A note on Escherichia coli O157:H7 serotype in Turkish meat products

255 minced beef, 101 soudjouk and 50 uncooked hamburger samples were analyzed for the presence of Escherichia coli O157:H7 serotype. m-EC and LST broths were used as selective enrichment media and SMAC agar was used as a selective isolation medium. A total of 3 E. coli O157 were isolated by conventional culture techniques; one from each of minced beef, uncooked hamburger and soudjouk but none were identified as the H7 serotype. For determination of selective media-growing cohabitant bacteria, 2645 isolates were obtained from SMAC agar. Results showed that E. coli type 1, Hafnia alvei and Citrobacter freundii were dominant competitive flora. As selective enrichment broth-growing cohabitant microflora existed at higher levels, it was too difficult to isolate E. coli O157 from these mixed flora. Therefore, conventional methods are not suitable for these types of products, because of isolation difficulties and failure to confirm.     

Bifidobacteria as indicators of faecal contamination in meat and meat products: detection, determination of origin and comparison with Escherichia coli

Faecal contamination of meat and meat products and its origin, whether human or animal, was determined by using the presence of bifidobacteria as an indicator. Enrichment of samples in Beerens liquid selective medium was followed by spreading onto Columbia agar containing paromomycin. In comparison with the detection of Escherichia coli (E. coli) the following results were obtained from 50 samples: E. coli. +; Bifidobacterium. +: 38; E. coli −; Bifidobacterium −: 9; E. coli, +; Bifidobacterium. −: 2; E. coli, −; Bifidobacterium. +: 1. From 39 positives samples, 50 strains of bifidobacteria were isolated. Two were of human origin, 48 of animal origin.     

Comparison of enrichment procedures for isolation of Escherichia coli O157:H7 from ground beef and radish sprouts

Enrichment procedures using Tryptic soy broth (TSB), modified TSB (mTSB), modified E. coli broth with novobiocin (mEC+n), mTSB (without novobiocin) with vancomycin, cefixime and cefsulodin (mTSB-VCC), or TSB with cefixime, tellurite and vancomycin (TSB-CTV) were evaluated by determining the rate of successful isolation of fifteen Escherichia coli O157:H7 strains from inoculated broth containing ground beef or radish sprout extract. E. coli O157:H7 tended to be isolated more efficiently after enrichment with TSB, mTSB and mEC+n than with the other broths. In order to identify the most efficient enrichmemt condition using these broths, E. coli O157:H7 were inoculated into 25 g ground beef or radish sprouts, which were then homogenized in 225 ml broth and incubated static at 37°C or 42°C for 6 h or 18 h. Attempts were made to isolate the inoculated bacteria by plating method in combination with the immunomagnetic separation method. The most effective enrichment condition was incubation in mTSB or mEC+n at 42°C for 18 h for ground beef, and in mEC+n at 42°C for 18 h for radish sprouts.     

纺织品中大肠杆菌O157:H7的检测方法

为建立一种灵敏、特异、快速、高效地鉴定纺织品基质中大肠杆菌O157:H7的方法,筛选出大肠杆菌O157:H7特有的rfbE基因保守序列,设计PCR特异性引物和荧光双标记探针,结合免疫磁珠技术,集成创新开发一种目标菌低浓度、不可培养情况下的样品中高效、快速富集分离大肠杆菌O157:H7的技术,建立了一种针对纺织品基质的免疫磁珠富集-实时荧光定量PCR方法,其检测下限可达8 CFU/mL。利用该方法对收集到的30份阳性样品进行鉴定,鉴定结果与传统方法结果100%吻合。结果表明,新建方法稳定性强,实现了纺织品中大肠杆菌O157:H7的快速灵敏鉴定。     

Models of the behavior of Escherichia coli O157:H7 in raw sterile ground beef stored at 5 to 46 degrees C

Escherichia coli O157:H7 can contaminate raw ground beef and cause serious human foodborne illness. Previous reports describe the behavior of E. coli O157:H7 in ground beef under different storage conditions; however, models are lacking for the pathogen's behavior in raw ground beef stored over a broad range of temperature. Using sterile irradiated raw ground beef, the behavioral kinetics of 10 individual E. coli O157:H7 strains and/or a 5- or 10-strain cocktail were measured at storage temperatures from 5° to 46 °C. Growth occurred from 6 to 45 °C. Although lag phase duration (LPD) decreased from 10.5 to 45 °C, no lag phase was observed at 6, 8, or 10 °C. The specific growth rate (SGR) increased from 6 to 42 °C then declined up to 45 °C. In contrast to these profiles, the maximum population density (MPD) declined with increasing temperature, from approximately 9.7 to 8.2 log cfu/g. Bias (B_f) and accuracy (A_f) factors for an E. coli O157:H7 broth-based aerobic growth model (10 to 42 °C) applied to the observations in ground beef were 1.05, 2.70, 1.00 and 1.29, 2.87, 1.03, for SGR, LPD and MPD, respectively. New secondary models increased the accuracy of predictions (5 to 45 °C), with B_f and A_f for SGR, LPD, and MPD of 1.00, 1.06, and 1.00 and 1.14, 1.33, and 1.02, respectively. These new models offer improved tools for designing and implementing food safety systems and assessing the impact of E. coli O157:H7 disease.     

Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland

A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes (n=200), Salmonella (n=200) and Campylobacter spp.(n=100). L. monocytogenes was not detected but Listeria innocua was found on five carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK.     

Survival of enterohaemorrhagic Escherichia coli O157:H7 strain in Turkish soudjouck during fermentation, drying and storage periods

Soudjouck (a kind of Turkish sausage) batter was inoculated with Escherichia coli O157:H7 at a level of 10⁵ colony-forming unit (CFUg) and kept overnight at 4°C. After stuffing the soudjouck batter into natural casing, fermentation was carried out at 24±2°C and 90–95% relative humidity (RH) for 3 days with subsequent drying at 22±2°C and 80–85% RH for 5 days. Then, half of soudjouck samples were vacuum-packed in polyethylene bags and the rest were kept open. All samples were stored at 4°C (55% RH) for 3 months. E. coli O157:H7 and lactic acid bacteria counts, moisture contents and pH values of the samples were determined during fermentation, drying and storage periods. Results showed that count of E. coli O157:H7 decreased by 3 log unit during fermentation and drying periods. It was observed that this pathogen survived longer in vacuum-packaged samples (more than 2 months) than non-vacuum samples (more than 1 month).     

The bacteriological quality of British beef 2. Frozen minced beef

Frozen minced beef was obtained from five commercial wholesale producers, and the aerobic plate count, and counts of coliforms, Escherichia coli type 1 and Staphylococcus aureus, determined. No E. coli O157:H7 or salmonellas were detected. The results were evaluated in relation to: (1) two earlier surveys involving fresh mince purchased from retail butchers and supermarkets in the UK (Roberts et al., 1980; Hudson et al., 1986); and (2) the standards set out in Annex II of the Minced Meat Directive 94/65/EC. In respect to the latter, none of the 99 samples of mince examined was unsatisfactory.     

Occurrence and survival of verocytotoxin-producing Escherichia coli O157 in meats obtained from retail outlets in The Netherlands.

In 1996 and 1997, 2,941 fresh and processed meat products obtained from supermarkets and butcher shops in The Netherlands were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC). Additionally, the fate of O157 VTEC in raw meat products stored at low temperatures and the effect of different additives were evaluated. O157 VTEC strains were isolated from 6 (1.1%) of 571 samples of raw minced beef, 2 (0.5%) of 402 samples of raw minced mixed beef and pork, 1 (1.3%) of 76 samples of raw minced pork, 1 (0.3%) of 393 samples of other raw pork products, and 1 (0.3%) of 328 samples of cooked or fermented ready-to-eat meats. Other raw beef products (n = 223) and meat samples originating from poultry (n = 819), sheep or lamb (n = 46), or wild animals (n = 83) were all found to be negative for O157 VTEC. For the survival experiments we used tartaar (minced beef with a fat content of less than 10%) and filet americain (tartaar mixed with a mayonnaise-based sauce [80 to 20%]). The O157 VTEC strain tested was able to survive in tartaar and filet americain stored at -20, 0, 5, or 7 degrees C for 3 days. At both 7 and at 15 degrees C, O157 VTEC counts in tartaar and filet americain remained virtually unchanged throughout a storage period of 5 days. Addition of acetic acid (to pH 4.0), sodium lactate (1 and 2% [wt/wt]), or components of the lactoperoxidase-thiocyanate-hydrogen peroxide system to filet americain did not result in a reduction of viable O157 VTEC cells during storage at 7 or 15 degrees C. It was concluded that raw meat contaminated with O157 VTEC will remain a hazard even if the meat is held at low or freezing temperatures.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

A quantitative assay for viable Escherichia coli O157:H7 in ground beef based on immunomagnetic separation (IMS) and the polymerase chain reaction (PCR) was developed. Bacterial cells inoculated into ground beef, were captured by immunomagnetic beads (IMB) after a 6 h non-selective enrichment. The percent capture of the target cells was consistent for the inoculation levels of 0.7 to 70 colony-forming-units (CFU)/g. Captured bacteria were lysed with PCR buffer containing 0.2 mg/mL proteinase K at 65°C for 30 min. DNA sequences of Shiga-like toxin 1 and 2 (Stx 1 and 2) were amplified independently. Log-linear relationships were observed between CFU of E. coli O157:H7 inoculated into ground beef and the integrated fluorescent image of the PCR products with Stx 1 and 2 primers after enrichment. The quantitative range was between 0.7 to 70 CFU/g.     

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 l min at 60 °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 °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 °C. From these cultures, 10 μ1 was heated at 95 °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.     

The persistence of infectious adenovirus (type 35) in mussels (Mytilus edulis) and oysters (Ostrea edulis)

Microbiological analyses of fruits and vegetables produced by farms in Minnesota and Wisconsin were conducted to determine the prevalence of Escherichia coli in pre-harvest fruits and vegetables. During the 2003 and 2004 harvest seasons, 14 organic (certified by accredited organic agencies), 30 semi-organic (used organic practices but not certified) and 19 conventional farms were sampled to analyze 2029 pre-harvest produce samples (473 organic, 911 semi-organic, 645 conventional). Before each harvest season, a farmer survey was conducted to collect relevant information on farm management practices that might affect the risk of E. coli contamination in fresh produce. The use of animal wastes for fertilization of produce plants increased the risk of E. coli contamination in organic (OR = 13.2, 95% CI = 2.2–61.2, P-value < 0.0001) and semi-organic (OR = 12.9, 95% CI = 2.9–56.3, P-value < 0.0001) produce significantly. Improper ageing of untreated animal manure significantly increased this risk in organic produce (OR = 4.2 95% CI = 1.7–12.3, P-value = 0.005) grown using such manure as a fertilizer. Organic growers who used cattle manure for fertilization of their crops showed significantly greater risk of contamination with the E. coli (OR = 7.4, 95% CI = 1.6–36.8, P-value = 0.003), compared to those who used other types of manure-based fertilizer. In Minnesota, organic and semi-organic produce collected from the southeastern (SE) part of the state were at a significantly greater risk of E. coli contamination (OR = 3.45, 95% CI = 1.8–35.2, P = 0.008), compared to those collected from farms located in the southern (S) regions of the state. In Wisconsin, organic and semi-organic produce collected from the southern (S) cluster of farms were at approximately 3-times greater risk of E. coli contamination (OR = 2.67, 95% CI = 1.3–9.4, P = 0.004), compared to those grown in the northern (N) cluster of farms.     

Modeling the effect of inoculum size and acid adaptation on growth/no growth interface of Escherichia coli O157:H7

The objective of this study was to model with logistic regression the growth/no growth interface of different initial inoculation levels (10¹, 10³ and 10⁵ CFU/ml; study 1), or nonadapted vs acid-adapted (study 2) Escherichia coli O157:H7 as influenced by pH, NaCl concentration and incubation temperature. Study 1 was conducted with a mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in tryptic soy broth (TSB). Study 2 was conducted with the same mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in glucose-free TSB with 1% added glucose (final pH 4.83), or in diluted lactic acid meat decontamination runoff fluids (washings; final pH 4.92), or nonadapted cultures prepared in glucose-free TSB (final pH 6.45), or in water washings (final pH 6.87). Parameters included incubation temperature (10–35 °C), pH (3.52–7.32), and NaCl concentration (0–10% w/v). Growth responses were evaluated for 60 days turbidimetrically (610 nm) every 5 days in 160 (study 1) and 360 (study 2) combinations in quadruplicate samples, with a microplate reader. The lower the initial inoculum the higher were the minimum pH and a_w values permitting growth. Differences in the pH and a_w growth limits among inoculum concentrations increased at 15 and 10 °C. Acid-adapted cultures were able to grow at lower pH than nonadapted cultures, while at temperatures below 25 °C, growth initiation of nonadapted cultures stopped at higher a_w compared to acid-adapted cultures for the whole pH range of 3.52 to 7.32. A comparison with available data indicated that our model for acid-adapted E. coli O157:H7 in different environments may provide representative growth probabilities covering both nonadapted and stress-adapted contaminants.     

Prevalence of Shiga toxin-producing Escherichia coli in beef

Over the past two decades, many human illness outbreaks were attributed to consumption of undercooked beef products containing Shiga toxin-producing Escherichia coli (STEC). The illnesses included mild or bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome (HUS). Tracing these outbreaks to O157 and an increasing number of non-O157 STEC strains suggests that beef safety concerns will continue to rise and may negatively affect the beef industry. To effectively address these concerns, it is critical to evaluate the role of beef in STEC infections. In this review, published reports on beef contamination were evaluated to assess prevalence rates and health risks of STEC isolates. Global testing of beef showed wide ranges of prevalence rates of O157 (from 0.01% to 54.2%) and non-O157 (from 1.7% to 62.5%) STEC. Of the 155 STEC serotypes found in beef, 31 and 25 are known to cause HUS and/or other illnesses, respectively.     

A survey of the incidence of E. coli O157 in the UK dairy industry

A survey of the incidence of Escherichia coli O157 in bovine faeces, pasteurised and unpasteurised dairy products, farm swabs and dairy factory environmental samples was undertaken. A single enrichment broth was employed, together with three detection methods, comprising a plating medium, a commercially available ELISA technique (Petrifilm) and an ELISA test under development (Organon-Teknika). For all detection methods, the efficiency of recovery, determined using artificially contaminated samples, was poor and this was attributed to the growth of competitors in the enrichment broth. Both the plating and the Organon-Teknika methods yielded a higher proportion of false positives than the Petrifilm method; however, the former two methods also detected a wider range of verotoxigenic E. coli than the Petrifilm method. Verotoxigenic E. coli were not detected in any of 1146 samples examined. The poor performance of the methodolgy may explain why the environmental incidence appears to be low although the incidence of infection by E. coli O157 is increasing.     

Staphylococcus aureus and Escherichia coli in nham (Thai-style fermented pork sausage)

The fate of Staphylococcus aureus and Escherichia coli was determined when they were introduced into ground pork made into nham (Thai-style fermented pork sausage) with or without 0.75 or 1.5% added starter culture. Without starter culture, the numbers of E. coli remained little changed but there was slow multiplication of S. aureus. With 0.75% starter culture, S. aureus was no longer detectable after 48 h and E. coli numbers decreased by 1 log after 96 h. No viable S. aureus or E. coli were recovered after 36 h and 96 h, respectively, when 1.5% starter culture was added. The addition of a starter culture is recommended when making nham.     

Comparison of surface sampling methods and cleanability assessment of stainless steel surfaces subjected or not to shot peening

The cleanability of AISI 304 stainless steel surfaces, indicated by the removal of Escherichia coli cells or Aspergillus niger spores was assessed by controlled inoculation and washing treatment of samples in standardised conditions. Two systems of recapture (Rodac plate technique and swabbing technique) were compared. Four industrial finishes, subjected or not to shot peening, contaminated at low concentration (1–10 cfu/cm²), and then washed with distilled water or alkaline detergent, were examined. The Rodac plate technique detected most of microorganisms inoculated (80% for E. coli cells and 67% for A. niger spores), whereas the swabbing technique recovered only 1% of the E. coli cells and 26% of the A. niger spores. Using the Rodac plate technique E. coli cells proved to be easily detachable from samples either with distilled water (98%) or alkaline detergent (>99%). For the surfaces contaminated with A. niger spores, the cleanability increased from 34% with distilled water to 77% with alkaline detergent. In these working conditions type of finish (shot treated or not) had no significant effect on cleanability of stainless steel.     

Prevalence and antibiotic resistance profiles of Escherichia coli O157:H7 in beef products from retail outlets in Gaborone, Botswana

Four hundred meat samples (134 meat cubes, 133 minced meat, 133 fresh sausages) were collected from 15 supermarkets and butcheries in Gaborone, Botswana, between the summer months of October 2002 and March 2003. Samples were assayed for Escherichia coli O157 by selective enrichment in modified E. coli broth containing novobiocin, followed by immunomagnetic separation and plating onto sorbitol MacConkey agar supplemented with potassium tellurite. The isolates were biochemically and serologically confirmed by API 20E and O157 antisera, respectively. The prevalence rates for E. coli O157 were 5.22% in meat cube samples, 3.76% in minced meat samples, and 2.26% in fresh sausages. The isolates showed single, double, and triple antibiotic resistance. Fifty-three percent of them were resistant to cephalothin. Resistance was also recorded for sulphatriad (33%), colistin sulphate (26%), streptomycin (0.7%), and tetracycline (26%). It is recommended that the cause for antibiotic resistance be investigated using a larger number of samples from cattle, especially from ranching areas of the country.     

The antimicrobial effect of thyme essential oil, nisin and their combination against Escherichia coli O157:H7 in minced beef during refrigerated storage

The antimicrobial effect of thyme essential oil (EO) at supplementation levels of 0.3%, 0.6% or 0.9%, nisin at 500 or 1000 IU/g, and their combination, on Escherichia coli O157:H7 was examined in both tryptic soy broth (TSB) and minced beef meat. EO at 0.3% possessed a weak antibacterial activity against the pathogen in TSB, whereas at 0.9% showed unacceptable organoleptic properties in minced meat. Thus, only the level of 0.6% of EO was further examined against the pathogens in minced meat. Treatment of minced beef meat with EO at 0.6% showed an inhibitory activity against E. coli O157:H7 during storage at 10 °C, but not at 4 °C. Treatment of minced beef meat or TSB with nisin at 500 or 1000 IU/g did not show any antibacterial activity against E. coli O157:H7. The combination of EO at 0.6% and nisin at 500 or 1000 IU/g showed an additive effect against the pathogen, which was higher during storage at 10 °C than at 4 °C.     

Decontamination of carcasses by vacuum-hot water cleaning and steam pasteurizing during routine operations at a beef packing plant

The microbiological effects of three operations for cleaning areas on dressed beef carcasses with vacuuming equipment which also applies hot water to the carcass, and of an operation for pasteurizing beef carcass sides with steam, were assessed. All four operations were routine in a commercial carcass dressing process. For each operation, swab samples were obtained from randomly selected carcasses, with a single sample being collected from each carcass, from a site selected at random from those affected by the operation. For the cleaning operations, 25 samples were obtained before and 25 after each operation. For the pasteurizing operation, 50 samples were obtained before and 50 after the operation. In addition, 50 samples were obtained from beef sides after the carcass cooling process which followed the pasteurizing operation. Total aerobic counts, coliforms and Escherichia coli from each sample were enumerated. The cleaning operations generally reduced the log mean numbers of bacteria on treated areas by ≤ 0.5 and had no discernible effect on the overall microbiological condition of the carcasses emerging from the process. The pasteurizing operation reduced the log mean numbers of total aerobic bacteria on carcasses by about 1, and the log mean numbers of coliforms and E. coli by > 2. The cooling process had no affect on the total counts, but further reduced the log mean numbers of coliforms and E. coli, apparently by about 1, to give beef sides from which E. coli were not recovered.