Evaluation of the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook methods for detection of Shiga toxin-producing Escherichia coli (STEC) and STEC and Salmonella simultaneously in ground beef.

AIMS: To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top seven Shiga toxin-producing Escherichia coli (STEC) (O157:H7, O26, O45, O103, O111, O121 and O145) in ground beef and both STEC and Salmonella in co-inoculated samples. METHODS AND RESULTS: Ground beef samples inoculated with ~10 CFU of STEC or both STEC and Salmonella Typhimurium were stored at 4°C for 72 h, followed by screening with the IQ-Check and BAX System kit (MLG) methods that employ different enrichment media. STEC and S. Typhimurium were detected after 12 and 18 h and their presence was confirmed by colony isolation. CONCLUSIONS: Both methods were able to detect STEC in ground beef after 12 h of enrichment in samples inoculated with low levels of the pathogen. STEC and S. Typhimurium can be detected and isolated in co-inoculated ground beef samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The IQ-Check methods are comparable to the MLG methods for detection of STEC and simultaneous detection of STEC and S. Typhimurium in seeded ground beef after a short enrichment time, thus the IQ-Check method can be useful for the food industry for rapid detection of these pathogens.

Shiga toxin-producing E. coli (STEC) in swine: prevalence over the finishing period and characteristics of the STEC isolates.

This descriptive longitudinal study was conducted to investigate the faecal shedding of Shiga toxin-producing E. coli (STEC) in finishing swine and to characterize the swine STEC isolates that were recovered. Three cohorts of finishing swine (n = 50/cohort; total 150 pigs) were included in the longitudinal study. Individual faecal samples were collected every 2 weeks (8 collections/pig) from the beginning (pig age 10 weeks) to the end (pig age 24 weeks) of the finishing period. STEC isolates were recovered in at least one sample from 65·3% (98/150) of the pigs, and the frequency distribution of first-time STEC detection during the finishing period resembled a point-source outbreak curve. Nineteen O:H serotypes were identified among the STEC isolates. Most STEC isolates (n = 148) belonged to serotype O59:H21 and carried the stx 2e gene. One O49:H21 STEC isolate carried the stx 2e and eae genes. High prevalence rates of STEC during the finishing period were observed, and STEC isolates in various non-O157 serogroups were recovered. These data enhance understanding of swine STEC epidemiology, and future research is needed to confirm whether or not swine STEC are of public health concern.

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching.

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.

A multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157:H7 in foods and bovine feces.

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was < or = 1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

Molecular characterization of an antibiotic resistance gene cluster of Salmonella typhimurium DT104.

Salmonella typhimurium phage type DT104 has become an important emerging pathogen. Isolates of this phage type often possess resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT resistance). The mechanism by which DT104 has accumulated resistance genes is of interest, since these genes interfere with treatment of DT104 infections and might be horizontally transferred to other bacteria, even to unrelated organisms. Previously, several laboratories have shown that the antibiotic resistance genes of DT104 are chromosomally encoded and involve integrons. The antibiotic resistance genes conferring the ACSSuT-resistant phenotype have been cloned and sequenced. These genes are grouped within two district integrons and intervening plasmid-derived sequences. This sequence is potentially useful for detection of multiresistant DT104.

Evaluation of an enzyme-linked immunosorbent assay, direct immunofluorescent filter technique, and multiplex polymerase chain reaction for detection of Escherichia coli O157:H7 seeded in beef carcass wash water.

In commercial beef processing, carcasses are customarily washed with water to remove physical and microbial contamination. Assaying the water that is shed from the carcasses after washing is a convenient method to determine whether the carcass is contaminated with Escherichia coli O157:H7 or other bacterial pathogens. E. coli O157:H7 was inoculated into carcass wash water at various levels and the bacteria were then concentrated by filtration. After collection of bacteria in the filter units, the nylon membranes were cut out and placed in tubes containing growth medium, and the tubes were mixed vigorously to dislodge the bacteria from the membranes. Prior to enrichment, samples were removed for testing by a multiplex polymerase chain reaction (PCR) and a direct immunofluorescent filter technique (DIFT). The remaining samples were subjected to 4-h enrichment culturing at 37 degrees C, after which aliquots were removed for testing by multiplex PCR, DIFT, and an enzyme-linked immunosorbent assay (ELISA). Following 4-h enrichment culturing, E. coli O157:H7 was detected in wash water samples initially inoculated with ca. 100, 0.1, and 1 CFU/ml by ELISA, DIFT, and multiplex PCR, respectively. Testing of the wash water using the ELISA and the DIFT can be accomplished in less than 8 h. On the basis of these results, assaying carcass wash water by ELISA, DIFT, or multiplex PCR can be useful for detection of E. coli O157:H7 beef carcass contamination and can potentially be employed to identify carcasses for further processing to inactivate the organism.

Enterotoxigenic Escherichia coli detected in foods by PCR and an enzyme-linked oligonucleotide probe.

A polymerase chain reaction (PCR) and an enzyme-linked oligonucleotide probe hybridization assay were developed for the detection of enterotoxigenic Escherichia coli (ETEC) in ground beef, chicken, pork and raw milk. Two synthetic primers, one of which was biotinylated, were used in the PCR to amplify a fragment of the E. coli heat-labile enterotoxin (LT) gene. The identity of the amplified products was confirmed by liquid hybridization using a horseradish peroxidase-linked internal oligonucleotide probe in a 96-well microplate coated with streptavidin. The final quantitation of the PCR products was performed by a colorimetric reaction. Under established conditions (including 1 min at 60 degrees C for primer annealing and extension in PCR cycles), this method detected all 7 LT-producing E. coli pathogenic for humans, but did not detect all 7 LT-positive E. coli of animal origin 3 E. coli strains that do not produce LT, and 9 other bacteria. Under less stringent PCR conditions (55 degrees C for annealing and extension), 2 strains of LT-producing E. coli of porcine origin were detected while the results of other bacterial strains remained unchanged. In pure cultures, the detection limit of the method was 1.4 colony forming units (CFU). Prior to PCR amplification, all food samples inoculated with an LT-producing ETEC, were subjected to enrichment in brain heart infusion broth for 8 h at 37 degrees C. From these cultures, 10 microliters was heated at 95 degrees C for 10 min and directly used in the PCR. An initial inoculum of as few as 1.2 to 12 CFU of the LT-producing ETEC per 25 g (or ml) of food sample gave a positive reaction.

Detection of Escherichia coli O157:H7 by multiplex PCR.

In order to develop a PCR assay for Escherichia coli O157:H7, a portion of the 60-MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then designed by employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxins I (SLT-I) and II (SLT-II), and the 60-MDa plasmid. PCR products of 1,087 bp (eaeA), 227 and/or 224 bp (SLT-I and/or SLT-II), and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC of serogroup O157.

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.

Studies on Escherichia coli serotype O157:H7 strains containing a 60-MDa plasmid and on 60-MDa plasmid-cured derivatives.

Seventeen strains of Escherichia coli serotype O157:H7 producing Shiga-like toxin were examined for the presence of plasmids and for the ability to adhere to HEp-2 and Intestine 407 cells. All of the strains possessed a common 60-MDa plasmid. To determine the role of the 60-MDa plasmid, plasmid-cured strains were compared with the parent strains for their ability to produce pili and to adhere to epithelial cells in culture. The total cell lysate protein and outer-membrane protein (OMP) profiles were also compared. Both the parent strains and their plasmid-cured derivatives produced pili. Immunofluorescence assay results indicated that the plasmid-cured and parent strains adhered equally well to HEp-2 and Intestine 407 cells; overall adherence was greater with intestinal cells than HEp-2 cells. SDS-PAGE of polypeptides synthesised in an E. coli system in vitro showed that plasmid DNA encodes c. 35 proteins. SDS-PAGE of OMP preparations demonstrated that the 60-MDa plasmid appears to be involved in the synthesis of a 33-kDa OMP. Two strains cured of the 60-MDa plasmid, one that possessed no plasmids and one that still contained a 2.2-MDa plasmid, produced exopolysaccharide (EPS) when cultured on solid medium at 25 degrees C. Two other strains, which were cured of the 60-MDa plasmid but contained a 4.5-MDa plasmid, did not produce visible amounts of EPS. Gas chromatography analysis showed that the EPS consisted of fucose, glucose and galactose in an approximate molar ratio of 2.0:0.9:1.1 and also had 7% of a uronic acid sugar as part of its structure.

Identification and characterization of an immunodominant 58-kilodalton antigen of Aspergillus fumigatus recognized by sera of patients with invasive aspergillosis.

Sera from 38 patients with invasive aspergillosis were tested by Western immunoblotting for the presence of antibodies to antigens of Aspergillus fumigatus present in a mycelial extract of the organism. All of the sera contained antibodies to an antigen of molecular weight 58,000, which was which was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was the only antigen recognized in approximately 90% of the sera tested. The 58-kDa antigen is an abundant component of mycelial extracts composing approximately 50% of the Coomassie blue-stained protein. The antigen also contains carbohydrate, since it is stained by the carbohydrate stain periodic acid-Schiff and it binds to the lectin concanavalin A. It was purified by immunoaffinity chromatography employing a monoclonal antibody directed against an epitope on the 58-kDa antigen. Analysis of the purified antigen by gas-liquid chromatography revealed the presence of mannose, galactose, and glucose residues in a 2:1:2 ratio. The ratio of protein to carbohydrate is 1.16:1. The protein is slightly acidic, containing relatively high quantities of glutamic and aspartic acids, glycine, alanine, serine, and threonine. The 58-kDa antigen also contains phosphate groups as part of its structure. Serological activity was totally destroyed after treatment with sodium metaperiodate and was partially destroyed after treatment with pronase. The 58-kDa antigen was not able to hydrolyze protein.

Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus.

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.