Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10% of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products - for example, adhesins and toxins - and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.

The relationship between enterotoxigenic {\it Escherichia coli\/} infection and environmental risk factors in Bilbeis, Egypt, 1981--1983

Diarrheal disease associated with enterotoxigenic Escherichia coli (ETEC) infection is one of the major public health problems in many developing countries, especially in infants and young children. Because tests suitable for field laboratories have been developed only relatively recently, the literature on the environmental risk factors associated with ETEC is not as complete as for many other pathogens or for diarrhea of unspecified etiology.^ Data from a diarrheal disease surveillance project in rural Egypt in which stool samples were tested for a variety of pathogens, and in which an environmental questionnaire was completed for the same study households, provided an opportunity to test for an association between ETEC and various risk factors present in those households. ETEC laboratory-positive specimens were compared with ETEC laboratory-negative specimens for both symptomatic and asymptomatic children less than three years of age at the individual and household level using a case-comparison design.^ Individual children more likely to have LT infection were those who lived in HHs that had cooked food stored for subsequent consumption at the time of the visit, where caretakers used water but not soap to clean an infant after a diarrheal stool, and that had an indoor, private water source. LT was more common in HHs where the caretaker did not clean an infant with soap after a diarrheal stool, and where a sleeping infant was not covered with a net. At both the individual and HH level, LT was significantly associated with good water supply in terms of quantity and storage.^ ST was isolated more frequently at the individual level where a sleeping infant was covered with a net, where large animals were kept in or around the house, where water was always available and was not potable, and where the water container was not covered. At the HH level, the absence of a toilet or latrine and the indiscriminate disposal of animal waste decreased risk. Using animal feces for fertilizer, the presence of large animals, and poor water quality were associated with ST at both the individual and HH level.^ These findings are mostly consistent with those of other studies, and/or are biologically plausible, with the obvious exception of those from this study where poorer water supplies are associated with less infection, at least in the case of LT. More direct observation of how animal ownership and feces disposal relates to different types of water supply and usage might clarify mechanisms through which some ETEC infection could be prevented in similar settings. ^

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca(2+) concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH's antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function.

O receptor de aerobactina IutA, uma proteína isolada em coluna de agarose, não é essencial para a infecção por Escherichia coli uropatogênica

Apenas alguns relatos na literatura demonstram que lectinas são importantes nos processos de colonização e infecção por Escherichia coli. A falta de compreensão clara dos mecanismos envolvendo lectinas, no processo de colonização por E. coli, motivou a realização deste estudo para se identificar a presença de outras lectinas não descritas em E. coli. Neste trabalho, isolou-se uma proteína de 75kDa de E. coli em coluna de Sepharose, correspondente ao receptor de aerobactina férrica (IutA). A associação de IutA com virulência de cepas de E. coli é controversa, principalmente em E. coli uropatogênica (UPEC), o que levou a se avaliar a presença do gene iutA em UPECs isoladas de pacientes com infecção urinária. O gene estava presente em 38% dos isolados, sugerindo fraca associação com virulência. Devido à existência de redundância nos sistemas de captura de ferro, sugere-se, aqui, que IutA possa ser vantajosa, mas não essencial para UPEC.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication.

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli as a cause of nosocomial infection or colonization: implications for control.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family are important nosocomial pathogens. Escherichia coli producing a specific family of ESBL (the CTX-M enzymes) are emerging worldwide. The epidemiology of these organisms as causes of nosocomial infection is poorly understood. The aims of this study were to investigate the clinical and molecular epidemiology of nosocomial infection or colonization due to ESBL-producing E. coli in hospitalized patients, consider the specific types of ESBLs produced, and identify the risk factors for infection and colonization with these organisms. METHODS: All patients with nosocomial colonization and/or infection due to ESBL-producing E. coli in 2 centers (a tertiary care hospital and a geriatric care center) identified between January 2001 and May 2002 were included. A double case-control study was performed. The clonal relatedness of the isolates was studied by repetitive extragenic palindromic-polymerase chain reaction and pulsed-field gel electrophoresis. ESBLs were characterized by isoelectric focusing, polymerase chain reaction, and sequencing. RESULTS: Forty-seven case patients were included. CTX-M-producing E. coli were clonally unrelated and more frequently susceptible to nonoxyimino-beta-lactams. Alternately, isolates producing SHV- and TEM-type ESBL were epidemic and multidrug resistant. Urinary catheterization was a risk factor for both CTX-M-producing and SHV-TEM-producing isolates. Previous oxyimino-beta-lactam use, diabetes, and ultimately fatal or nonfatal underlying diseases were independent risk factors for infection or colonization with CTX-M-producing isolates, whereas previous fluoroquinolone use was associated with infection or colonization with SHV-TEM-producing isolates. CONCLUSIONS: The epidemiology of ESBL-producing E. coli as a cause of nosocomial infection is complex. Sporadic CTX-M-producing isolates coexisted with epidemic multidrug-resistant SHV-TEM-producing isolates. These data should be taken into account for the design of control measures.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

Activities of fosfomycin, tigecycline, colistin, and gentamicin against extended-spectrum-β-lactamase-producing Escherichia coli in a foreign-body infection model.

Limited antimicrobial agents are available for the treatment of implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli. We compared the activities of fosfomycin, tigecycline, colistin, and gentamicin (alone and in combination) against a CTX-M15-producing strain of Escherichia coli (Bj HDE-1) in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration in logarithmic phase (MBC(log)) and stationary phase (MBC(stat)) were 0.12, 0.12, and 8 μg/ml for fosfomycin, 0.25, 32, and 32 μg/ml for tigecycline, 0.25, 0.5, and 2 μg/ml for colistin, and 2, 8, and 16 μg/ml for gentamicin, respectively. In time-kill studies, colistin showed concentration-dependent activity, but regrowth occurred after 24 h. Fosfomycin demonstrated rapid bactericidal activity at the MIC, and no regrowth occurred. Synergistic activity between fosfomycin and colistin in vitro was observed, with no detectable bacterial counts after 6 h. In animal studies, fosfomycin reduced planktonic counts by 4 log(10) CFU/ml, whereas in combination with colistin, tigecycline, or gentamicin, it reduced counts by >6 log(10) CFU/ml. Fosfomycin was the only single agent which was able to eradicate E. coli biofilms (cure rate, 17% of implanted, infected cages). In combination, colistin plus tigecycline (50%) and fosfomycin plus gentamicin (42%) cured significantly more infected cages than colistin plus gentamicin (33%) or fosfomycin plus tigecycline (25%) (P < 0.05). The combination of fosfomycin plus colistin showed the highest cure rate (67%), which was significantly better than that of fosfomycin alone (P < 0.05). In conclusion, the combination of fosfomycin plus colistin is a promising treatment option for implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli.

Effect of the Escherichia coli EMO strain on experimental infection by Salmonella enterica serovar Typhimurium in gnotobiotic mice

An experimental infection with Salmonella enterica subsp. enterica serovar Typhimurium was evaluated in gnotobiotic mice previously exposed to a plasmid-free non-pathogenic Escherichia coli (EMO strain). Mice were exposed to EMO (experimental) or not (control) 10 days before challenge with Salmonella Typhimurium (10² colony forming units (CFU)/mouse). Survival after challenge was higher (P < 0.05) in the experimental group (16%) than in the control animals (0%). Histopathological examination of the colon and ileum mucosa of the experimental group showed less extensive lesions such as edema, cell inflammatory infiltration and hyperemia. The epithelial cells of the mucosal surface and the production of the mucous layer were also better preserved in the experimental group. The population levels of Salmonella Typhimurium in the feces were initially 10-fold lower (P < 0.05) in the experimental groups. However, 3 days after challenge both experimental and control groups showed similar population levels ranging from 10(8) to()10(9) CFU/g of feces. The intestinal contents of total and anti-Salmonella Typhimurium sIgA were higher in the experimental groups 10 days after inoculation of E. coli EMO strain. Translocation of Salmonella Typhimurium to the spleen was 10-fold lower (P < 0.05) in the experimental group only on day 3 after infection. This was not related to an increase in the bacterial blood clearance of the animals, as shown by experimental venous challenge with E. coli B41. In conclusion, treatment of mice with E. coli EMO strain promoted a relative protection against experimental infection with Salmonella Typhimurium. This protection was not due to the reduction of the population of pathogens in the intestine but was probably related to stimulation of the immune response.

Identification des gènes de Escherichia coli entérohémorragique exprimés pendant l'infection de macrophages humains

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Non-curliation of Escherichia coli O78 : K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when groan on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E, coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Attaching and effacing lesions in the large intestine of an eight-month-old heifer associated with Escherichia coli O26 infection in a group of animals with dysentery

Escherichia coli O26:K60, with genetic attributes consistent with a potentially human enterohaemorrhagic E coli was isolated from the faeces of an eight-month-old heifer with dysentery. Attaching and effacing lesions were identified in the colon of a similarly affected heifer examined postmortem, and shown to be associated with E coli O26 by specific immunolabelling.

Infection of gnotobiotic calves with Escherichia coli O157 : H7 strain A84

Six colostrum-deprived, hysterotomy-derived calves were maintained under sterile conditions and fed a milk replacer diet. At five days of age, five of the calves were dosed orally with 10(9)cfu of Escherichia coli O157: H7 strain A84. They were killed after, one, two, six, 12 and 24 days, and samples were taken for bacteriological and pathological examination. The sixth uninfected control calf was killed at seven days of age and matched samples were taken for pathological comparison. The animals remained normal throughout the observation period. Bacteriological data indicated a heavy bacterial load of strain A84 throughout the gastrointestinal tract but the bacterium was not found in liver, kidney or muscle. No evidence of attaching and effacing' lesions in the small or large intestine was found although there was a mild inflammatory response in the intestinal tract, consisting mainly of infiltrating eosinophils.