Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin

Norfloxacin (NFLX) caused induction of prophages VT1 and VT2 of enterohemorrhagic Escherichia coli O157 at subinhibitory concentrations. In time course experiments, we observed the following sequential events: upon induction, the phage genomes underwent multiplication; the amount of stx genes increased; and subsequently, large quantities of toxins VT1 and VT2 were produced. Further studies showed that the molecular mechanism of prophage induction is closely related to the RecA system since the prophage VT2 was not induced with NFLX in a recA mutant strain.     

Interaction of enterohemorrhagic Escherichia coli O157:H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.     

Experimental infection of specific-pathogen-free mice with enterohemorrhagic Escherichia coli O157:H7.

By subcutaneous inoculation of 10(8) CFU of enterohemorrhagic E. coli O157:H7, specific-pathogen-free mice revealed most of the symptoms and histological changes observed in patients. The histological changes in intestine were mainly seen in the distal parts of small intestine and the cecum. Vacuolation of villi in the cecum was also observed. The histological changes in the kidneys of the infected mice were featured as the swollen epithelial cells of glomeruli and the marked thickening of glomerular capillaries with barely visible lumens. Unexpected findings in the bronchiole were characterized by sloughing of the epithelial cells of bronchiolar wall, leading to partial or complete obstruction of the lumens. Histological changes in the spleen, liver and lymphnodes were also observed. The bacteria were recovered from the feces, contents of small intestine, and samples taken from kidney, liver, heart, spleen, different parts of small intestine, cecum, and colon. By using peroxidase-antiperoxidase (PAP) assay with polyclonal antibodies against "O" antigen of E. coli O157:H7, it was observed that the samples taken from the brain, kidney, ileum, cecum, spleen, and liver gave positive reactions. Feces and contents of small intestine obtained from all of the infected animals were positive by occult blood test. These results show that the experimental infection of E. coli O157:H7 in this model is systemic in nature.     

Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces.

Dairy cattle have been identified as a principal reservoir of Escherichia coli O157:H7. The fate of this pathogen in bovine feces at 5, 22, and 37 degrees C was determined. Two levels of inocula (10(3) and 10(5) CFU/g) of a mixture of five nalidixic acid-resistant E. coli O157:H7 strains were used. E. coli O157:H7 survived at 37 degrees C for 42 and 49 days with low and high inocula, respectively, and at 22 degrees C for 49 and 56 days with low and high inocula, respectively. Fecal samples at both temperatures had low moisture contents (about 10%) and water activities ( < 0.5) near the end of the study. E. coli O157:H7 at 5 degrees C survived for 63 to 70 days, with the moisture content (74%) of feces remaining high through the study. Chromosomal DNA fingerprinting of E. coli O157:H7 isolates surviving near the completion of the study revealed that the human isolate strain 932 was the only surviving strain at 22 or 37 degrees C. All five strains were isolated near the end of incubation from feces held at 5 degrees C. Isolates at each temperature were still capable of producing both verotoxin 1 and verotoxin 2. Results indicate that E. coli O157:H7 can survive in feces for a long period of time and retain its ability to produce verotoxins. Hence, bovine feces are a potential vehicle for transmitting E. coli O157:H7 to cattle, food, and the environment. Appropriate handling of bovine feces is important to control the spread of this pathogen.     

Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.     

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

Background

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

Results

We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.

Conclusions

Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.     

Clonal turnover of enterohemorrhagic Escherichia coli O157:H7 in experimentally infected cattle

A total of 401 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates from two experimentally infected calves were analyzed using molecular biological methods. Genetic differences detected by pulsed-field gel electrophoresis were observed between the inoculated and recovered strains as early as 1 day post inoculation. The loss of the inoculated clone was observed in one calf. Replication and dissemination of the EHEC O157:H7 strains that mutated in cattle may result in the diversification of this organism among cattle populations.     

A carboxy-terminal domain of Tir from enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) required for efficient type III secretion

The type III secreted protein Tir from Enterohemorrhagic Escherichia coli (EHEC O157:H7) plays a central role in adherence and pedestal formation during infection. Little is known about how Tir domains outside of the amino-terminus contribute to efficient Tir secretion and translocation. We found a 6 amino acid (519-524) carboxy-terminal region which was required for efficient Tir secretion and translocation. Interestingly, EHEC O157:H7 Tir(Delta)519-524 was efficiently secreted when expressed in the related pathogen enteropathogenic E. coli. These data suggest that this region may play a role in maintaining EHEC O157:H7 Tir in a secretion-competent conformation.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

Bile salts induce resistance to polymyxin in enterohemorrhagic Escherichia coli O157:H7

Many enteric bacteria use bile as an environmental cue to signal resistance and virulence gene expression. Microarray analysis of enterohemorrhagic Escherichia coli O157:H7 (EHEC) treated with bile salts revealed upregulation of genes for an efflux system (acrAB), a two-component signal transduction system (basRS/pmrAB), and lipid A modification (arnBCADTEF and ugd). Bile salt treatment of EHEC produced a basS- and arnT-dependent resistance to polymyxin.     

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.     

Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives.

A strain of enterohemorrhagic Escherichia coli serotype O157:H7 isolated from a patient in an apple cider-related outbreak was used to study the fate of E. coli O157:H7 in six different lots of unpasteurized apple cider. In addition, the efficacy of two preservatives, 0.1% sodium benzoate and 0.1% potassium sorbate, used separately and in combination was evaluated for antimicrobial effects on the bacterium. Studies were done at 8 or 25 degrees C with ciders having pH values of 3.6 to 4.0. The results revealed that E. coli O157:H7 populations increased slightly (ca. 1 log10 CFU/ml) and then remained stable for approximately 12 days in lots inoculated with an initial population of 10(5) E. coli O157:H7 organisms per ml and held at 8 degrees C. The bacterium survived from 10 to 31 days or 2 to 3 days at 8 or 25 degrees C, respectively, depending on the lot. Potassium sorbate had minimal effect on E. coli O157:H7 populations, with survivors detected for 15 to 20 days or 1 to 3 days at 8 or 25 degrees C, respectively. In contrast, survivors in cider containing sodium benzoate were detected for only 2 to 10 days or less than 1 to 2 days at 8 or 25 degrees C, respectively. The highest rates of inactivation occurred in the presence of a combination of 0.1% sodium benzoate and 0.1% potassium sorbate. The use of 0.1% sodium benzoate, an approved preservative used by some cider processors, will substantially increase the safety of apple cider in terms of E. coli O157:H7, in addition to suppressing the growth of yeasts and molds.     

The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been considered as an agent responsible for outbreak of hemorrhagic colitis and the hemolytic uremic syndrome. We examined the effect of the probiotic agent Clostridium butyricum MIYAIRI strain 588 on EHEC O157:H7 infections in vitro and in vivo using gnotobiotic mice. The growth of EHEC O157:H7 and the production of Shiga-like toxins in broth cultures were inhibited by co-incubation with C. butyricum. The antibacterial effects of butyric and lactic acid were demonstrated in a dose-dependent manner. In addition, the inhibitory effect of butyric acid on the viability of EHEC was demonstrated not only at low pH, but also at neutral pH adjusted to 7.0. Flowcytometric analysis showed that pre-incubation of Caco-2 cells with C. butyricum and E. coli K12 inhibited the adhesion of EHEC O157:H7. However, the effect of C. butyricum on adhesion of EHEC to Caco-2 cells was more inhibitory than that of E. coli K12. Gnotobiotic mice mono-associated with EHEC O157:H7 died within 4-7 days after the infection. On the other hand, all gnotobiotic mice prophylactically pre-treated with C. butyricum survived exposure to EHEC O157:H7 and of the gnotobiotic mice therapeutically post-treated with C. butyricum, 50% survived. Both counts of EHEC O157:H7 and the amounts of shiga-like toxins (Stx1 and Stx2) in fecal contents of gnotobiotic mice di-associated with EHEC O157:H7 and C. butyricum were less than those of gnotobiotic mice mono-associated with EHEC O157:H7. These results indicated that the probiotic bacterium C. butyricum MIYAIRI strain 588 has preventive and therapeutic effects on EHEC O157:H7 infection in gnotobiotic mice.