Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and β-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

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.     

Production of slime polysaccharide by EHEC and STEC as well as the influence of culture conditions on slime production in Escherichia coli O157:H7

AIMS: To quantify the slime polysaccharide, composed of colanic acid (CA), produced by enterohaemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) and to determine the influence of culture conditions on CA production in E. coli O157:H7. METHODS AND RESULTS: The study examined the amounts of CA produced by EHEC and STEC, and evaluated the production of CA in E. coli O157:H7 as influenced by medium pH and incubation temperatures. The results indicated that the amounts of CA produced by EHEC and STEC vary to a great extent and CA production in E. coli O157:H7 is influenced by the tested culture conditions. CONCLUSIONS: The abilities of EHEC and STEC to produce CA differ. Medium pH and incubation temperature are among the important factors affecting CA production in E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Slime polysaccharide can affect the abilities of E. coli O157:H7 cells to combat environmental stress. This study contributes to a better understanding of the physiological factors influencing slime polysaccharide production in EHEC and STEC.     

EHEC O157:H7 z3276基因缺失株构建及其生物学特性

【目的】肠出血性大肠杆菌O157:H7是世界范围内重要的动物源性致病菌之一,可感染人。I型菌毛是多种致病性大肠杆菌(如肾盂肾炎型大肠杆菌等)可表达的一种黏附结构,与细菌吸附黏膜表面密切相关。然而,O157:H7 fim操纵子上几个核苷酸的缺失却导致其不能表达I型菌毛。BLAST比对结果表明O157:H7独有的开放阅读框z3276编码的氨基酸序列与其他大肠杆菌I型菌毛高度同源,这可能是对O157:H7不能表达I型菌毛的补偿机制,但确切功能尚不清楚。本文探究z3276基因的生物学功能。【方法】利用O157:H7 86-24参考菌株构建z3276基因缺失株(?z3276),并构建其互补株(C?z3276),进而比较亲本株、?z3276与C?z3276的生物学特性及对小鼠致病性差异。【结果】与亲本株相比,?z3276进入对数生长期的时间延后,在半固体琼脂平板上的迁移直径明显缩小,生物被膜形成能力显著减弱。?z3276对HEp-2细胞的黏附和侵袭能力并无明显变化,但对IPEC-J2细胞的侵袭能力明显减弱。在小鼠攻毒试验中,?z3276组排菌数量减少、排菌持续时间缩短。C?z3276各项特性均能回复到与亲本株一致的水平。【结论】z3276基因可能是O157:H7重要的毒力相关因子。     

长春地区家禽（家畜）中E．coli O157：H7调查

为了通过对宿主动物EHEC O157∶H7监测,了解长春地区肠出血性大肠埃希菌O157∶H7的污染状况,建立流行病学监测网,以便为疾病预防控制提供良好的科学依据.结果在采集的639份家禽、家畜粪便样品中共检出7株EHEC O157∶H7.     

长春地区家禽(家畜)中E.coli O157∶H7调查

为了通过对宿主动物EHEC O157∶H7监测,了解长春地区肠出血性大肠埃希菌O157∶H7的污染状况,建立流行病学监测网,以便为疾病预防控制提供良好的科学依据。结果在采集的639份家禽、家畜粪便样品中共检出7株EHEC O157∶H7。     

Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.     

出血性大肠杆菌O157基因缺失疫苗株的构建及其免疫

出血性大肠杆菌O157感染是重要的新发食物源性传染病,主要致病特征之一是能引起人肠上皮细胞特征性的A/E损伤,A/E损伤主要是由LEE致病岛所编码的毒力因子所引起,ler是LEE致病岛毒力基因群的中心调节基因,对LEE致病岛所编码的毒力因子有正调控作用。O157:H7另一个毒力因子是由整合到染色体上的原噬菌体编码的Stx毒素。以O157:H786-24为始发菌株,利用自杀性质粒pCVD442和同源重组的原理构建了O157:H7的ler基因缺失突变菌株(缺失了ler基因中第73-351位的碱基,共279bp),并利用噬菌体消除技术筛选到消除了编码Stx的原噬菌体DNA的菌株,构建出了O157:H7ler/stx基因缺失突变弱毒菌株,并对该菌株的Vero细胞毒性、小鼠模型的安全性以及乳鼠的被动免疫保护作用进行了研究。结果表明,O157:H7ler/stx基因缺失突变菌株丧失了对Vero细胞的毒性作用,并丧失了对实验小鼠的致病性,具有良好的安全性。乳鼠被动免疫保护性实验表明,用该菌株免疫母鼠后,乳鼠通过吸吮母乳可以获得良好的被动免疫保护作用。因此本研究所构建的O157:H7ler/stx基因缺失突变弱毒菌株可作为预防EHEC O157:H7感染的疫苗候选株,为最终研究制出O157的基因工程菌苗奠定基础。     

Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.     

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.     

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.     

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.     

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.     

Analysis of fimbrial gene clusters and their expression in enterohaemorrhagic Escherichia coli O157:H7

The sequence of two enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains reveals the possession of at least 16 fimbrial gene clusters, many of the chaperone/usher class. The first part of this study examined the distribution of these clusters in a selection of EHEC/EPEC (enteropathogenic E. coli) serotypes to determine if any were likely to be unique to E. coli O157:H7. Six of the clusters, as determined by the presence of amplified main subunit or usher gene sequences, were detected only in the E. coli O157 and O145 serotypes tested. With the exception of one serotype O103 strain that contained an lpf2 cluster, lpf sequences were only detected in E. coli O157 of the serotypes tested. Expression from each cluster was measured by the construction of chromosomally integrated lacZ promoter fusions and plasmid-based eGFP fusions in E. coli O157:H7. This analysis demonstrated that the majority (11/15) of main fimbrial subunit genes were not expressed under the majority of conditions tested in vitro. One of the clusters showing promoter activity, loc8, has a temperature expression optimum indicating a possible role outside the host. From the presence of pseudogenes in three of the clusters, the lack of FimH-like minor adhesins in the clusters and their limited expression in vitro, it would appear that E. coli O157:H7 has a limited repertoire of expressed functional fimbriae. This restricted selection of fimbriae may be important in bringing about the tropism E. coli O157:H7 demonstrates for the terminal rectum of cattle.     

Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli

It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.     

Fate of Escherichia coli O157:H7 in unpasteurized milk stored at 8 degrees C

Escherichia coli O157:H7 and verocytotoxins were not found in any of 100 unpasteurized milk samples obtained from the bulk tanks of eight dairy farms located in the Puglia and Basilicata areas. Seven E. coli O157:H7 (EHEC) strains were inoculated separately into raw milk samples and then examined periodically to determine the fate of EHEC as influenced by the storage temperature (8 degrees C) and time. There was essentially no change in the viable population of three EHEC strains for up to 14 d. The remaining four strains showed an increase in population from < 2 log to 3 log cfu ml-1 in a time period of between 9 and 17 d. The results indicate good survival or even multiplication of E. coli O157:H7 in raw milk when stored at 8 degrees C and reaffirm the need for pasteurization and holding the milk at < or = 5 degrees C.     

Presence and characterization of a mosaic genomic island which distinguishes sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H(-) from EHEC O157:H7.     

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.