Resolution of Escherichia coli O157:H7 That Contaminated Radish Sprouts in Two Outbreaks by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) was performed to examine exoproteins and periplasmic proteins of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated from cases associated with radish sprouts in two outbreaks. We found that STEC O157:H7 released a large number of proteins into the medium during the stationary phase of growth, as observed with 2-DE. Although pulsed-field gel electrophoresis (PFGE) patterns of STECs NGY9 (RIMD0509894), a Sakai isolate; NGY33, a Gamagoori isolate; and NGY120, a Kanagawa isolate, were all the same, comparison of 2-DE patterns of exoproteins and periplasmic proteins clarified that NGY9 was distinct from NGY33, whereas NGY33 and NGY120 were of close lineage. We therefore suggest that 2-DE analysis of exoproteins and periplasmic proteins is a powerful epidemiological method with high resolution. Received: 21 August 2000 / Accepted: 9 March 2001     

Role for Salmonella enterica enterobacterial common antigen in bile resistance and virulence

Passage through the digestive tract exposes Salmonella enterica to high concentrations of bile salts, powerful detergents that disrupt biological membranes. Mutations in the wecD or wecA gene, both of which are involved in the synthesis of enterobacterial common antigen (ECA), render S. enterica serovar Typhimurium sensitive to the bile salt deoxycholate. Competitive infectivity analysis of wecD and wecA mutants in the mouse model indicates that ECA is an important virulence factor for oral infection. In contrast, lack of ECA causes only a slight decrease in Salmonella virulence during intraperitoneal infection. A tentative interpretation is that ECA may contribute to Salmonella virulence by protecting the pathogen from bile salts.     

Mouse model for hemolytic uremic syndrome induced by outer membrane vesicles of Escherichia coli O157:H7

Hemolytic uremic syndrome (HUS) is characterized by acute renal failure in children and is typically complicated with thrombocytopenia and hemolytic anemia. Although mouse models of HUS have been evaluated using Shiga toxin (STx) combined with or without lipopolysaccharide (LPS), no HUS model has been tested using purified outer membrane vesicles (OMVs) from STx-producing Escherichia coli (STEC) O157:H7. Accordingly, we investigated whether OMVs of STEC O157:H7 conveying STx2 and LPS can cause HUS-like symptoms in mice inoculated intraperitoneally. Three types of OMVs differing in LPS acylation status and STx2 amount were used to compare their ability to induce HUS-like symptoms. Native OMVs (nOMV) with fully hexa-acylated LPS caused HUS-like symptoms at 72-96?h after dually divided injections of 1?μg nOMV per animal. However, elevated doses of modified OMVs (mOMV) carrying mainly penta-acylated LPS were required to induce similar HUS signs. Moreover, mitomycin-C-induced OMVs (mcOMV) that were enriched with STx2 induced HUS-like symptoms similar to those of nOMV at the same dose. These results suggest that the OMVs of STEC O157:H7 potentiated with STx2 and fully hexa-acylated LPS can be utilized for inducing HUS-like symptoms in mice and could be the causative material involved in the development of HUS.     

Structure of the O-antigen of Providencia stuartii O20, a new polysaccharide containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid

The O-polysaccharide chain of the lipopolysaccharide (LPS) of Providencia stuartii O20 was found to contain d-glucuronic acid, N-acetyl-d-glucosamine, and a rarely occurring higher sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). Degradation of the LPS with dilute acetic acid caused depolymerization of the polysaccharide chain by the ketosidic linkage to give a tetrasaccharide corresponding to the repeating unit of the polysaccharide. Based on sugar and methylation analyses of the tetrasaccharide and O-deacylated LPS as well as ESIMS, (1)H and (13)C NMR spectroscopy data, the structure of the O-polysaccharide of P. stuartii O20 was established.     

Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli O157:H7 as a function of growth rate and growth-limiting nutrient

Escherichia coli O157:H7 was grown in chemostats as continuous cultures at different controlled growth rates and under different nutrient limitations to determine the effects on lipopolysaccharide (LPS) structure. LPS from whole cells and extracted using the hot aqueous phenol method was examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration after hydrolysis with acetic acid. At low growth rates under glucose limitation (D = 0.1 h-1, doubling time (td), approx. 416 min; or D = 0.4 h-1, td, approx. 104 min), E. coli O157 produced high molecular weight LPS identical to that previously characterized from cells grown in batch culture. At a high growth rate (D = 0.8 h-1, td, approx. 52 min), the ratio of high molecular weight LPS to low molecular weight LPS produced greatly decreased. A small amount of high molecular weight LPS, containing O-polysaccharide which lacked amino sugars, and which thus was chemically different from that previously characterized, was produced by the cells at high growth rates. The predominant form of LPS from these cells was of slightly higher molecular weight than rough LPS, probably S-R LPS, and it consistently formed aggregates on SDS-PAGE. This form of LPS was also predominant when E. coli O157 was grown under Mg2+ limitation at an intermediate growth rate (D = 0.4 h-1, td, approx. 104 min).     

Identification of three oligo-/polysaccharide-specific ligases in Yersinia enterocolitica

In lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria the lipid A-core oligosaccharide (LA-core) and O-polysaccharide (O-PS) biosynthesis pathways proceed separately and converge in periplasmic space where the waaL-encoded ligase joins O-PS onto LA-core. Enterobacterial common antigen (ECA) biosynthesis follows that of O-PS except that ECA is usually ligated to phosphatidylglycerol (PG) and only rarely to LA-core. In Yersinia enterocolitica serotype O:3 LPS is composed of LA-inner core (IC) onto which a homopolymeric O-PS, a hexasaccharide called outer core (OC), and/or ECA are ligated. We found that an individual O:3 LPS molecule carries either OC or O-PS substitution but not both. Related to this, we identified three genes in Y. enterocolitica O:3 that all expressed O-PS ligase activity in the Escherichia coliΔwaaL mutant. The LPS phenotypes of Y. enterocolitica O:3 single, double and triple ligase mutants indicated that two of ligases, named as WaaL(os) and WaaL(ps) , had a preferred substrate specificity for OC and O-PS, respectively, although with some promiscuity between the ligases; the third ligase named as WaaL(xs) was not involved in LPS or ECA biosynthesis. In Y. enterocolitica O:8 the WaaL(os) homologue (Ye1727) ligated a single pentasaccharide O-unit to LA-IC suggesting that in both Y. enterocolitica O:3 and O:8 WaaL(os) is an oligosaccharide (OS)-specific ligase. Finally, Yersinia pestis and Y. pseudotuberculosis carry only the waaL(ps) gene, while either waaL(os) or waaL(xs) or both are additionally present in other Yersinia species. This is the first report on the presence of three different oligo-/polysaccharide-specific ligases in a single bacterium.     

Structure of supported bilayers composed of lipopolysaccharides and bacterial phospholipids: raft formation and implications for bacterial resistance

Lipopolysaccharide (LPS), the major lipid on the surface of Gram-negative bacteria, plays a key role in bacterial resistance to hydrophobic antibiotics and antimicrobial peptides. Using atomic force microscopy (AFM) we characterized supported bilayers composed of LPSs from two bacterial chemotypes with different sensitivities to such antibiotics and peptides. Rd LPS, from more sensitive "deep rough" mutants, contains only an inner saccharide core, whereas Ra LPS, from "rough" mutants, contains a longer polysaccharide region. A vesicle fusion technique was used to deposit LPS onto either freshly cleaved mica or polyethylenimine-coated mica substrates. The thickness of the supported bilayers measured with contact-mode AFM was 7 nm for Rd LPS and 9 nm for Ra LPS, consistent with previous x-ray diffraction measurements. In water the Ra LPS bilayer surface was more disordered than Rd LPS bilayers, likely due to the greater volume occupied by the longer Ra LPS polysaccharide region. Since deep rough mutants contain bacterial phospholipid (BPL) as well as LPS on their surfaces, we also investigated the organization of Rd LPS/BPL bilayers. Differential scanning calorimetry and x-ray diffraction indicated that incorporation of BPL reduced the phase transition temperature, enthalpy, and average bilayer thickness of Rd LPS. For Rd LPS/BPL mixtures, AFM showed irregularly shaped regions thinner than Rd LPS bilayers by 2 nm (the difference in thickness between Rd LPS and BPL bilayers), whose area increased with increasing BPL concentration. We argue that the increased permeability of deep rough mutants is due to structural modifications caused by BPL to the LPS membrane, in LPS hydrocarbon chain packing and in the formation of BPL-enriched microdomains.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7

Aims:  To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans.
Methods and Results:  Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004–0·006, P  < 0·0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0·002–0·04, P  < 0·0001) than did phage wV8 (25–29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81·0%), 321 (76·1%) and 407 (96·4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype.
Conclusions:  Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7.
Significance and Impact of the Study:  The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy.     

Prevalence and characterization of non-O157 Shiga toxin-producing Escherichia coli on carcasses in commercial beef cattle processing plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples

This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157 for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.     

Comparison of four enrichment broths for the detection of non-O157 Shiga-toxin-producing Escherichia coli O91, O103, O111, O119, O121, O145 and O165 from pure culture and food samples

Aims: We compared the efficiency of universal pre‐enrichment broth (UPB), modified Escherichia coli broth containing novobiocin (mEC + n), modified Tryptic Soy Broth (mTSB) and mTSB with novobiocin (mTSB + n) for the enrichment of non‐O157 Shiga‐toxin‐producing E. coli (STEC). Methods and Results: Freeze‐injured and control non‐O157 STEC (O91, O103, O111, O119, O121, O145 and O165) strains were used to artificially contaminate beef and radish sprout samples, which were then cultivated in each of the four enrichment media. After incubation, STEC strains were detected by loop‐mediated isothermal amplification (LAMP) and plating assays. Enrichment in mEC + n was least effective for facilitating the detection of uninjured STEC strains in radish sprouts, while mTSB + n was least effective for enriching freeze‐injured non‐O157 STEC strains from beef samples for detection by LAMP assay. UPB and mTSB were superior to mEC + n and mTSB + n for the enrichment of non‐O157 STEC from food samples. Conclusions: The enrichment of non‐O157 STEC was negatively affected by the addition of novobiocin to enrichment broths. Significance and Impact of the study: Novobiocin should not be added to media used for the enrichment of non‐O157 STEC in food when cell injury is anticipated.     

Phylogenetic classification of Escherichia coli O157:H7 strains of human and bovine origin using a novel set of nucleotide polymorphisms

Background

Cattle are a reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157), and are known to harbor subtypes not typically found in clinically ill humans. Consequently, nucleotide polymorphisms previously discovered via strains originating from human outbreaks may be restricted in their ability to distinguish STEC O157 genetic subtypes present in cattle. The objectives of this study were firstly to identify nucleotide polymorphisms in a diverse sampling of human and bovine STEC O157 strains, secondly to classify strains of either bovine or human origin by polymorphism-derived genotypes, and finally to compare the genotype diversity with pulsed-field gel electrophoresis (PFGE), a method currently used for assessing STEC O157 diversity.

Results

High-throughput 454 sequencing of pooled STEC O157 strain DNAs from human clinical cases (n = 91) and cattle (n = 102) identified 16,218 putative polymorphisms. From those, 178 were selected primarily within genomic regions conserved across E. coli serotypes and genotyped in 261 STEC O157 strains. Forty-two unique genotypes were observed that are tagged by a minimal set of 32 polymorphisms. Phylogenetic trees of the genotypes are divided into clades that represent strains of cattle origin, or cattle and human origin. Although PFGE diversity surpassed genotype diversity overall, ten PFGE patterns each occurred with multiple strains having different genotypes.

Conclusions

Deep sequencing of pooled STEC O157 DNAs proved highly effective in polymorphism discovery. A polymorphism set has been identified that characterizes genetic diversity within STEC O157 strains of bovine origin, and a subset observed in human strains. The set may complement current techniques used to classify strains implicated in disease outbreaks.     

Development of a Robust Method for Isolation of Shiga Toxin-Positive Escherichia coli (STEC) from Fecal,Plant, Soil and Water Samples from a Leafy Greens Production Region in California

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the “Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.     

Chemical characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.     

Structure of a teichoic acid-like O-polysaccharide of Escherichia coli O29

A teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O29. The O-polysaccharide and an oligosaccharide obtained by dephosphorylation of the O-polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The following structure of the branched oligosaccharide repeating unit, containing five monosaccharide residues and glycerol 1-phosphate (D-Gro-1-P), was established: [carbohydrate structure: see text].     

The Escherichia coli ycbQRST operon encodes fimbriae with laminin-binding and epithelial cell adherence properties in Shiga-toxigenic E. coli O157:H7

The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E. coli . We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18 kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E . coli YcbQ l aminin-binding f imbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro . Deletion in the fimbrin subunit gene elfA (or ycbQ ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.     

Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

Comparison of different PCR tests for detecting Shiga toxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria

In an attempt to develop a standard for ELISA-PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3' end of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5' end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO(157) locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157.     

Alteration of the rugose phenotype in waaG and ddhC mutants of Salmonella enterica serovar Typhimurium DT104 is associated with inverse production of curli and cellulose

The rugose (also known as wrinkled or rdar) phenotype in Salmonella enterica serovar Typhimurium DT104 Rv has been associated with cell aggregation and the ability, at low temperature under low-osmolarity conditions, to form pellicles and biofilms. Two Tn5 insertion mutations in genes that are involved in lipopolysaccharide (LPS) synthesis, ddhC (A1-8) and waaG (A1-9), of Rv resulted in diminished expression of colony rugosity. Scanning electron micrographs revealed that the ddhC mutant showed reduced amounts of extracellular matrix, while there was relatively more, profuse matrix production in the waaG mutant, compared to Rv. Both mutants appeared to produce decreased levels of curli, as judged by Western blot assays probed with anti-AgfA (curli) antibodies but, surprisingly, were observed to have increased amounts of cellulose relative to Rv. Comparison with a non-curli-producing mutant suggested that the alteration in curli production may have engendered the increased presence of cellulose. While both mutants had impaired biofilm formation when grown in rich medium with low osmolarity, they constitutively formed larger amounts of biofilms when the growth medium was supplemented with either glucose or a combination of glucose and NaCl. These observations indicated that LPS alterations may have opposing effects on biofilm formation in these mutants, depending upon either the presence or the absence of these osmolytes. The phenotypes of the waaG mutant were further confirmed in a constructed, nonpolar deletion mutant of S. enterica serovar Typhimurium LT2, where restoration to the wild-type phenotypes was accomplished by complementation. These results highlight the importance of an integral LPS, at both the O-antigen and core polysaccharide levels, in the modulation of curli protein and cellulose production, as well as in biofilm formation, thereby adding another potential component to the complex regulatory system which governs multicellular behaviors in S. enterica serovar Typhimurium.