Three different routes of EHEC O157:H7 infection were used to establish EHEC broiler model.

In order to study the prevention and control EHEC disease measures in poultry, the infection process and development of this disease and the pathological changes of various organs were to be observed. In this study, chickens were infected with different doses of enterohemorrhagic Escherichia coli (EHEC) O157:H7 using different routes of administration to establish EHEC broiler model. A total of 195 14-day-old broilers were randomly divided into 13 groups: including control group, Enema-drip groups (1010, 1011, 1012, 1013 CFUs E. coli O157:H7), gavage groups (P.O) (1011, 1012, 1013, 1014 CFUs E. coli O157:H7), and intraperitoneal injection group (I.P.) (108, 109, 1010, 1011 CFUs E. coli O157:H7). Escherichia coli (E. coli) was given using enema-drip, gavage or intraperitoneal infection. Then the feed intake, weight changes, stool and clinical symptoms of the chicks were recorded during the experiment. 7 d after E. coli infection, blood was collected from the jugular vein and serological tests were carried out. The liver, spleen, and colon of the chicks were extracted to get the organ index, bacteria load, and their histopathological changes. After infection with E. coli, some chicks feces were green or red watery stool, sometimes accompanied by foam, and the material to weight ratio of broilers in I.P. group increased significantly (P < 0.05), the 108 CFUs group were 1.3 times as large as control group. Three modeling methods can result in abnormal serum lipid metabolism and liver function indexes (increase of AST, TBA, T-Bil and TC level; decrease of ALB, TG, and TP level). Infection of chicks with O157:H7 by all 3 methods resulted in its detection in the liver, spleen, and colon. Three modeling methods significantly decreased liver index, and inflammatory cell infiltration and hyperemia were observed in liver. The spleen index in E. coli broilers by gavage and enema-drip was significantly decreased, splenic hyperemia and periarteriolar hyalinosis were observed. The spleen was enlarged with purplish-black spheroids in I.P. group broilers, and the spleen histological changes was more serious. The colon villi of broilers in gavage and enema-drip groups were thinner, more prone to rupture, intestinal lamina propria hyperemia, and inflammatory cell infiltration. Moreover, the number of goblet cells in the mucosal epithelium increased. E. coli O157:H7 can induce liver, spleen and intestinal damage and reduce growth performance of chicks. By comparing these 3 methods, we found that chicks infected with O157:H7 by gavage had more severe liver and intestinal damage, the enema-drip method caused most serious intestinal damage, and I.P. method significantly damaged the liver and spleen of chickens.

Non-canonical transcriptional start sites in E. coli O157:H7 EDL933 are regulated and appear in surprisingly high numbers.

Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized that many such signals represent noise or pervasive transcription, not associated with a biological function. Here, a modified Cappable-seq protocol allows determining the primary transcriptome of the enterohemorrhagic E. coli O157:H7 EDL933 (EHEC). We used four different growth media, both in exponential and stationary growth phase, replicated each thrice. This yielded 19,975 EHEC canonical and non-canonical TSS, which reproducibly occurring in three biological replicates. This questions the hypothesis of experimental noise or pervasive transcription. Accordingly, conserved promoter motifs were found upstream indicating proper TSSs. More than 50% of 5,567 canonical and between 32% and 47% of 10,355 non-canonical TSS were differentially expressed in different media and growth phases, providing evidence for a potential biological function also of non-canonical TSS. Thus, reproducible and environmentally regulated expression suggests that a substantial number of the non-canonical TSSs may be of unknown function rather than being the result of noise or pervasive transcription.

Enterohemorrhagic Escherichia coli senses microbiota-derived nicotinamide to increase its virulence and colonization in the large intestine.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen that specifically colonizes and infects the human large intestine. EHEC O157:H7 engages intricate regulatory pathways to detect host intestinal signals and regulate virulence-related gene expression during colonization and infection. However, the overall EHEC O157:H7 virulence regulatory network in the human large intestine remains incompletely understood. Here, we report a complete signal regulatory pathway where the EvgSA two-component system responds to high-nicotinamide levels produced by microbiota in the large intestine and directly activates loci of enterocyte effacement genes to promote EHEC O157:H7 adherence and colonization. This EvgSA-mediated nicotinamide signaling regulatory pathway is conserved and widespread among several other EHEC serotypes. Moreover, disruption of this virulence-regulating pathway by the deletion of evgS or evgA significantly decreased EHEC O157:H7 adherence and colonization in the mouse intestinal tract, indicating that these genes could be potential targets for the development of new therapeutics for EHEC O157:H7 infection.

Enterohemorrhagic Escherichia coli O157:H7 - Xuzhou City, Jiangsu Province, China, 2001-2021.

What is already known about this topic?: The largest and longest outbreak of diarrhea, which was complicated with hemolytic uremic syndrome (HUS) caused by enterohemorrhagic Escherichia coli (EHEC) O157:H7, occurred in Xuzhou City and its adjacent areas from 1999 to 2000 in China. What is added by this report?: According to surveillance results from 2001 to 2021, there was a significant decrease in the isolation rate of O157:H7, and cattle and sheep remained the primary hosts. However, non-Shiga toxin-producing O157:H7 emerged as the dominant strain, with stx2+stx1- strains following closely behind. What are the implications for public health practice?: National surveillance of O157:H7 effectively serves as an early warning system and guidance for assessing the intensity and trend of disease epidemics. It is crucial to raise awareness of the public health risks associated with Shiga toxin-producing E. coli.

A Novel Role of the Two-Component System Response Regulator UvrY in Enterohemorrhagic Escherichia coli O157:H7 Pathogenicity Regulation.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen causing severe diseases, such as hemorrhagic colitis and lethal hemolytic uremic syndrome. The signal-sensing capability of EHEC O157:H7 at specific host colonization sites via different two-component systems (TCSs) is closely related to its pathogenicity during infection. However, the types of systems involved and the regulatory mechanisms are not fully understood. Here, we investigated the function of the TCS BarA/UvrY regulator UvrY in the pathogenicity regulation of EHEC O157:H7. Our results showed that UvrY acts as a positive regulator of EHEC O157:H7 for cellular adherence and mouse colonization through the transcriptional activation of the locus for enterocyte effacement (LEE) pathogenic genes. Furthermore, this regulation is mediated by the LEE island master regulator, Ler. Our results highlight the significance of UvrY in EHEC O157:H7 pathogenicity and underline the unknown importance of BarA/UvrY in colonization establishment and intestinal adaptability during infection.

Three Novel Antisense Overlapping Genes in E. coli O157:H7 EDL933.

The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated. To date, only a few examples of such genes are fully established. Using RNA sequencing and ribosome profiling, we found expression of novel overlapping open reading frames in Escherichia coli O157:H7 EDL933 (EHEC). Indeed, the overlapping candidate genes are equipped with typical structural elements required for transcription and translation, i.e., promoters, transcription start sites, as well as terminators, all of which were experimentally verified. Translationally arrested mutants, unable to produce the overlapping encoded protein, were found to have a growth disadvantage when grown competitively against the wild type. Thus, the phenotypes found imply biological functionality of the genes at the level of proteins produced. The addition of 3 more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes emphasizes the underestimated coding capacity of bacterial genomes. IMPORTANCE The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated, since such genes are not allowed in genome annotations. However, ribosome profiling catches mRNA in the moment of being template for protein production. Using this technique and subsequent experiments, we verified 3 novel overlapping genes encoded in antisense of known genes. This adds more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes.

Human cathelicidin LL-37 exerts amelioration effects against EHEC O157:H7 infection regarding inflammation, enteric dysbacteriosis, and impairment of gut barrier function.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection impairs intestinal barrier function, causing intestinal inflammation and enteric dysbacteriosis. The human cathelicidin LL-37 can regulate excessive inflammatory responses, barrier function, and balance the intestinal microbial community; however, little is known about its effects on inflammation, intestinal barrier function, and microbiota disorders in EHEC O157:H7-infected mice. In this study, we investigated the protective effect of LL-37 against EHEC O157:H7 infection and elucidated the underlying mechanism using a mouse model. LL-37 treatment was found to inhibit body weight loss, restore edema and destruction of the intestinal villi, and significantly reduce epithelial apoptosis (P < 0.05) in EHEC O157:H7-infected mice. Furthermore, inflammatory infiltration of macrophages and neutrophils into the jejunum and colon was significantly decreased (P < 0.05). LL-37 significantly downregulated the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) (P < 0.05) and upregulated the anti-inflammatory cytokine (IL-10) during EHEC O157:H7 infection. LL-37 increased the expression of tight junction proteins (ZO-1, ZO-2, claudin-1, and occludin), which are associated with intestinal barrier function, and had a positive effect on EHEC O157:H7-induced microbial disorders, particularly in terms of the inflammation-related microbiota. LL-37 also significantly decreased the E. coli load in the liver and spleen (P < 0.01) and restored the structure of the liver and kidney. Taken together, LL-37 conferred protection in a EHEC O157:H7-induced mouse model by reducing intestinal inflammation, enhancing intestinal barrier function, and restoring the balance of the intestinal microbiota, which indicates the therapeutic potential of LL-37 against pathogen infection.

Application of a novel lytic phage vB_EcoM_SQ17 for the biocontrol of Enterohemorrhagic Escherichia coli O157:H7 and Enterotoxigenic E. coli in food matrices.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and Enterotoxigenic E. coli (ETEC) are important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages, as novel antibacterial agents, have been increasingly exploited to control foodborne pathogens. In this study, a novel broad-host range lytic phage vB_EcoM_SQ17 (SQ17), was isolated, characterized, and evaluated for its potential to control bacterial counts in vitro and in three different food matrices (milk, raw beef, and fresh lettuce). Phage SQ17 was capable of infecting EHEC O157:H7, ETEC, and other E. coli strains. Morphology, one-step growth, and stability assay showed that phage SQ17 belongs to the Caudovirales order, Myoviridae family, and Mosigvirus genus. It has a short latent period of 10 min, a burst size of 71 PFU/infected cell, high stability between pH 4 to 12 as well as thermostability between 30°C and 60°C for 60 min. Genome sequencing analysis revealed that the genome of SQ17 does not contain any genes associated with antibiotic resistance, toxins, lysogeny, or virulence factors, indicating the potential safe application of phage SQ17 in the food industry. In Luria-Bertani (LB) medium, phage SQ17 significantly decreased the viable counts of EHEC O157:H7 by more than 2.40 log CFU/ml (p < 0.05) after 6 h of incubation at 37°C. Phage SQ17 showed great potential to be applied for biocontrol of EHEC O157:H7 in milk and raw beef. In fresh lettuce, treatment with SQ17 also resulted in significant reduction of viable cell counts of EHEC O157:H7 and ETEC at both 4°C and 25°C. Our results demonstrate that SQ17 is a good candidate for application as an EHEC O157:H7 and ETEC biocontrol agent in the processing stages of food production and food preservation.

Regulation of flagellar motility and biosynthesis in enterohemorrhagic Escherichia coli O157:H7.

ABSTARCTEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen that causes a variety of diseases, such as hemorrhagic colitis and lethal hemolytic uremic syndrome. Flagellum-dependent motility plays diverse roles in the pathogenesis of EHEC O157:H7, including its migration to an optimal host site, adherence and colonization, survival at the infection site, and post-infection dispersal. However, it is very expensive for cellular economy in terms of the number of genes and the energy required for flagellar biosynthesis and functioning. Furthermore, the flagellar filament bears strong antigenic properties that induce a strong host immune response. Consequently, the flagellar gene expression and biosynthesis are highly regulated to occur at the appropriate time and place by different regulatory influences. The present review focuses on the regulatory mechanisms of EHEC O157:H7 motility and flagellar biosynthesis, especially in terms of flagellar gene regulation by environmental factors, regulatory proteins, and small regulatory RNAs.

Pediococcus pentosaceus IM96 Exerts Protective Effects against Enterohemorrhagic Escherichia coli O157:H7 Infection In Vivo.

Enterohemorrhagic Escherichia coli (EHEC) is a notorious and prevalent foodborne pathogen which can cause serious intestinal diseases. The antagonistic activity of probiotics against EHEC is promising, but most of the studies concerning this subject have been carried out in vitro. Specifically, the interaction between Pediococcus pentosaceus and EHEC O157:H7 in vivo has not been reported yet. In this study, we investigated the protective effect of P. pentosaceus IM96 on EHEC O157:H7-infected female mice in vivo. The results demonstrated that P. pentosaceus IM96 reduced the level of pro-inflammatory factors and increased the level of anti-inflammatory factors of EHEC O157:H7-infected mice. Furthermore, P. pentosaceus IM96 alleviated intestinal mucosal damage and increased the level of MUC-2, tight junction (TJ) proteins, and short chain fatty acids (SCFAs). The intestinal microbial community structure and the diversity and richness of the microbiota were also changed by P. pentosaceus IM96 treatment. In summary, P. pentosaceus IM96 exerted protective effects against EHEC O157:H7 via alleviating intestinal inflammation, strengthening the intestinal barrier function, and regulating intestinal microbiota, suggesting that P. pentosaceus IM96 might serve as a potential microbial agent to prevent and treat intestinal diseases caused by EHEC O157:H7 infection in the future.

Microfluidics for the rapid detection of Escherichia coli O157:H7 using antibody-coated microspheres.

This study developed a novel method for the rapid detection of Escherichia coli (E. coli) O157:H7 on a microfluidic platform. First, the concentration of bacteria in a sample was determined with the adenosine triphosphate (ATP) method. Then, the specific detection of E. coli was achieved in a microfluidic chip by the immune-microsphere technique. The influences of the culture time, flow rate and capture time on the detection of the target bacteria were investigated systematically. Generally, with increasing capture time, more bacteria could be captured by the microspheres, which had a positive effect on bacterial detection. Furthermore, the sensitivity and specificity of the method were also tested. The results showed that this method could specifically detect E. coli with a sensitivity as high as 49.1 cfu/µL; the consumption of bacteria was 1 µL, and the reagent was at the microliter level. The testing time can be controlled within one and a half hours, and the cost of testing was approximately RMB 10. The method described in this article is simple and accurate and has great application value in bacterial detection for medical diagnostics.

Dps Protects Enterohemorrhagic Escherichia coli against Acid-Induced Antimicrobial Peptide Killing.

Dps, a DNA-binding protein from starved cells in Escherichia coli, is part of the bacterial defense system that protects DNA against various cellular stresses. Our lab previously demonstrated that a novel antimicrobial peptide, WRWYCR, enhances acid-induced killing of enterohemorrhagic Escherichia coli (EHEC) and ameliorates infection in a Citrobacter rodentium mouse model of EHEC infection. WRWYCR has previously been shown to compromise DNA damage repair and to increase chelatable iron within the cell. These findings, combined with the effects of peptide and acid stress on DNA damage, suggest a key defense role for Dps in peptide-induced killing of EHEC. The goal of this study is to evaluate the role of Dps in peptide-induced killing of EHEC through survival assays and flow cytometric analyses of DNA damage and hydroxyl radical formation. Our results demonstrate that disruption of the dps gene in stationary-phase EHEC O157:H7 cells, but not in exponential-phase cells, enhances acid-, peptide-, and peptide-acid-induced killing relative to that of wild-type (WT) EHEC. Using flow cytometric analysis, we have also demonstrated increased levels of hydroxyl radicals in peptide-treated wild-type EHEC relative to those in the untreated control. Disruption of the dps gene further increases this. These findings indicate that peptide treatment of EHEC enhances the formation of hydroxyl radicals, likely through the Fenton reaction, thereby contributing to the killing action of the peptide, and that dps protects against peptide killing of EHEC. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials.IMPORTANCE The research presented in this paper explores the role of the DNA-binding protein Dps as a key defense mechanism of enterohemorrhagic Escherichia coli (EHEC) strains in protecting against killing by the novel antimicrobial peptide WRWYCR. Our results demonstrate that Dps protects against peptide-induced killing of EHEC through direct protection against acid stress and hydroxyl radical formation, both of which are mechanisms targeted by the antimicrobial peptide. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials through specific targeting of Dps in order to allow a more potent response to the antimicrobial WRWYCR.

Expression and Functional Characterization of Various Chaperon-Usher Fimbriae, Curli Fimbriae, and Type 4 Pili of Enterohemorrhagic Escherichia coli O157:H7 Sakai.

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic strain leading to hemorrhagic colitis and to the hemolytic-uremic syndrome (HUS) in humans. The mechanisms by which pathogenic E. coli infect and colonize humans leading to the typical disease pattern are in focus of many investigations. The adhesion of EHEC to epithelial cells by the coordinated translocation of receptor Tir and surface expression of corresponding adhesin intimin is a key event in host-pathogen-interaction. However, less is known about other adhesins encoded by EHEC, especially about the complex set of fimbrial adhesins varying among various serotypes. Here, we investigate EHEC serotype O157:H7 strain Sakai possessing at least 16 putative fimbrial gene clusters. Using a synthetic heterologous expression system in a non-pathogenic E. coli strain, a subset of 6 gene clusters for fimbrial adhesins was analyzed. We were able to visualize surface expression of two γ1 class fimbriae (Fim and Ycb), two γ4 class fimbriae (Yad and Yeh), and two fimbrial adhesins which are assembled by the nucleation/precipitation pathway (Curli fimbriae), and by a type 2 secretion system (type 4 pili). Further, we elucidated the impact of these fimbrial adhesins in adhesion to various epithelial cells lines (HeLa, MDCK, and CaCo2), and the contribution on biofilm formation. We demonstrate the ultrastructure of Fim fimbriae and Yad fimbriae of EHEC Sakai, and Yeh fimbriae of E. coli in general. The involvement of Fim fimbriae of EHEC Sakai to adhesion to various epithelial cell lines, and contribution to biofilm formation is reported here. Our approach provides first ultrastructural and functional data for novel EHEC adhesins, and enables further understanding of the involvement of fimbrial adhesins in pathogenesis of EHEC Sakai.

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC).

Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. An active σ70 promoter, transcription start site (TSS), Shine-Dalgarno motif and rho-independent terminator were experimentally validated, providing evidence that this open reading frame has all the structural features of a functional gene. Furthermore, ribosomal profiling revealed translation of the mRNA, the protein was detected in Western blots and a pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene), thus adding another example to the growing list of overlapping, protein coding genes in bacteria.

Transcriptional Activator OvrA Encoded in O Island 19 Modulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7.

The human intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes bloody diarrhea, hemorrhagic colitis, and fatal hemolytic uremic syndrome. Its genome contains 177 unique O islands (OIs), which contribute largely to the high virulence and pathogenicity although most OI genes remain uncharacterized. In the current study, we demonstrated that OI-19 is required for EHEC O157:H7 adherence to host cells. Z0442 (OI-encoded virulence regulator A [OvrA]) encoded in OI-19 positively regulated bacterial adherence by activating locus of enterocyte effacement (LEE) gene expression through direct OvrA binding to the gene promoter region of the LEE gene master regulator Ler. Mouse colonization experiments revealed that OvrA promotes EHEC O157:H7 adherence in mouse intestine, preferentially the colon. Finally, OvrA also regulated virulence in other non-O157 pathogenic E. coli, including EHEC strains O145:H28 and O157:H16 and enteropathogenic E. coli strain O55:H7. Our work markedly enriches the understanding of bacterial adherence control and provides another example of laterally acquired regulators that mediate LEE gene expression.

Transcriptomic analysis reveals specific metabolic pathways of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents.

BACKGROUND: The cattle gastrointestinal tract (GIT) is the main enterohemorrhagic Escherichia coli (EHEC) reservoir. In order to identify nutrients required for the survival or multiplication of EHEC in the bovine GIT, we compared the transcriptomes of the EHEC O157:H7 reference strain EDL933 cultured in vitro in bovine digestive contents (DCs) (rumen, small intestine and rectum) using RNA-sequencing. RESULTS: Gene expression profiles showed that EHEC EDL933 activated common but also specific metabolic pathways to survive in the different bovine DCs. Mucus-derived carbohydrates seem important in EHEC nutrition in posterior DCs (small intestine and rectum) but not in rumen content. Additional carbohydrates (xylose, ribose, mannitol, galactitol) as well as gluconeogenic substrates (aspartate, serine, glycerol) would also be used by EHEC as carbon and/or nitrogen sources all along the bovine GIT including the rumen. However, xylose, GalNac, ribose and fucose transport and/or assimilation encoding genes were over-expressed during incubation in rectum content compared with rumen and intestine contents, and genes coding for maltose transport were only induced in rectum. This suggests a role for these carbohydrates in the colonization of the cattle rectum, considered as the major site for EHEC multiplication. In contrast, the transcription of the genes associated with the assimilation of ethanolamine, an important nitrogen source for EHEC, was poorly induced in EHEC growing in rectum content, suggesting that ethanolamine is mainly assimilated in the cattle rumen and small intestine. Respiratory flexibility would also be required for EHEC survival because of the redundancy of dehydrogenases and reductases simultaneously induced in the bovine DCs, probably in response to the availability of electron donors and acceptors. CONCLUSION: EHEC EDL933 showed a high flexibility in the activation of genes involved in respiratory pathways and assimilation of carbon and nitrogen sources, most of them from animal origin. This may allow the bacterium to adapt and survive in the various bovine GIT compartments. Obtaining a better understanding of EHEC physiology in bovine GIT is a key step to ultimately propose strategies to limit EHEC carriage and shedding by cattle.

The Fimbrial Gene z3276 in Enterohemorrhagic Escherichia coli O157:H7 Contributes to Bacterial Pathogenicity.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. It is not capable of expressing type I fimbrial because of base deletion in the fim operon. BLAST analysis shows that the open reading frame z3276, a specific genetic marker of EHEC O157:H7, encodes a sequence with high amino acid identity to other E. coli type I fimbrial, but its definitive function in EHEC O157:H7 remains unclear. We are here to report that a z3276 mutant (Δz3276) was constructed using the reference EHEC O157:H7, the mutant Δz3276 was biologically characterized, and the pathogenicity of Δz3276 was assessed in mice in comparison with the wild-type (WT) strain. Motility and biofilm formation assays revealed a smaller twitching motility zone for Δz3276 on the agar surface and significantly decreased biofilm formation by Δz3276 compared with the parental strain. The adhesion and invasion ability of Δz3276 to HEp-2 cells showed no significant change, but the invasion ability of Δz3276 to IPEC-J2 cells was attenuated. Furthermore, in the animal study, we observed shortened and lower fecal shedding among the Δz3276 mutant-infected animals compared with those infected WT strain. The data in this study indicate that this unique gene of z3276 in EHEC O157:H7 seems to play an important role in bacterial pathogenicity.

The Novel Anaerobiosis-Responsive Overlapping Gene ano Is Overlapping Antisense to the Annotated Gene ECs2385 of Escherichia coli O157:H7 Sakai.

Current notion presumes that only one protein is encoded at a given bacterial genetic locus. However, transcription and translation of an overlapping open reading frame (ORF) of 186 bp length were discovered by RNAseq and RIBOseq experiments. This ORF is almost completely embedded in the annotated L,D-transpeptidase gene ECs2385 of Escherichia coli O157:H7 Sakai in the antisense reading frame -3. The ORF is transcribed as part of a bicistronic mRNA, which includes the annotated upstream gene ECs2384, encoding a murein lipoprotein. The transcriptional start site of the operon resides 38 bp upstream of the ECs2384 start codon and is driven by a predicted σ70 promoter, which is constitutively active under different growth conditions. The bicistronic operon contains a ρ-independent terminator just upstream of the novel gene, significantly decreasing its transcription. The novel gene can be stably expressed as an EGFP-fusion protein and a translationally arrested mutant of ano, unable to produce the protein, shows a growth advantage in competitive growth experiments compared to the wild type under anaerobiosis. Therefore, the novel antisense overlapping gene is named ano (anaerobiosis responsive overlapping gene). A phylostratigraphic analysis indicates that ano originated very recently de novo by overprinting after the Escherichia/Shigella clade separated from other enterobacteria. Therefore, ano is one of the very rare cases of overlapping genes known in the genus Escherichia.

Proteomic changes in EHEC O157:H7 under catechin intervention.

Catechin exhibits antimicrobial activity against various microorganisms, such as EHEC O157:H7. This study reports the bactericidal effect of catechin on EHEC O157:H7 in simulated human gastrointestinal environment and the underlying antibacterial mechanism. Bacteriostasis test results showed that the minimum bactericidal concentration of catechin for EHEC O157:H7 was 5 g/L. The bactericidal effect of catechin in the gastrointestinal juice became more significant with increased culture time, and catechin exhibited a synergistic effect with bile salt in inhibiting EHEC O157:H7. Changes in the profile of protein expression in EHEC O157:H7 in response to catechin intervention were investigated. Two-dimensional electrophoresis identified 34 proteins with significantly altered expression. A total of 2 and 12 proteins were upregulated and downregulated, respectively. However, 20 proteins disappeared. No new protein was expressed compared with the control. Hence, catechin intervention resulted in diverse changes in the expression of proteins associated with cell structure and genetic information processing. Catechin could cause the disappearance of certain proteins or the destruction of certain peptides. These processes lead to the inhibition of EHEC O157: H7 cells.

The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7.

The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics.