Effects of fermentation products of the commensal bacterium Clostridium ramosum on motility, intracellular pH, and flagellar synthesis of enterohemorrhagic Escherichia coli.

The flagellum and motility are crucial virulence factors for many pathogenic bacteria. In general, pathogens invade and translocate through motility and adhere to specific tissue via flagella. Therefore, the motility and flagella of pathogens are effectual targets for attenuation. Here, we show that the fermentation products of Clostridium ramosum, a commensal intestinal bacterium, decrease the intracellular pH of enterohemorrhagic Escherichia coli (EHEC) and influence its swimming motility. Quantifications of flagellar rotation in individual EHEC cells showed an increase in reversal frequency and a decrease in rotation rate in the presence of C. ramosum fermentation products. Furthermore, the C. ramosum fermentation products affected synthesis of flagellar filaments. The results were reproduced by a combination of organic acids under acidic conditions. Short-chain fatty acids produced by microbes in the gut flora are beneficial for the host, e.g. they prevent infection. Thus, C. ramosum could affect the physiologies of other enteric microbes and host tissues.

Strand-specific transcriptomes of Enterohemorrhagic Escherichia coli in response to interactions with ground beef microbiota: interactions between microorganisms in raw meat.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are zoonotic agents associated with outbreaks worldwide. Growth of EHEC strains in ground beef could be inhibited by background microbiota that is present initially at levels greater than that of the pathogen E. coli. However, how the microbiota outcompetes the pathogenic bacteria is unknown. Our objective was to identify metabolic pathways of EHEC that were altered by natural microbiota in order to improve our understanding of the mechanisms controlling the growth and survival of EHECs in ground beef. RESULTS: Based on 16S metagenomics analysis, we identified the microbial community structure in our beef samples which was an essential preliminary for subtractively analyzing the gene expression of the EHEC strains. Then, we applied strand-specific RNA-seq to investigate the effects of this microbiota on the global gene expression of EHEC O2621765 and O157EDL933 strains by comparison with their behavior in beef meat without microbiota. In strain O2621765, the expression of genes connected with nitrate metabolism and nitrite detoxification, DNA repair, iron and nickel acquisition and carbohydrate metabolism, and numerous genes involved in amino acid metabolism were down-regulated. Further, the observed repression of ftsL and murF, involved respectively in building the cytokinetic ring apparatus and in synthesizing the cytoplasmic precursor of cell wall peptidoglycan, might help to explain the microbiota's inhibitory effect on EHECs. For strain O157EDL933, the induced expression of the genes implicated in detoxification and the general stress response and the repressed expression of the peR gene, a gene negatively associated with the virulence phenotype, might be linked to the survival and virulence of O157:H7 in ground beef with microbiota. CONCLUSION: In the present study, we show how RNA-Seq coupled with a 16S metagenomics analysis can be used to identify the effects of a complex microbial community on relevant functions of an individual microbe within it. These findings add to our understanding of the behavior of EHECs in ground beef. By measuring transcriptional responses of EHEC, we could identify putative targets which may be useful to develop new strategies to limit their shedding in ground meat thus reducing the risk of human illnesses.

[The characteristics of biological properties of E. coli O104:H4--the causative agent of large-scale alimentary ictus in Germany may 2001].

The review presents the characteristics of E. coli O104:H4, the causative agent of large-scale alimentary ictus in Germany in spring time 2011. The antigenic characteristics and factors of E. coli pathogenicity are taken into account. The causative agent has a combination of pathogenic factors of two groups of diarrheigenic Escherichia: shigella similar toxin, specific for entero-hemorrhagic E. coli and adhesins of enteroaggregative E. coli.

High stability of Stx2 phage in food and under food-processing conditions.

Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δstx::cat) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ. The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions.

Spontaneous and specific binding of enterohemorrhagic Escherichia coli to overoxidized polypyrrole-coated microspheres.

Specific identification of enterohemorrhagic Escherichia coli was achieved using microspheres coated with overoxidized polypyrrole. The microspheres are well dispersed in aqueous media, and they specifically, spontaneously, and efficiently bind E. coli O157:H7 through surface area effects. In addition, we found that light-scattering by a single microsphere depended linearly on the number of bound cells.

Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx) and the type III secretion system (T3SS), including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3-4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut.

Microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

Enterohaemorrhagic and enteropathogenic Escherichia coli Tir proteins trigger a common Nck-independent actin assembly pathway.

The Tir proteins of enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are each translocated into the host plasma membrane where they promote F-actin pedestals in epithelial cells beneath adherent bacteria, but the two proteins act by different means. The canonical EPEC Tir becomes phosphorylated on tyrosine residue 474 (Y474) to recruit the host adaptor protein Nck, and also stimulates an inefficient, Nck-independent pathway utilizing tyrosine residue 454 (Y454). In contrast, the canonical EHEC Tir lacks Y474 and instead utilizes residues 452-463 to recruit EspF(U), an EHEC-specific effector that stimulates robust Nck-independent actin assembly. EHEC Tir Y458 and EPEC Tir Y454 are both part of an asparagine-proline-tyrosine (NPY) sequence. We report that each of the EHEC Tir NPY residues is required for EspF(U) recruitment and pedestal formation, and each of the EPEC Tir NPY residues is critical for inefficient, Nck-independent pedestal formation. Introduction of EspF(U) into EPEC dramatically enhanced Nck-independent actin assembly by EPEC Tir in a manner dependent on NPY(454). These results suggest that EPEC and EHEC Tir trigger a common Nck-independent actin assembly pathway and are both derived from an ancestral Tir molecule that utilized NPY to stimulate low-level pedestal formation.