Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Modelling time to growth of Escherichia coli as a function of water activity and undissociated lactic acid

Aims: To model the effect of water activity (a_w) and concentration of undissociated lactic acid (HLac) on the time to growth (TTG) and the growth/no growth boundary of acid‐adapted generic Escherichia coli, used as model organisms for Shiga toxin‐producing E. coli (STEC). Methods and Results: For each of two E. coli strains, the TTG in brain heart infusion broth at 27°C was estimated at 30 combinations of a_w (range 0·945–0·995) and concentration of HLac (range 0–6·9 mol m^?3) by using an automated turbidity reader. Survival analysis was used to develop a model predicting the TTG and the growth/no growth boundary. Conclusions: The present model can be used to predict the TTG and to indicate the growth/no growth boundary of acid‐adapted E. coli strains as a function of a_w and concentration of HLac. Significance and Impact of the Study: Fermented food products have been implicated as sources of STEC in several outbreaks. The study results are relevant for modelling of growth of STEC in fermented food and can be used in microbiological risk assessments or in the design and validation of food‐production processes.     

Heat and lime-treatment as effective control methods for E. coli O157:H7 in organic wastes

Land-application of abattoir wastes is economically appealing and may provide an effective means of closing the nutrient cycling loop. This practise is constrained, however, by legislation which necessitates pre-treatment to remove pathogenic micro-organisms prior to land-spreading. Here we investigated whether heat-treatment or lime addition could eliminate Escherichia coli O157:H7 from three contrasting abattoir wastes. We found that treatment at 60 °C for 10 min effectively eradicated the organism while treatment for the same length of time at 50 °C led to 2–4 log reductions, but not a complete kill. Temperatures of 72 °C induced waste solidification rendering its use impractical. The potential for re-growth in heat-treated and untreated wastes was also investigated. Survival was significantly greater in heat-treated wastes, although the difference was less than half a log unit in magnitude. This effect of heat-treatment on pathogen survival appeared to be ameliorated when wastes were mixed with soil. No viable E. coli O157:H7 cells were recovered from any waste after application of lime (CaO) at a rate of 10 g l⁻¹, even after enrichment. Our results indicate that pasteurisation-style or liming treatments may provide a suitable alternative method for reducing pathogen loads in abattoir wastes, so that they can be applied to land with minimal biological risk.     

Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.     

Methods available for the sub-typing of Escherichia coli O157

The epidemiological investigation of Escherichia coli O157 is complicated by the lack of heterogeneity between strains responsible for the majority of cases of infection. As a consequence it is difficult to reliably cluster together an outbreak strain and differentiate it from other sporadically occurring isolates. The methods available for the sub-typing of E. coli O157 vary in their speed, technical complexity, cost and ability to discriminate reliably between strains, with many of the recently developed methods targeting the genome to provide differentiation. No single typing method is individually superior, and ideally a combination of techniques should be employed depending on the level of discrimination required and time or resources available. The aim of this review is to consider the relative merits of the available typing methodologies with particular emphasis on those which may find application in a diagnostic laboratory. This revised version was published online in November 2006 with corrections to the Cover Date.     

Occurrence of Escherichia coli O157 on hides of slaughtered cattle

AIMS: To obtain the first information on the occurrence of Escherichia coli O157 on hides of slaughtered cattle in Serbia. METHODS AND RESULTS: A total of 355 swabs were taken on the slaughterline from five areas of hide of each of the 71 cattle in a single commercial abattoir in Serbia. Using an ISO method incorporating enrichment and immunomagnetic separation steps, E. coli O157 was isolated from the hides of 20 animals (28 x 2%). With respect to different areas of the hides, the occurrence of the pathogen was, in decreasing order: hooves (11 x 3%), brisket (8 x 4%), rump (7 x 0%), neck (4 x 2) and flank (2 x 8%). In addition, factors that had more or less effects on the occurrence included visible dirtiness of the hide, cattle's age category, geographical origin of the animals and season. CONCLUSIONS: This study revealed the presence of E. coli O157 in the beef chain in Serbia and confirmed hide as an important potential source of related contamination of beef carcasses. Therefore, incorporation of preskinning hide decontamination treatments into HACCP-based slaughterline hygiene control measures could be very useful. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will enable further optimization of necessary measures along the beef chain to reduce the E. coli O157 risks in Serbia.     

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques

AIMS: The aim of this study was to isolate Escherichia coli O26, O103, O111 and O145 from 745 samples of bovine faeces using (i) immunomagnetic separation (IMS) beads coated with antibodies to lipopolysaccharide, and slide agglutination (SA) tests and (ii) PCR and DNA probes for the detection of the Verocytotoxin (VT) genes. METHODS AND RESULTS: IMS-SA tests detected 132 isolates of presumptive E. coli O26, 112 (85%) were confirmed as serogroup O26 and 102 had the VT genes. One hundred and twenty-two strains of presumptive E. coli O103 were isolated by IMS-SA, 45 (37%) were confirmed as serogroup O103 but only one of these strains was identified as Verocytotoxin-producing E. coli (VTEC). Using the PCR/DNA probe method, 40 strains of VTEC O26 and three strains of VTEC O103 were isolated. IMS-SA identified 21 strains of presumptive E. coli O145, of which only four (19%) were confirmed as serogroup O145. VTEC of this serogroup was not detected by either IMS-SA or PCR/DNA probes. E. coli O111 was not isolated by either method. CONCLUSION: IMS beads were 2.5 times more sensitive than PCR/DNA probe methods for the detection of VTEC O26 in bovine faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: IMS-SA is a sensitive method for detecting specific E. coli serogroups. However, the specificity of this method would be enhanced by the introduction of selective media and the use of tube agglutination tests for confirmation of the preliminary SA results.