Dual-Serotype Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and O26:H11 Strains

Escherichia coli O26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms. E. coli O157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore, E. coli O26:H11 remains a potential risk in food safety.     

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS⁺ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH₂O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log₁₀ in sterile ddH₂O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log₁₀ in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS⁺ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.     

Influence of Plasmid pO157 on Escherichia coli O157:H7 Sakai Biofilm Formation

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.Enterohemorrhagic Escherichia coli serotype O157:H7 is a major food-borne pathogen causing hemorrhagic colitis and the hemolytic-uremic syndrome (17). Many E. coli O157:H7 outbreaks have been associated with contaminated undercooked ground beef, vegetables, fruits, and sprouts (20, 31). One of the largest disease outbreaks occurred in Sakai City, Japan, in 1996 with nearly 8,000 confirmed cases. The E. coli isolate responsible for this outbreak, referred to as “Sakai,” is one of the best-characterized isolates and one of only three O157 strains for which the genome has been fully sequenced (8, 16). Because of its importance as a human pathogen and its characterization, Sakai was the focus of this investigation.There is significant phenotypic diversity among E. coli O157:H7 strains, including the ability to form biofilm. Previous studies show that certain E. coli O157:H7 strains form biofilm on various surfaces, and biofilm on food or food-processing surfaces can serve as a source or vehicle of contamination that may result in human infection (6, 18, 25). Biofilm is an organized and structured community of microorganisms that attaches to solid surfaces and contains cells embedded in an extracellular polymer matrix (4, 26). Exopolysaccharide (EPS) is a major component of the biofilm matrix and is required for the development of characteristic biofilm architecture (5, 29). Bacteria gain a variety of advantages from biofilm formation that include attachment, colonization, and protection from adverse environments (4, 11).E. coli O157:H7 carries a 92-kb virulence plasmid (pO157) encoding a number of putative virulence determinants, including ehxA, etpC to etpO, espP, katP, toxB, ecf, and stcE (31). However, the biological role of pO157 is not fully understood, and only 19 genes among the 100 open reading frames (ORFs) in pO157 have been characterized (2, 15). Our previous work indicates that pO157 is a colonization factor in cattle and may regulate several chromosomal genes (14, 24, 31).To investigate the role of pO157 in biofilm formation, we characterized the biofilm of wild-type E. coli O157:H7 strain Sakai and an isogenic pO157-cured Sakai (Sakai-Cu). Both strains were kindly provided by C. Sasakawa (University of Tokyo). Sakai-Cu was generated using a plasmid incompatibility method (27). This method is not prone to secondary mutations and requires minimal passage in laboratory medium. The mini-R plasmid pK2368, harboring a chloramphenicol (CM) resistance gene and being in the same plasmid incompatibility group as pO157, was introduced into wild-type Sakai by transformation. Transformants were isolated on LB agar containing CM and selected for loss of pO157 by agarose gel electrophoresis analysis. CM-resistant transformants were cured of pKP2368 by subculturing in LB broth without CM. The absence of pO157 was confirmed by Southern blot hybridization with a pO157-specific gene probe (derived from ecf1), and chromosomal DNA integrity was confirmed by pulsed-field gel electrophoresis (data not shown).Because E. coli O157:H7 strains are generally not strong biofilm producers, the condition most conducive to biofilm production, a fluorometric flow cell method, was used to compare separately grown Sakai and Sakai-CU (3). The biofilm cultivation systems consisted of seven parts: (i) medium reservoir, (ii) multichannel pump (205S; Watson Marlow, United Kingdom), (iii) bubble trap (BioSurface Technologies Co., Bozeman, MT), (iv) flow cell, (v) outflow reservoir, (vi) air pump (DrsFosterSmith, Rhinelander, WI), and (vii) flow meter (Gilmont, BC Group, St. Louis, MO). The flow cell was constructed from two rectangular acrylic plates that were 104 by 48 mm. Sidewalls (62 by 26 by 5 mm) were glued to the top plate to form an elongated hexagonal growth chamber. There were 56- by 20-mm square openings in the top and bottom rectangular plates that were sealed with 60- by 24-mm glass slides (Fisher, Pittsburgh, PA). The upper and lower plates were assembled with screws and sealed using a microseal B film (MJ Research, Waltham, MA). The flow cell volume was about 10.4 ml, the medium flow rate was 10.5 ml/h, and the hydraulic retention time was 1 h. Under these conditions, the linear surface velocity was about 80 mm/h at the center of the flow cell. The biofilm was grown with BGM2 medium (21). To prepare the inoculums, Sakai and Sakai-Cu were grown at 37°C in BGM2 medium to mid-exponential phase, and cells were harvested by centrifugation and resuspended in 0.85% NaCl. One hundred μl of the resuspended cell solution was inoculated from the effluent side of flow cells through a long stainless steel needle (Fisher, Pittsburgh, PA). The cells were incubated for at least 3 h without supplying fresh medium, and then fresh medium was supplied to the biofilm cultivation system at 30°C.At various times, the resulting biofilms were stained with a green fluorescent dye, wheat germ agglutinin (WGA)- Alexa Fluor 488 (Invitrogen, Carlsbad, CA), and analyzed using the Olympus FluoView confocal laser scanning microscopy system (Olympus, Tokyo, Japan). Using the Olympus FluoView software program, version 1.7b, for analysis, the fluorescence intensities of Sakai and Sakai-Cu biofilm matrices were each analyzed from >20 three-dimensional-complexity images. Fluorescence was greater for Sakai than for Sakai-Cu, with average values of 2,448 ± 668 and 2,022 ± 619, respectively (Student''s t test; P < 0.05). Overhead images from the Sakai-Cu strain biofilm revealed more-compact cell clusters than images from wild-type Sakai (Fig. 1A and B). Comparisons of images taken sideways indicated that the Sakai-Cu biofilms were not as thick as those of wild-type Sakai (Fig. 1C and D), and typical ratios were consistently 9:11, respectively (P < 0.05). A previous study demonstrated that the biofilm of a wcaF::can mutant of E. coli K-12, which is deficient in EPS production, lacked depth and complex architecture (5). Sakai-Cu showed a similar but less dramatic phenomenon. These observations indicated that pO157 influenced biofilm formation and architecture.Open in a separate windowFIG. 1.Wild-type Sakai (A and C) or Sakai-Cu (B and D) biofilms after 3 days of incubation. Both strains were grown at 30°C in an individual flow cell apparatus. The biofilm was stained with WGA-Alexa Fluor 488 and examined by confocal microscopy. Representative overhead (A and B) or sagittal (C and D) images are shown and were generated using the deconvolution software. Bar, 50 μm.To quantitatively compare Sakai and Sakai-Cu biofilms, the contents of each flow cell apparatus were collected at various times and analyzed for bacterial cell number, viscosity, and EPS production. Biofilms were harvested by a standard technique that preserves cell numbers and minimizes viscosity changes (9). Briefly, floating cells in the biofilm were carefully collected with a pipet, and the remaining cells were scraped from the flow cell apparatus with sterilized applicator sticks. Biofilm samples were collected on days 1, 3, 5, 8, and 12, and measurements were means ± standard deviations (SD) of at least triplicate measurements from separately grown biofilms. There was no significant difference in bacterial number (CFU/ml) from Sakai and Sakai-Cu biofilms at any of the times measured (data not shown). A Cannon-Fenske routine viscometer (Size 100; Cannon Instrument Co., Pennsylvania) was used to determine biofilm viscosity. The conversion constant was 0.015 cSt/s (mm²/s²), and viscosities were measured according to the manufacturer''s instructions. Briefly, the viscometer was aligned vertically in the holder, and the sample was charged into the viscometer tube until the sample reached the “F” mark in the tube. A suction bulb was used to draw the sample slightly above mark “E.” The sample was allowed to flow freely, and the efflux time was measured as the time for the meniscus to pass from mark “E” to mark “F.” Measurements were repeated at least six times, and the kinematic viscosity in mm²/s (cSt) of the samples was calculated by multiplying the efflux time in seconds by the viscometer constant. The viscosity of Sakai biofilm was dramatically increased after 8 days (P < 0.001), while there was no significant change in the viscosities of Sakai-Cu biofilms through day 12 (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Comparison of Sakai and Sakai-Cu biofilm viscosity. Three or four separately grown biofilms were each harvested on the days indicated, and viscosity was measured using a Cannon-Fenske Routine viscometer.Bacterial EPS are associated with attachment to both inanimate surfaces and host cells (29). EPS can be categorized as extracellular carbohydrate complexes (ECC) that are loosely associated with cells and easily removed, referred to as slime (fraction I), or ECC that are closely associated with cells and removed only after heat treatment, referred to as capsule (fraction II) (22). No significant difference in ECC was observed until days eight and 12, when the level of total ECC produced from Sakai biofilms was significantly higher than that from the Sakai-Cu biofilms (P < 0.05) (Fig. ​(Fig.3).3). Also, by days eight and 12, levels of Sakai ECC fraction I, representing primarily secreted slime carbohydrates, were 5 and 10 times higher than Sakai-Cu ECC fraction I, respectively. These results correlated with the results of increased viscosity in Sakai biofilm samples that had aged for 8 or 12 days.Open in a separate windowFIG. 3.Comparison of Sakai and Sakai-Cu biofilm extracellular carbohydrate (ECC) production. ECC I was collected from cells by centrifugation, and ECC II was collected by centrifugation after heat treatment on each indicated day. Bar height represents total ECC production from each biofilm sample. The proportion of total ECC that was either ECC I (dark gray) or ECC II (light gray) is shown. Asterisks indicate significant differences between wild-type Sakai (Wt) and Sakai-Cu (Cu); day 8, P < 0.05; day 12, P < 0.001.Interestingly, during biofilm sampling, two colony morphology variants were isolated that are referred to here as sticky and mucoid. These variants were found only in wild-type Sakai biofilms that had aged for ≥8 days and were not found in Sakai-Cu biofilms even after screening of 10⁴ colonies and even among biofilms aged for 18 days. The percentages of sticky and mucoid variants in Sakai biofilms ranged from 5 to 30% and 0 to 5%, respectively. The differences in colony morphology were readily distinguished, as shown in Fig. ​Fig.4.4. The sticky variant was raised in elevation and shinier than the Sakai parent strain but was not difference in size. When single bacterial colonies grown on agar plates were touched with a sterilized toothpick and that toothpick was gently lifted up, the colonies had a hyperadherence phenotype and elongated to approximately 1 cm between the plate and the toothpick. This phenomenon was unique to the sticky colony variants and was not observed among colonies of the parent Sakai strain (Fig. ​(Fig.4D).4D). The mucoid colony variants were convex in elevation and shiny in texture, had irregular colony shapes, and were larger than the Sakai parent strain but were not hyperadherent. The motility of variants was determined using 0.3% soft agar, and both sticky and mucoid variants exhibited 30- to 90%-reduced motility compared to the parent Sakai strain (data not shown). The characteristics of both sticky and mucoid variants were inherited, and the variant characteristics were maintained in laboratory subculture through 15 generations.Open in a separate windowFIG. 4.Colony morphologies of wild-type, mucoid, and sticky variants. The wild-type E. coli O157:H7 Sakai strain formed small, flat, and nonsticky colonies on LB agar (A). The mucoid variant formed irregular, large, shiny, mucoid, convex, and nonsticky colonies (B). The sticky variant formed small, slightly raised, and sticky colonies (C). The sticky variant adheres to a toothpick touched to the colony surface (D). Bar, 1 cm.It is known that mutation is a powerful mechanism of adaptation when bacteria are faced with environmental change (1). Like other bacterial variants, the sticky and mucoid phenotypic biofilm variants may provide a survival advantage in specific niches (10, 19). Pseudomonas aeruginosa is a well-known biofilm model, and colony morphology variants are a common biofilm-related phenomenon. Both reduced-motility and hyperadherence variants have been described (10) and have characteristics similar to those of the E. coli O157:H7 biofilm variants described here. However, unlike the P. aeruginosa biofilm variants, the sticky and mucoid Sakai variants were not smaller, rougher, or more wrinkled than the parent colony.Although it is possible that the changes measured in biofilm formation and the generation of hyperadherent variants were not due to the plasmid, it is highly unlikely. The method of plasmid curing by incompatibility is gentle and is not prone to secondary mutation. A powerful and common approach to address possible secondary mutations is complementation; however, it was not used here because reintroduction of the plasmid requires the manipulation of a very large piece of DNA (92 kb) and the procedure itself is likely to introduce mutation. Also, reintroduction of the large 92-kb pO157 plasmid would require antibiotic resistance for efficient selection, and this may influence biofilm formation.Many regulatory mechanisms are involved in biofilm formation (7, 12, 13, 28, 30, 32). Among those mechanisms, the relationship between biofilm formation and acid resistance is well known. Biofilm formation is upregulated after the deletion of the gad or hde gene, which allows bacteria to survive under acidic conditions (12). Previously we showed that an isogenic pO157-cured strain of E. coli O157:H7, ATCC 43894, enhanced acid resistance through increased expression of Gad (14). Similarly, Sakai-Cu has enhanced acid resistance compared to wild-type Sakai (data not shown and J. Y. Lim, B. Hong, H. Sheng, S. Shringi, R. Kaul, and C. J. Hovde, submitted for publication). The link between increased acid resistance and reduced biofilm formation, reduced ESP production, reduced viscosity, and lack of colony morphology variants was not explored here. Comparisons of biofilm formation were not made between these two strains because neither wild-type E. coli O157:H7 ATCC 43894 nor its plasmid-cured strain form significant biofilm under the laboratory conditions tested (data not shown).Two pO157-cured E. coli O157 strains (ATCC 43894 and Sakai) do not colonize cattle as well as their wild-type counterpart (14, 24). The mechanism for this difference may be related to pO157 encoding a set of putative type II secretion genes, etpC to etpM, etpO, and etpS, and these etp genes may be associated with protein secretion required for efficient adherence (23). Tatsuno et al. reported that the toxB gene encoded on pO157 is required for the full epithelial cell adherence phenotype (27). These results may relate to the defect of Sakai-Cu in biofilm formation.In conclusion, this is the first report that pO157 affects biofilm formation of E. coli O157:H7 Sakai through increased EPS production and generation of hyperadherent variants. Further study of biofilm formation under a variety of conditions and comparisons of Sakai with other E. coli O157:H7 strains will be important for understanding the relationship between biofilm formation and E. coli O157:H7 virulence and survival on foods and in the farm environment.     

Derivation of Escherichia coli O157:H7 from Its O55:H7 Precursor

There are 29 E. coli genome sequences available, mostly related to studies of species diversity or mode of pathogenicity, including two genomes of the well-known O157:H7 clone. However, there have been no genome studies of closely related clones aimed at exposing the details of evolutionary change. Here we sequenced the genome of an O55:H7 strain, closely related to the major pathogenic O157:H7 clone, with published genome sequences, and undertook comparative genomic and proteomic analysis. We were able to allocate most differences between the genomes to individual mutations, recombination events, or lateral gene transfer events, in specific lineages. Major differences include a type II secretion system present only in the O55:H7 chromosome, fewer type III secretion system effectors in O55:H7, and 19 phage genomes or phagelike elements in O55:H7 compared to 23 in O157:H7, with only three common to both. Many other changes were found in both O55:H7 and O157:H7 lineages, but in general there has been more change in the O157:H7 lineages. For example, we found 50% more synonymous mutational substitutions in O157:H7 compared to O55:H7. The two strains also diverged at the proteomic level. Mutational synonymous SNPs were used to estimate a divergence time of 400 years using a new clock rate, in contrast to 14,000 to 70,000 years using the traditional clock rates. The same approaches were applied to three closely related extraintestinal pathogenic E. coli genomes, and similar levels of mutation and recombination were found. This study revealed for the first time the full range of events involved in the evolution of the O157:H7 clone from its O55:H7 ancestor, and suggested that O157:H7 arose quite recently. Our findings also suggest that E. coli has a much lower frequency of recombination relative to mutation than was observed in a comparable study of a Vibrio cholerae lineage.     

Analyses of the Red-Dry-Rough Phenotype of an Escherichia coli O157:H7 Strain and Its Role in Biofilm Formation and Resistance to Antibacterial Agents

In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.     

Functional Metagenomics of Escherichia coli O157:H7 Interactions with Spinach Indigenous Microorganisms during Biofilm Formation

The increase in foodborne outbreaks worldwide attributed to fresh fruit and vegetables suggests that produce may serve as an ecological niche for enteric pathogens. Here we examined the interaction of E. coli O157:H7 (EcO157) with spinach leaf indigenous microorganisms during co-colonization and establishment of a mixed biofilm on a stainless steel surface. Stainless steel surface was selected to mimic the surface of produce-processing equipment, where retention of foodborne pathogens such as EcO157 could serve as a potential source for transmission. We observed a positive effect of spinach-associated microbes on the initial attachment of EcO157, but an antagonistic effect on the EcO157 population at the later stage of biofilm formation. Metagenomic analyses of the biofilm community with the GeoChip revealed an extremely diverse community (gene richness, 23409; Shannon-Weiner index H, 9.55). Presence of EcO157 in the mixed biofilm resulted in a significant decrease in the community α-diversity (t test, P<0.05), indicating a putative competition between the pathogen and indigenous spinach microbes. The decrease in the β-diversity of the EcO157-inoculated biofilm at 48 h (ANOVA, P<0.05) suggested a convergent shift in functional composition in response to EcO157 invasion. The success of EcO157 in the mixed biofilm is likely associated with its metabolic potential in utilizing spinach nutrients: the generation time of EcO157 in spinach lysates at 28°C is ～ 38 min, which is comparable to that in rich broth. The significant decrease in the abundance of many genes involved in carbon, nitrogen, and phosphorus cycling in the EcO157-inoculated biofilms (t test, P<0.05) further support our conclusion that competition for essential macronutrients is likely the primary interaction between the EcO157 and indigenous spinach-biofilm species.     

In Vitro Evolution of an Archetypal Enteropathogenic Escherichia coli Strain

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal^r) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal^r strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str^r) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK. Another, in gyrA, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD, the lysogenization regulator, to purB. The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal^r clone has a lower growth rate than the Str^r isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal^r clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro.     

Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log₁₀ CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 μg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12°C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Correlation between In Vivo Biofilm Formation and Virulence Gene Expression in Escherichia coli O104:H4

The emergence of novel pathogens poses a major public health threat causing widespread epidemics in susceptible populations. The Escherichia coli O104:H4 strain implicated in a 2011 outbreak in northern Germany caused the highest frequency of hemolytic uremic syndrome (HUS) and death ever recorded in a single E. coli outbreak. Therefore, it has been suggested that this strain is more virulent than other pathogenic E. coli (e.g., E. coli O157:H7). The E. coli O104:H4 outbreak strain possesses multiple virulence factors from both Shiga toxin (Stx)-producing E. coli (STEC) and enteroaggregative E. coli (EAEC), though the mechanism of pathogenesis is not known. Here, we demonstrate that E. coli O104:H4 produces a stable biofilm in vitro and that in vivo virulence gene expression is highest when E. coli O104:H4 overexpresses genes required for aggregation and exopolysaccharide production, a characteristic of bacterial cells residing within an established biofilm. Interrupting exopolysaccharide production and biofilm formation may therefore represent effective strategies for combating future E. coli O104:H4 infections.     

Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain

The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.Shiga-toxigenic Escherichia coli (STEC) serotype O157:H7 carries O157 and H7 antigens, so these traits are extensively used in identification (1). Strain MA6, isolated from beef in Malaysia (8), carries the O157:H7 virulence factor genes, including the Shiga toxin 2 gene (stx₂), the γ intimin allele (γ-eae), the enterohemolysin gene (ehxA), and the +93 uidA single nucleotide polymorphism (SNP) found only in O157:H7 strains (1). Multilocus sequence typing also showed MA6 to have the most common sequence type (ST-66) for O157:H7 strains. However, and in spite the fact that MA6 had per gene sequences essential for O157 antigen synthesis (2), no O157 antigen is expressed (O rough), and therefore, it is undetectable with serological assays used in O157:H7 analysis.The biosynthesis and assembly of E. coli O antigen are highly complex (9). The rfb operon (12 genes) (16), along with 3 ancillary genes outside of the rfb, is required for the biosynthesis of the 4 sugar nucleotide precursors and the assembly of the O unit (11). This is then linked to the core antigen, comprising an inner and an outer component, which require 3 other operons for biosynthesis and assembly (9). As defects in any of these genes could produce the O-null phenotype (13), we systematically examined these genes (Table ​(Table1)1) to elucidate the cause of the absence of O157 expression in MA6.

TABLE 1.

rfb operon genes, ancillary genes, and waa cluster genes examined in this study

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.     

Tir细胞骨架偶联蛋白:大肠杆菌O157：H7的一种新的毒力因子

肠出血性大肠杆菌O157:H7是一种重要的传染性病原菌,可引起多种致死性疾病的爆发流行。Tir细胞骨架偶联蛋白(TccP)是近年来发现的O157:H7的一种新的重要的毒力因子,在O157:H7黏附宿主细胞造成黏附擦拭(A/E)损伤过程中起重要作用,TccP相关研究对阐明O157:H7致病机制具有重要意义。     

Production of an autoinducer of growth by norepinephrine cultured Escherichia coli O157:H7

Abstract Escherichia coli O157:H7 were cultured in the presence or absence of norepinephrine to generate conditioned media. The presence of a growth-inducing factoKs) in the conditioned media was examined by measurement of the ability of conditioned media to support the growth of fresh cultures of E. coli O157:H7. Supplementation of fresh cultures with as little as 0.024% (v/v) norepinephrine conditioned medium resulted in increased growth as compared to controls, thereby indicating the presence of an autoinducer of growth. Analysis of the production kinetics for the autoinducer during the generation of conditioned media indicates that it differs from other more well characterized autoinducers. It is proposed that the neurohumoral environment of the host may contribute to the production of bacterial growth factors.     

Escherichia coli O157:H7 Strain Origin,Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10³ to 10⁵ CFU and maximum colonization at 10⁷ CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx₁) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx₂). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx₂ expression in modulating the amount of E. coli O157:H7 colonization of cattle.Enterohemorrhagic Escherichia coli O157:H7 is a bacterium that causes serious human disease outbreaks through the consumption of contaminated food or water (39). Mature cattle are considered the primary reservoir for E. coli O157:H7 and historically were reported to have no symptoms or pathologies (17, 23, 38); this was attributed both to a lack of receptors for a critical E. coli O157:H7 virulence factor, Shiga toxin 1 (Stx1 [29]), and to a differential expression of type III protein secretion system effector molecules such as EspA, EspD, and Iha (25, 30) in cattle compared to humans. In 2008, it was established for the first time that E. coli O157:H7 causes mild to severe intestinal pathology in persistent shedding cattle (5, 26) and that the secreted cytotoxins enhanced E. coli O157:H7 colonization of intestinal tissues of cattle (6). This suggested that cattle were susceptible to E. coli O157:H7 infection and that previously discounted virulence factors could influence the amount of colonization in cattle.Three distinct E. coli O157:H7 lineages have been identified based on the lineage specific polymorphism assay (LSPA-6) that suggests both the evolutionary history of the strain and their propensity to be present among animals, the environment, and clinical human isolates (21, 22, 24, 33, 40, 42). Typically, two predominant lineages have been described, lineages I and II (22, 40) and, more recently, intermediate lineages that have characteristics of lineage I and/or II have been reported at higher frequency among cattle (34). Although all E. coli O157:H7 lineages have been isolated from feedlot cattle, the predominant recovery of lineage I from clinical human illnesses suggests that this particular lineage type has unique expression patterns that may contribute to its preferential colonization of humans. There is some evidence to suggest that lineage I strains do not express certain virulence factors in bovine hosts, whereas other factors such as cytotoxins are expressed equally irrespective of host (30). One virulence factor associated with all lineages is the bacterium''s ability to form intimate attaching-and-effacing lesions or colonization sites in the ilea of susceptible animals (28). The amount of colonization is enhanced by the expression of Shiga toxin 2 (Stx2) through both an increase in the expression of alternative non-TIR (translocated intimin receptor) colonization sites (31) and toxicity to the absorptive epithelial cells (32). In cattle, attaching-and-effacing lesions are also formed (5), and Stx2 increases colonization but is not cytotoxic to epithelial cells from the jejuna and descending colons of cattle (4). Differential expression of stx₂ among E. coli O157:H7 lineages is also linked to the increased pathogenicity of lineage I strains in humans (25), and this may affect cattle similarly. Together, this information suggests that at least some similar virulence factors affecting E. coli O157:H7 colonization in humans also function in cattle.In order to gain a better understanding of the factors modulating E. coli O157:H7 colonization in cattle, we compared the ability of lineage I, lineage II, and intermediate lineages isolated from human sources to colonize the jejunum tissue and a colonic cell line from cattle. We hypothesized that the bovine colonic cell line could be used as a model system to reflect E. coli O157:H7 colonization of tissue. To confirm the value of this model, the role of strain origin in colonization of cattle was examined. In order to understand the differences in colonization associated with lineage and strain origins, we assessed cytotoxin expression, secreted cytotoxin activity, and cytotoxin-induced changes in E. coli O157:H7 colonization. Given the known lack of Stx1 activity in cattle, we examined the effects of LSPA-6 genotype, strain origin (human versus bovine), and cytotoxin activity on E. coli O157:H7 colonization of cattle.