Cefixime Tellurite Sorbitol MacConkey Research Articles

Atypical strains of verocytotoxin-producing Escherichia coli O157 in beef cattle at slaughter in Veneto region, Italy.

Cattle arriving for slaughter at abattoirs in the Veneto region of N. Italy were examined for intestinal carriage of Escherichia coli O157. Rectal swabs were cultured in modified buffered peptone water and E. coli O157 was concentrated by an immunomagnetic separation technique; the magnetic beads were cultured onto cefixime tellurite sorbitol MacConkey agar. Sorbitol non-fermenting E. coli O157 was isolated from 15 (3.6%) of 419 feedlot cattle but not from 437 veal calves or 65 culled cows. All strains of E. coli O157 hybridized with DNA probes specific for the VT1 or VT2 genes, but two strains did not produce toxin detectable by Vero cell assay. Six different plasmid profiles were observed with all strains harbouring the large 93 kb plasmid characteristic of VTEC. Six strains produced urease but otherwise strains were biochemically typical of E. coli O157. One strain was resistant to streptomycin, tetracycline and sulphonamides but the remainder were sensitive to all antimicrobials tested. This is the first description of the isolation of verocytotoxin-producing E. coli O157 from cattle in Italy. As the contamination of bovine carcasses with E. coli O157 during slaughter and processing has been demonstrated, the risk of transmission of this organism from beef cattle to the human population in the Veneto region, through foods of bovine origin or by other routes, should not be overlooked.

Specific identification of Escherichia coli O157:H7 using a multiplex PCR assay.

Entero-hemorrhagic Escherichia coli (EHEC) are associated with hemorrhagic colitis and the hemolytic uremic syndrome in humans. EHEC produce one or more antigenic type of Vero toxin (VT) and possess a gene designated eae, which is associated with attachment to- and effacement of the microvilli of enterocytes. E. coli O157:H7 is the most common EHEC serotype isolated and this organism has been associated with a number of serious outbreaks of food-borne disease. Cultural methods for identification of E. coli O157:H7 rely on selective media such as Cefixime Tellurite Sorbitol MacConkey Agar (Chapman et al., 1993), serotyping and toxin testing. This approach is labor intensive and may take a week or more to complete. In addition, certain other bacteria do not ferment sorbitol, grow on this selective media and cross-react in slide agglutination reactions with antiserum to the O157 lipopolysaccharide. Sorbitol-fermenting E. coli O157 belonging to the EHEC group have also been reported (Karch et al., 1993). We have previously described methods which allow rapid identification of E. coli O157:H7 and other EHEC using polymerase chain reaction (PCR) assays which target vt and eae genes (Gannon et al., 1992, Gannon et al., 1993). In these studies, so called “generic” EHEC PCR primer sets were developed which target vt genes and a 5′ conserved area of the eae gene. These PCR primers allow identification of all EHEC. In addition, a PCR assay was devised which is highly specific for E. coli O157:H7. This PCR assay uses the 3′ end of the eae gene of E. coli O157:H7 (eae O157) as a target for amplification. Unfortunately, strains of the enteropathogenic E. coli (EPEC) serotype O55:H7 and the EHEC serotype O145:H- are also positive in PCR assays with eae O157 oligonucleotide primers.KeywordsPolymerase Chain Reaction AssayHemolytic Uremic SyndromeMultiplex Polymerase Chain ReactionAntigenic TypeMultiplex Polymerase Chain Reaction AssayThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Evaluation of a commercial enzyme immunoassay (EHEC-Tek) for detecting Escherichia coliO157 in beef and beef products

A commercial enzyme immunoassay system (EHEC-Tek) was compared with immunomagnetic separation followed by culture to cefixime–tellurite–sorbitol MacConkey agar (IMS/C) for detectingEscherichia coli O157 in artificially inoculated beef samples and naturally contaminated beefburgers. The EHEC-Tek assay used modifiedE. coli broth supplemented with novobiocin (mECn) as a primary enrichment medium, followed by immunomagnetic separation, secondary enrichment of magnetic beads in tryptone soya broth supplemented with acriflavine, followed by a standard monoclonal antibody-based immunoassay to detect O157 antigen. Twelve diverse strains ofE. coli O157 were used to inoculate samples of minced beef in triplicate at each of three different concentrations (50, 5 and 0.5 cfu 25 g −1). When the 108 samples of minced beef were examined, there was complete agreement between EHEC-Tek and IMS/C on 87 samples. Ten samples were positive only by IMS/C and 11 samples were positive only by EHEC-Tek, such discrepant results occurring mainly at lower inoculum levels; of the latter 11, eight were confirmed culturally as containing E. coli O157. Five beefburgers naturally contaminated withE. coli O157 atc . 2 g −1were negative by both EHEC-Tek and IMS/C after primary enrichment in mECn, but when the primary enrichment medium was buffered peptone water supplemented with vancomycin, cefixime and cefsulodin (BPW-VCC) E. coli O157 was detected in four out of five samples by EHEC-Tek and five out of five samples by IMS/C. The EHEC-Tek immunoassay is a technically simple and sensitive method for detectingE. coli O157 in beef, and requires a minimum of microbiological expertise for its performance: the assay is improved by use of BPW-VCC in place of mECn as the primary enrichment medium.

Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture.

We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.

Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0.002-0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody. During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies.