also the methicillin resistant S. aureus (MRSA) to humans via the food chain is one of the greatest public health problems being confronted today. Therefore, we focused on the bacterial safety of fruit, vegetables and sprouts on sale in the Czech Republic. One strain (0.3%) of Salmonella Enteritidis phage type PT8, one strain (0.3%) of MRSA and 17 strains (5.0%) of L. monocytogenes were isolated from a total of 339 collected samples. The most problematic commodities were frozen fruit and vegetables (packed and unpacked) and fresh-cut vegetables. Our findings indicate deficiencies in hygiene practices during harvesting, processing and distribution of these commodities. Although sprouts and berries are the most likely to be contaminated by human pathogens, only two samples were positive for the presence of L. monocytogenes.

• MeSH
• Bacteria izolace a purifikace   MeSH
• bezpečnost potravin * MeSH
• Escherichia coli O157 izolace a purifikace   MeSH
• lidé MeSH
• Listeria monocytogenes izolace a purifikace   MeSH
• methicilin rezistentní Staphylococcus aureus izolace a purifikace   MeSH
• ovoce mikrobiologie   MeSH
• počet mikrobiálních kolonií MeSH
• polymerázová řetězová reakce MeSH
• potravinářská mikrobiologie MeSH
• Salmonella enteritidis izolace a purifikace   MeSH
• semenáček mikrobiologie   MeSH
• shiga-toxigenní Escherichia coli izolace a purifikace   MeSH
• techniky typizace bakterií MeSH
• zelenina mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H- strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H- strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H- strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H- strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology.IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H- patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

• MeSH
• Escherichia coli O157 klasifikace   izolace a purifikace   metabolismus   MeSH
• faktory virulence genetika   metabolismus   MeSH
• fermentace MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• lidé MeSH
• plazmidy genetika   metabolismus   MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• sorbitol metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Německo MeSH