Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

Occurrence of Escherichia coli O157:H7 in faeces, skin and carcasses from sheep and goats in Ethiopia

Aims:  To determine the occurrence and proportion of Escherichia coli O157:H7 in faeces, skin swabs and carcasses before and after washing, from sheep and goats in Ethiopia.
Method and Results:  Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx ₁, stx ₂ and eae genes. Escherichia coli O157:H7 was isolated from faeces (4·7%), skin swabs (8·7%) and carcasses before washing (8·1%) and after washing (8·7%) and on water samples (4·2%). The proportion of carcasses contaminated with E. coli O157:H7 was strongly associated with those recovered from faecal and skin samples. Both stx ₁ and stx ₂ genes were identified from one E. coli O157:H7 isolate from a goat carcass.
Conclusions:  Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E.   coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations.
Significance and Impact of the Study:  Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins.     

Evaluation of methods for the isolation and detection of Escherichia coli O157 in minced beef

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157 inoculated into minced beef. The use of a 24 h enrichment in modified EC broth containing novobiocin allowed low numbers of contaminating cells to multiply to levels detectable on culture media and by ELISA test kits. Total analysis time was reduced by the use of the Dynabead^TM immunomagnetic separation system. The use of the Petrifilm^TM Test Kit-HEC for E. coli O157: H7 and Organon Teknika EHEC-TEK system detected low numbers of contaminating cells following enrichment and reduced analysis time by 1 d. The incorporation of cefixime and tellurite into Sorbitol MacConkey Agar increased the rate and ease of isolation of E. coli O157 and its use is therefore recommended.     

Prevalence and potential link between E. coli O157:H7 isolated from drinking water, meat and vegetables and stools of diarrhoeic confirmed and non-confirmed HIV/AIDS patients in the Amathole District - South Africa

Aim:  The current study investigated the prevalence and molecular relatedness between Escherichia coli O157:H7 isolated from water, meat and meat products and vegetables and from stools of confirmed and non-confirmed Human Immune Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) patients with diarrhoea.
Methods and Results:  Culture-based and polymerase chain reaction techniques were used to identify E . coli O157:H7. Thirty-five per cent of meat products, 25·5% of water, 21·7% of vegetables as well as 56·5% and 43·5% of stools of confirmed and non-confirmed HIV/AIDS patients, respectively, were presumptively positive with E . coli O157. Molecular results indicated that 10·3%, 8·6% and 7·8% of the vegetables, water and meat products examined carried E . coli O157:H7, which had homologous fliC _H7 , rfbE _O157 and eaeA genetic loci to the genes of some E . coli O157:H7 isolated from 12·2% and 8·8% of the stools of confirmed and non-confirmed HIV/AIDS patients, respectively.
Conclusions:  Water, meat and meat products and vegetables are potential sources of E . coli O157:H7 that are potentially capable of causing diarrhoea in humans especially HIV/AIDS patients.
Significance and Impact of the Study:  Great care should be exercised to ensure that water and foods consumed by HIV/AIDS patients are safe, as contaminated water and foods can cause secondary infections in these patients.     

Electrophoretic profile of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains

Escherichia coli O157 : H7 (O157) has unusual acid tolerance. The influence of heat shock on acid tolerance of O157 was studied. Seven strains of O157 and E. coli K-12 were tested for their ability to survive in minimum glucose medium (pH 2·5) at 37 °C. The survival of heat-shocked (10 min at 48 °C) cells was about 10–100 times greater compared with untreated cells depending on the strain. No significant difference ( P > 0·05) for O157 strain 932 was observed between heat shock-induced and acid adaptation-induced (pH 5·0) acid tolerance. Chloramphenicol prevented heat shock-induced acid tolerance, indicating the requirement of newly synthesized protein(s). Two outer membrane proteins (OMP) (22 and 15 kDa) were synthesized within 10 min of heat shock and were expressed for at least 6 h by cells held at 37 °C. N-terminal amino acid sequence analysis suggested that the 22 kDa OMP is a component of an alkyl hydroperoxide reductase. This protein contains a redox active disulphide, which is probably involved in H⁺ transport. Results indicate that sublethal heat treatment of O157 cells substantially increases their tolerance to acidic conditions. This could have practical implications for foods that receive a mild heat treatment and rely on acid as a preservative.     

Effectiveness of a steam-vacuum sanitizer for reducing Escherichia coli O157:H7 inoculated to beef carcass surface tissue

A steam-vacuum sanitizer reduced aerobic plate counts associated with bovine faecal contamination from 5.5 log₁₀ cfu cm⁻² to 3.0 ± 0.21 log₁₀ cfu cm⁻² on beef carcass short plates. The same beef carcass short plates inoculated wiht 7.6 ± 0.09 log₁₀ cfu cm⁻² Escherichia coli O157: H7 in faeces, yielded an average residual level of E. coli O157: H7 of 2.1 ± 0.21 log₁₀ cfu cm⁻² after steam-vacuum treatments. This study demonstrates the effectiveness of a steam-vacuum sanitizer for removing E. coli O157: H7 from beef carcasses.     

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti- E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10g of ground beef when samples were enriched in modified EC broth for 7 h at 37°C. The minimum detectable number of cells for the apple cider samples was calculated to be ∼0.5 cfu ml^-1 The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.     

Persistence of Escherichia coli O157:H7 on the rhizosphere and phyllosphere of lettuce

Aims:  The major objective of this study was to determine the effects of low levels of Escherichia coli O157:H7 contamination on plant by monitoring the survival of the pathogen on the rhizosphere and leaf surfaces of lettuce during the growth process.
Methods and Results:  Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the rhizosphere and leaf surfaces after planting. Real-time PCR assays were designed to amplify the stx 1, stx 2 and the eae genes of E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real-time PCR was 2·4 × 10³ CFU g⁻¹ of starting DNA in rhizosphere and phyllosphere samples and about 10² CFU g⁻¹ by plate count. The time for pathogens to reach detection limits on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere. However, real-time PCR continued to detect stx 1, stx 2 and the eae genes throughout the experimental period.
Conclusion:  Escherichia coli O157:H7 survived throughout the growth period as was determined by real-time PCR and by subsequent enrichment and immunomagnetic separation of edible part of plants.
Significance and impact of the Study:  The potential presence of human pathogens in vegetables grown in soils contaminated with E. coli O157:H7 is a serious problem to our national food supply as the pathogen may survive on the leaf surface as they come in contact with contaminated soil during germination.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

THE USE OF STREPTAVIDIN COATED MAGNETIC BEADS FOR DETECTING PATHOGENIC BACTERIA BY LIGHT ADDRESSABLE POTENTIOMETRIC SENSOR (LAPS)

A modified procedure for magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor (LAPS) detection using the Threshold System was developed. Streptavidin coated magnetic beads, partially labeled with biotinylated anti Escherichia coli O157 antibodies, were used to capture Escherichia coli O157:H7. Captured bacteria were further labeled with fluorescein-conjugated anti -E. coli O157:H7 antibodies and urease-labeled. anti-fluorescein antibody. Magnetically concentrated bacteria-containing complexes were then immobilized through streptavidin-biotin interactions on 0.45 μ biotinylated nitro-cellulose membranes assembled as sample sticks for the Threshold instrument. The rate of pH change associated with the production of NH₃ by the urease in urea-containing solution was measured by a LAPS incorporated in the Threshold instrument. This approach allowed us to detect 10³ to 10⁴ CPU of cultured E. coli O157:H7 in PBS solutions. Furthermore, detectable LAPS signals of the sample sticks remained relatively constant for at least 24 h at 4C. The developed approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-h enrichment at 37C, as low as 1 CFU/g of E. coli O157:H7 in beef hamburger could be detected.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

Growth, diet and metabolism of common wolf–fish in the North Sea, a fast–growing population

The von Bertalanffy growth parameters for common wolf–fish Anarhichas lupus in the North Sea were: male: L ∞=111·2 cm, t ₀=–0·43 and K =0·12; and female: L ∞=115·1 cm, t ₀=–0·39 and K =0·11, making this the fastest growing stock reported. Resting metabolic rates (RMR±S.E.) and maximum metabolic rates (MMR±S.E.) for six adult common wolf–fish (mean weight, 1·39 kg) at 5° C were 12·18±1·6 mg O₂ kg^–1 h^–1 and 70·65±7·63 mg O₂ kg^–1 h^–1 respectively, and at 10° C were 25·43±1·31 mg O₂ kg^–1 h^–1 and 113·84±16·26 mg O₂ kg^–1 h^–1. Absolute metabolic scope was 53% greater at 10° C than at 5° C. The diet was dominated by Decapoda (39% overall by relative occurrence), Bivalvia (20%) and Gastropoda (12%). Sea urchins, typically of low energy value, occupied only 7% of the diet. The fast growth probably resulted from summer temperatures approximating to the optimum for food processing and growth, but may have been influenced by diet, and reduced competition following high fishing intensity.     

EVALUATION OF 5'NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

Two 5'nuclease-based PCR methods (PCR-LS-50B and PCR-7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR-7200 method was able to detect E. coli O157:H7 at ± 10² CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR-7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR-LS-50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR-based detection techniques for E. coli O157:H7, however, the 5'nuclease-based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.     

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157 : H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean^® purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g⁻¹ faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157 : H7 in less than 8 h, but with a sensitivity of approximately 10³ cfu g⁻¹ faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.     

Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef.

Artificially inoculated Escherichia coli O157:H7 was directly enumerated in ground beef and beef exudate, without enrichment or selection, by the antibody-direct epifluorescent filter technique (Ab-DEFT). The total assay time of the Ab-DEFT was less than 1 h. The beef was homogenized, treated for 15 min with trypsin and Triton X-100, and passed through a 5-microns-pore-size prefilter and then through a 0.2-microns-pore-size black polycarbonate filter. The final filter was stained directly with fluorescein-labeled anti-O157 polyclonal antibody, rinsed, and examined by epifluorescence microscopy. The sensitivity of the Ab-DEFT was compared with that of a standard enrichment culture technique. Both methods reliably determined the presence of the pathogen in beef at 16 CFU/g. The Ab-DEFT was also useful for quantifying the pathogen and monitoring its growth in beef.     

A MEMBRANE SEPARATOR/BIOREACTOR COUPLED WITH ABSORBANCE MEASUREMENT FOR DETECTION OF Escherichia coli O157:H7

A membrane separator/bioreactor system was developed for rapid detection of Escherichia coli O157:H7. The system consisted of a membrane separator/bioreactor (0.45 μm of the pore size) to separate the-complexes of E. coli O157:H7 and alkaline phosphatase-conjugated anti-E. coli O157:H7 antibodies from the sample and to produce p-nitrophenol through the enzymatic reaction (p-nitrophenyl phosphate hydrolysis), and an optical detector for measuring the p-nitrophenol absorbance at 400 nm. The membrane material and the flow rate of the substrate for the enzymatic hydrolysis had great effects on the absorbance of p-nitrophenol. The optimum conditions for the enzymatic reaction were determined as 1.0 M Tris buffer, pH 8.0, and 0.1 M MgCl₂ for this system. The detection range was 10⁴± 10⁷ CFU/mL with a relative standard deviation of 4.3 ± 14.2%, and whole procedure could be completed in 50 min without any enrichment and culture. Other bacteria such as Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes had no significant interference with the detection of E. coli O157:H7.     

Direct inoculation into media containing bile salts and antibiotics is unsuitable for the detection of acid/salt stressed Escherichia coli O157:H7

The efficiency of selective enrichment broths for the recovery of low numbers of acid/salt stressed Escherichia coli O157:H7 was determined. Stressed cultures were diluted to low levels and recovered in tryptone soya broth with added bile salts, to make modified tryptone soya broth, and buffered peptone water with various combinations of antibiotic supplementation including novobiocin, acriflavine and a mixture of vancomycin, cefsulodin and cefixime (VCC) at 37 °C and 42 °C. Significantly fewer stressed cells, in some cases as little as 0·3% of the starting population, were recovered by all the selective enrichment broths containing bile salts or VCC antibiotics compared to the non-selective controls. The use of such enrichments to recover low numbers of stressed E. coli O157:H7 may result in failure to detect the organism. Parallels with salmonella methodology are made and the need for a non-selective pre-enrichment stage in E. coli O157:H7 methods discussed.