Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Mechanism of translation inhibition by type II GNAT toxin AtaT2.

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.

The sRNA DicF integrates oxygen sensing to enhance enterohemorrhagic Escherichia coli virulence via distinctive RNA control mechanisms.

To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract (GIT) and precisely control expression of virulence determinants. During passage through the GIT, pathogens encounter relatively high levels of oxygen in the small intestine before transit to the oxygen-limited environment of the colon. However, how bacterial pathogens sense oxygen availability and coordinate expression of virulence traits is not resolved. Here, we demonstrate that enterohemorrhagic Escherichia coli O157:H7 (EHEC) regulates virulence via the oxygen-responsive small RNA DicF. Under oxygen-limited conditions, DicF enhances global expression of the EHEC type three secretion system, which is a key virulence factor required for host colonization, through the transcriptional activator PchA. Mechanistically, the pchA coding sequence (CDS) base pairs with the 5' untranslated region of the mRNA to sequester the ribosome binding site (RBS) and inhibit translation. DicF disrupts pchA cis-interactions by binding to the pchA CDS, thereby unmasking the pchA RBS and promoting PchA expression. These findings uncover a feed-forward regulatory pathway that involves distinctive mechanisms of RNA-based regulation and that provides spatiotemporal control of EHEC virulence.

Differential Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.

Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes.

BACKGROUND: Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. MATERIALS AND METHODS: A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. RESULTS: Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. CONCLUSION: It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.

Characterization of a non-glycosylated fraction from honey proteins of Melipona beecheii with antimicrobial activity against Escherichia coli O157:H7.

AIMS: To analyse the non-glycosylated protein fraction from Melipona beecheii honey for antimicrobial activity against Escherichia coli O157:H7. METHODS AND RESULTS: The proteins from M. beecheii honey were separated according to their degree of glycosylation using Concanavalin A-affinity chromatography. The total protein extract and its fractions were analysed by 1D and 2D electrophoresis. We also determined the antimicrobial and antihaemolytic activities of the total protein extract and the non-glycosylated fraction. Furthermore, we evaluated the effect of this non-glycosylated fraction for the expression of the Stx1, Stx2, EAE and HlyA pathogen genes. Melipona beecheii honey contained at least 24 proteins with molecular weights ranging between 7·6 and 95 kDa and isoelectric points between 3 and 10, three proteins from the 24 are non-glycosylated. The non-glycosylated fraction had an MIC90 of 1·128 µg ml-1 , and this fraction inhibited the haemolytic activity of the pathogen, as well as reduced the expression of Stx1, Stx2 and HlyA. The MbF1-2 protein from the non-glycosylated fraction was sequenced and identified as a homologue of the royal jelly-like protein of Melipona quadrifasciata. CONCLUSIONS: The non-glycosylated protein fraction from M. beecheii honey greatly contributes to antibacterial activity and it is composed of at least three proteins, of which MbF1-2 provided over 50% of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed significant antimicrobial activity from several proteins present in the honey of M. beecheii. Interestingly, the non-glycosylated protein fraction demonstrated antihaemolytic activity and adversely affected the expression of virulence genes in Escherichia coli O157:H7; these proteins have the potential to be used in developing therapeutic agents against this bacterium.

Occurrence, Molecular Characteristics, and Antimicrobial Resistance of Escherichia coli O157 in Cattle, Beef, and Humans in Bishoftu Town, Central Ethiopia.

Escherichia coli O157 is a Shiga toxin-producing E. coli causing disease in humans. Cattle are the primary reservoir of the pathogen. Information regarding the contribution of cattle to diarrheal illnesses in humans through consumption of contaminated beef is scarce in Ethiopia. We collected samples from 240 cattle, 127 beef, and 216 diarrheic patients in Bishoftu town in Ethiopia to assess the occurrence and determine the virulence genes, genetic relatedness, and antimicrobial resistance of E. coli O157. E. coli O157 was detected in 7.1% of the rectal content samples from cattle in slaughterhouses, in 6.3% (n = 127) of the beef samples, and in 2.8% of the diarrheic patients' stool samples. All isolates were positive for eae gene, 24 (77%) of them were positive for stx2 gene (21 stx2c and 3 stx2a), whereas stx1 gene was not detected. Molecular typing grouped the isolates into eight pulsed-field gel electrophoresis pulsotypes with three pulsotypes containing isolates from all three sources, one pulsotype containing one isolate from human origin and one isolate from beef. The remaining four pulsotypes contained isolates unique either to beef or to humans. With the exception of 1 multidrug-resistant isolate from beef, which was resistant to 8 antimicrobial drugs, the remaining 30 isolates were susceptible to the 14 antimicrobials tested. In conclusion, the finding of genetically similar isolates in cattle, beef, and humans may indicate a potential transmission of E. coli O157 from cattle to humans through beef. However, more robust studies are required to confirm this epidemiological link.

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.

Novel molecular components involved in callose-mediated Arabidopsis defense against Salmonella enterica and Escherichia coli O157:H7.

BACKGROUND: Food contamination with Salmonella enterica and enterohemorrhagic Escherichia coli is among the leading causes of foodborne illnesses worldwide and crop plants are associated with > 50% of the disease outbreaks. However, the mechanisms underlying the interaction of these human pathogens with plants remain elusive. In this study, we have explored plant resistance mechanisms against these enterobacteria and the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3118, as an opportunity to improve food safety. RESULTS: We found that S. enterica serovar Typhimurium (STm) transcriptionally modulates stress responses in Arabidopsis leaves, including induction of two hallmark processes of plant defense: ROS burst and cell wall modifications. Analyses of plants with a mutation in the potentially STm-induced gene EXO70H4 revealed that its encoded protein is required for stomatal defense against STm and E. coli O157:H7, but not against Pst DC3118. In the apoplast however, EXO70H4 is required for defense against STm and Pst DC3118, but not against E. coli O157:H7. Moreover, EXO70H4 is required for callose deposition, but had no function in ROS burst, triggered by all three bacteria. The salicylic acid (SA) signaling and biosynthesis proteins NPR1 and ICS1, respectively, were involved in stomatal and apoplastic defense, as well as callose deposition, against human and plant pathogens. CONCLUSIONS: The results show that EXO70H4 is involved in stomatal and apoplastic defenses in Arabidopsis and suggest that EXO70H4-mediated defense play a distinct role in guard cells and leaf mesophyll cells in a bacteria-dependent manner. Nonetheless, EXO70H4 contributes to callose deposition in response to both human and plant pathogens. NPR1 and ICS1, two proteins involved in the SA signaling pathway, are important to inhibit leaf internalization and apoplastic persistence of enterobacteria and proliferation of phytopathogens. These findings highlight the existence of unique and shared plant genetic components to fight off diverse bacterial pathogens providing specific targets for the prevention of foodborne diseases.

Vitamin K Analogs Influence the Growth and Virulence Potential of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H2O2), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H2O2 or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H2O2 and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest.IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

Bifidobacterium mongoliense genome seems particularly adapted to milk oligosaccharide digestion leading to production of antivirulent metabolites.

BACKGROUND: Human milk oligosaccharides (HMO) could promote the growth of bifidobacteria, improving young children's health. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulent activity against intestinal pathogens. Bovine milk oligosaccharides (BMO), structurally similar to HMO, are found at high concentration in cow whey. This is particularly observed for 3'-sialyllactose (3'SL). This study focused on enzymes and transport systems involved in HMO/BMO metabolism contained in B. crudilactis and B. mongoliense genomes, two species from bovine milk origin. The ability of B. mongoliense to grow in media supplemented with whey or 3'SL was assessed. Next, the effects of cell-free spent media (CFSM) were tested against the virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. RESULTS: Due to the presence of genes encoding ß-galactosidases, ß-hexosaminidases, α-sialidases and α-fucosidases, B. mongoliense presents a genome more sophisticated and more adapted to the digestion of BMO/HMO than B. crudilactis (which contains only ß-galactosidases). In addition, HMO/BMO digestion involves genes encoding oligosaccharide transport systems found in B. mongoliense but not in B. crudilactis. B. mongoliense seemed able to grow on media supplemented with whey or 3'SL as main source of carbon (8.3 ± 1.0 and 6.7 ± 0.3 log cfu/mL, respectively). CFSM obtained from whey resulted in a significant under-expression of ler, fliC, luxS, stx1 and qseA genes (- 2.2, - 5.3, - 2.4, - 2.5 and - 4.8, respectively; P < 0.05) of E. coli O157:H7. CFSM from 3'SL resulted in a significant up-regulation of luxS (2.0; P < 0.05) gene and a down-regulation of fliC (- 5.0; P < 0.05) gene. CFSM obtained from whey resulted in significant up-regulations of sopD and hil genes (2.9 and 3.5, respectively; P < 0.05) of S. Typhimurium, while CFSM obtained from 3'SL fermentation down-regulated hil and sopD genes (- 2.7 and - 4.2, respectively; P < 0.05). CONCLUSION: From enzymes and transporters highlighted in the genome of B. mongoliense and its potential ability to metabolise 3'SL and whey, B. mongoliense seems well able to digest HMO/BMO. The exact nature of the metabolites contained in CFSM has to be identified still. These results suggest that BMO associated with B. mongoliense could be an interesting synbiotic formulation to maintain or restore intestinal health of young children.

Mechanisms involved in the adaptation of Escherichia coli O157:H7 to the host intestinal microenvironment.

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.

Screen Printed Based Impedimetric Immunosensor for Rapid Detection of Escherichia coli in Drinking Water.

The development of a simple and low cost electrochemical impedance immunosensor based on screen printed gold electrode for rapid detection of Escherichia coli in water is reported. The immunosensor is fabricated by immobilizing anti-E. coli antibodies onto a gold surface in a covalent way by the photochemical immobilization technique, a simple procedure able to bind antibodies upright onto gold surfaces. Impedance spectra are recorded in 0.01 M phosphate buffer solution (PBS) containing 10 mM Fe(CN)63-/Fe(CN)64- as redox probe. The Nyquist plots can be modelled with a modified Randles circuit, identifying the charge transfer resistance Rct as the relevant parameter after the immobilization of antibodies, the blocking with BSA and the binding of E. coli. The introduction of a standard amplification procedure leads to a significant enhancement of the impedance increase, which allows one to measure E. coli in drinking water with a limit of detection of 3 × 101 CFU mL-1 while preserving the rapidity of the method that requires only 1 h to provide a "yes/no" response. Additionally, by applying the Langmuir adsorption model, we are able to describe the change of Rct in terms of the "effective" electrode, which is modified by the detection of the analyte whose microscopic conducting properties can be quantified.

Prevalence and antimicrobial resistance of Shiga toxin-producing Escherichia coli in milk and dairy products in Egypt.

Food contaminated with Shiga toxin-producing Escherichia coli (STEC) represents a hazardous public health problem worldwide. Therefore, the present study was performed to elucidate the virulent and antimicrobial resistance characteristics of STEC isolated from milk and dairy products marketed in Egypt. A total of 125 samples (raw market milk, bulk tank milk, Kareish cheese, white soft cheese, and small scale-produced ice cream, 25 each) were collected for determination the prevalence and antimicrobial resistance profiling of STEC. Thirty-six STEC isolates were recovered from milk and dairy products. Serological analysis illustrated that three isolates were E. coli O157:H7 and 33 isolates belonged to different serotypes. Molecular examination indicated that all isolates harboured stx1 and/or stx2 genes, 14 isolates expressed eaeA gene and 3 isolates possessed rfbE gene. Antimicrobial resistance profiling of the isolates was both phenotypically and genetically examined. Interestingly, 31 out of 36 (86.11%) isolates were multidrug-resistant and harboured the extended-spectrum ß-lactamase encoding genes, namely, blaCTX-M-15, blaSHV-12 and blaCTX-M-14. Moreover, 12 isolates (33.33%) harboured plasmid-mediated quinolone resistant gene, qnrS. The overall conclusion of the current investigation indicated insufficient hygienic measures adopted during milking, handling, and processing leading to development of pathogenic and multidrug-resistant STEC.

A Putative Microcin Amplifies Shiga Toxin 2a Production of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a foodborne pathogen implicated in various multistate outbreaks. It encodes Shiga toxin on a prophage, and Shiga toxin production is linked to phage induction. An E. coli strain, designated 0.1229, that amplified Stx2a production when cocultured with E. coli O157:H7 strain PA2 was identified. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10 min, but not after treatment with proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2-kb region of a 12.8-kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two other E. coli strains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggest that E. coli 0.1229 and other E. coli strains produce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced by E. coli, this study provides an additional mechanism by which stx2a expression is increased in response to the gut microflora.IMPORTANCE How the gut microflora influences the progression of bacterial infections is only beginning to be understood. Antibiotics are counterindicated for E. coli O157:H7 infections, limiting treatment options. An increased understanding of how the gut microflora directs O157:H7 virulence gene expression may lead to additional treatment options. This work identified E. coli strains that enhance the production of Shiga toxin by O157:H7 through the secretion of a proposed microcin. Microcins are natural antimicrobial peptides that target specific species, can act as alternatives to antibiotics, and mediate microbial competition. This work demonstrates another mechanism by which non-O157 E. coli strains may increase Shiga toxin production and adds to our understanding of microcins, a group of antimicrobials less well understood than colicins.

Phylogeographic Analysis Reveals Multiple International transmission Events Have Driven the Global Emergence of Escherichia coli O157:H7.

BACKGROUND: Shiga toxin-producing Escherchia coli (STEC) O157:H7 is a zoonotic pathogen that causes numerous food and waterborne disease outbreaks. It is globally distributed, but its origin and the temporal sequence of its geographical spread are unknown. METHODS: We analyzed whole-genome sequencing data of 757 isolates from 4 continents, and performed a pan-genome analysis to identify the core genome and, from this, extracted single-nucleotide polymorphisms. A timed phylogeographic analysis was performed on a subset of the isolates to investigate its worldwide spread. RESULTS: The common ancestor of this set of isolates occurred around 1890 (1845-1925) and originated from the Netherlands. Phylogeographic analysis identified 34 major transmission events. The earliest were predominantly intercontinental, moving from Europe to Australia around 1937 (1909-1958), to the United States in 1941 (1921-1962), to Canada in 1960 (1943-1979), and from Australia to New Zealand in 1966 (1943-1982). This pre-dates the first reported human case of E. coli O157:H7, which was in 1975 from the United States. CONCLUSIONS: Inter- and intra-continental transmission events have resulted in the current international distribution of E. coli O157:H7, and it is likely that these events were facilitated by animal movements (eg, Holstein Friesian cattle). These findings will inform policy on action that is crucial to reduce the further spread of E. coli O157:H7 and other (emerging) STEC strains globally.

Effects of Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde Adaptation on Virulence Properties of Escherichia coli O157:H7.

Essential oils (EOs) have demonstrated wide-spectrum antimicrobial activities and have been actively studied for their application in foods as alternative natural preservatives. However, information regarding microbial adaptive responses and changes in virulence properties following sublethal EO exposure is still scarce. The present study investigated the effect of sublethal thymol (Thy), carvacrol (Car), or trans-cinnamaldehyde (TC) adaptation on virulence gene expression and virulence properties of Escherichia coli O157:H7. The results demonstrated that E. coli O157:H7 grown to the early stationary phase in the presence of sublethal EO showed significantly (P < 0.05) reduced motility (reversible after stress removal), biofilm-forming ability, and efflux pump activity, with no induction of antibiotic resistance and no significant changes to its adhesion and invasion ability on a human colon adenocarcinoma (Caco-2) cell line. Reverse transcription-quantitative PCR revealed reduced expression of relevant virulence genes, including those encoding flagellar biosynthesis and function, biofilm formation regulators, multidrug efflux pumps, and type III secretion system components. This study demonstrated that Thy, Car, and TC at sublethal concentrations did not potentiate virulence in adapted E. coli O157:H7, which could benefit to their application in the food industry.IMPORTANCE The present study was conducted to evaluate changes in virulence properties in Escherichia coli O157:H7 adapted to sublethal essential oils (EOs). The results demonstrated reduced motility, biofilm-forming ability, and efflux pump activities in EO-adapted E. coli O157:H7, with no induction of antibiotic resistance or infection (adhesion and invasion) on Caco-2 cells. Reverse transcription-quantitative PCR results revealed changes in the expression of related virulence genes. Thus, the present study provides new insights into microbial virulence behavior following EO adaptation and suggests that Thy, Car, and TC sublethal exposure did not constitute a significant risk in inducing microbial virulence.

Activation of lpxR gene through enterohaemorrhagic Escherichia coli virulence regulators mediates lipid A modification to attenuate innate immune response.

During the course of infection, pathogens must overcome a variety of host defence systems. Modulation of lipid A, which is a strong stimulant for host immune systems, is one of the strategies used by microorganisms to evade the host response. The lpxR gene, which encodes a lipid A 3'-O-deacylase, is commonly found in several pathogens and has been shown to reduce the inflammatory response. Here, we demonstrated that the lpxR gene of enterohaemorrhagic Escherichia coli (EHEC) was positively regulated by two virulence regulators, Pch and Ler, and that this regulation was coordinated with the locus of enterocyte effacement genes, which encode major virulence factors for colonisation. The lpxR promoter was repressed by the binding of H-NS, but the competitive binding of both regulators resulted in transcription activation. Next, we showed that lipid A from the lpxR mutant was more stimulatory of the inflammatory response in macrophage-like cells than lipid A from wild-type EHEC. Furthermore, phagocytic activity and phagosome maturation in host cells infected with the lpxR mutant were increased in a p38 mitogen-activated protein kinase-dependent manner in comparison with wild-type EHEC infection. Finally, we demonstrated that the pch mutant, which is deficient in activation of the locus of enterocyte effacement genes, was phagocytised more efficiently than the wild type. Thus, EHEC modulates lipid A to dampen the host immune response when activating virulence genes for colonisation.

Long polar fimbriae contribute to pathogenic Escherichia coli infection to host cells.

Long polar fimbria (LPF) is one of the few fimbrial adhesins of enterohemorrhagic Escherichia coli (E. coli) O157:H7 associated with colonization on host intestine, and both two types of LPF (including LPF1 and LPF2) play essential roles during the bacterial infection process. Though the fimbriae had been well studied in intestinal pathogenic E. coli strains, new evidences from our research revealed that it might be the key virulence for bovine mastitis pathogenic E. coli (MPEC) as well. This article summarizes the current knowledge on the LPF in E. coli, focusing on its genetic characteristics, prevalence, expression regulation, and adherence mechanism in different pathotypes of E. coli strains.

Anti-adhesion of probiotic Enterococcus faecium WEFA23 against five pathogens and the beneficial effect of its S-layer proteins against Listeria monocytogenes.

Enterococcus faecium WEFA23 is a potential probiotic strain isolated from Chinese infant feces. In this study, the antagonistic activity of E. faecium WEFA23 on adhesion to pathogens was investigated. Enterococcus faecium WEFA23 was able to compete, exclude, and displace the adhesion of Escherichia coli O157:H7, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes CMCC54007, Staphylococcus aureus CMCC26003, and Shigella sonnei ATCC 25931 to Caco-2 cells. Among them, L. monocytogenes achieved the strongest inhibition rate in both competition and displacement assays. Those anti-adhesion capacities were related to the bacterial physicochemical properties (hydrophobicity, auto-aggregation, and co-aggregation) of the bacterial surface. For L. monocytogenes, the anti-adhesion capacity was affected by the heat treatment, cell density, and growth phase of E. faecium WEFA23; 108 colony-forming units of viable cells per millilitre at the stationary phase exhibited the strongest anti-adhesion activity. In addition, removal of S-layer proteins of E. faecium WEFA23 by treatment with 5 mol/L LiCl significantly decreased its adhesion capacity, and those S-layer proteins were able to compete, displace, and exclude L. monocytogenes at different levels. Both cells and S-layer proteins of E. faecium WEFA23 significantly reduced the apoptosis of Caco-2 cells induced by L. monocytogenes, which was mediated by caspase-3 activation. This study might be helpful in understanding the anti-adhesion mechanism of probiotics against pathogens.