Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef

AIM: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. METHODS AND RESULTS: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. CONCLUSIONS: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. SIGNIFICANCE AND IMPACT OF THE STUDY: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

The prevalence and spread of Escherichia coli O157:H7 at a commercial beef abattoir

AIMS: To investigate the prevalence and virulence characteristics of Escherichia coli O157:H7 after a number of beef process operations at a commercial Irish abattoir. METHODS AND RESULTS: Two 12-month studies were carried out. The first study (study 1) examined the prevalence of E. coli O157:H7 at up to six sites on carcasses at eight stages of the dressing, washing, chilling and boning process. The second study (study 2) examined the prevalence of E. coli O157:H7 in bovine faeces and rumen contents post-slaughter and on dressed, washed carcasses. Isolates from both studies were phage-typed and the presence of genes encoding verocytotoxin, enterohaemolysin and intimin production was determined. E. coli O157:H7 was isolated from four of 36 carcasses in study 1. E. coli O157:H7 was detected during hide removal and was detected at multiple carcass sites and multiple process stages, including boning. On two carcasses, contamination was first detected at the bung following its freeing and tying. All isolates from study 1 were phage type (PT) 2, eaeAO157 and ehlyA positive, but were verocytotoxin 1 (VT1) and verocytotoxin 2 (VT2) negative. In study 2, E. coli O157:H7 was isolated from 2.4% of faecal, 0.8% of rumen and 3.2% of carcass samples. In some cases, isolates recovered from the faeces of a particular animal, the resulting carcass and adjacent carcasses on the line had the same phage typing and virulence characteristic profile patterns. All isolates from study 2 were eaeAO157 and ehlyA positive and only one isolate was VT1 and VT2 negative. Most isolates were PT 32. A higher frequency of positive isolations was noted from samples taken during spring and late summer. CONCLUSION: These studies show that in a typical Irish beef abattoir, carcass contamination with E. coli O157:H7 can occur during hide removal and bung tying and this contamination can remain on the carcass during subsequent processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data that is necessary for the understanding of how E. coli O157:H7 contamination of beef occurs.     

Clonal turnover of enterohemorrhagic Escherichia coli O157:H7 in experimentally infected cattle

A total of 401 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates from two experimentally infected calves were analyzed using molecular biological methods. Genetic differences detected by pulsed-field gel electrophoresis were observed between the inoculated and recovered strains as early as 1 day post inoculation. The loss of the inoculated clone was observed in one calf. Replication and dissemination of the EHEC O157:H7 strains that mutated in cattle may result in the diversification of this organism among cattle populations.     

Studies of steam decontamination of beef inoculated with Escherichia coli O157:H7 and its effect on subsequent storage

AIM: This study was carried out to determine the survival of Escherichia coli O157:H7 and subsequent shelf life of beef subjected to subatmospheric steam at differing temperatures. METHODS AND RESULTS: A specifically built, laboratory scale decontamination apparatus was used in decontamination trials to examine the effect of condensing steam at differing subatmospheric pressures on the survival of E. coli O157:H7 on meat. Beef slices were inoculated with a nontoxigenic E. coli O157:H7 strain and subjected to condensing steam at temperatures of 55, 65 and 75 degrees C. Following treatment, the decontaminated meat was packaged and stored in air or under vacuum at temperatures of 10 or 0 degrees C for up to 42 days. Microbiological analysis of the decontaminated and a control product (not subjected to any heat treatment) was carried out at regular intervals over the storage time of the product. Overall, significant reductions (ca 1.5 log(10) CFU cm(-2)) in pathogen numbers were observed at a steam treatment temperature of 75 degrees C, however, postprocess storage conditions were important in ensuring no re-growth of the pathogen and this was best achieved by storage under vacuum at 0 degrees C. CONCLUSIONS: Steam had a significant impact in reducing E. coli O157:H7 populations, but storage conditions post-treatment were important for ensuring inhibition of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that subatmospheric steam could have significant application in the decontamination of meat primals postfabrication, immediately prior to packaging thus ensuring a safer product for consumers.     

Escherichia coli O157:H7 colonization in small domestic ruminants

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.     

Sequence analysis of Escherichia coli O157:H7 bacteriophage ΦV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen

Bacteriophage ΦV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the ΦV10 genome is 39 104 bp long and contains 55 predicted genes. ΦV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage ɛ15 and a prophage from E. coli APEC O1. The attachment site of ΦV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. ΦV10 encodes an O -acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block ΦV10 superinfection, indicating that the O157 antigen is most likely the ΦV10 receptor.     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌O157抗原的rfbE基因、 编码H7抗原的fliC基因以及编码毒力因子的eaeA基因为靶基因, 选择3对引物, 建立并优化了检测大肠杆菌O157:H7的多重PCR体系, 扩增产物分别为291 bp、625 bp、368 bp, 采用30株细菌验证了该多重PCR具有特异性。PCR检测的灵敏度在DNA水平上达到91.35 pg; 在存在干扰菌鼠伤寒沙门氏菌(Salmonella?typhimurium)的情况下, 当起始污染量为1.4 CFU/mL时, 37 ℃培养6 h 即可检出。在30份肉类样品中, 有3份检出了大肠杆菌O157:H7。本研究建立的多重PCR方法可特异、灵敏地实现对大肠杆菌O157:H7的检测。     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌 O157 抗原的 rfbE 基因、编码 H7 抗原的 fliC 基因以及编码毒力因子的eaeA 基因为靶基因,选择3对引物,建立并优化了检测大肠杆菌 O157:H7 的多重 PCR 体系,扩增产物分别为291 bp、625 bp,368 bp,采用30株细菌验证了该多重 PCR 具有特异性.PCR 检测的灵敏度在 DNA 水平上达到91.35 Pg;在存在干扰菌鼠伤寒沙门氏(Salmonella typhimurium)的情况下,当起始污染量为1.4 CFU/mL时,37℃培养6 h即可检出.在30份肉类样品中,有3份检出了大肠杆菌 O157:H7.本研究建立的多重 PCR 方法可特异、灵敏地实现对大肠杆菌 O157:H7 的检测.     

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.     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry

An exponential linear destruction was observed for Escherichia coli O157:H7 and Salmonella typhimurium in cattle manure and manure slurry stored at 4, 20 or 37 degrees C. The resulting decimal reduction times ranged from 6 days to 3 weeks in manure and from 2 days to 5 weeks in manure slurry. The main effects of time as well as temperature were pronounced with the most rapid destruction at 37 degrees C. The ammonia concentration in manure increased slightly during storage but did not exceed 0.1%. pH values in the deeper layers of manure remained constant except at 37 degrees C when the pH increased by 1 unit in 60 days. In the surface layers of manure, pH increased by 1.5-2 units, the oxidation-reduction potential of the manure declined rapidly to values below -200 mV. These changes do not seem to be reflected in changing rates of bacterial destruction. The observed order of destruction makes it possible to predict storage conditions (temperature and time) that will lead to a predetermined level of reduction of the two pathogens.     

可视化抗体阵列快速联合检测大肠杆菌O157:H7和鼠伤寒沙门氏菌

【目的】建立一种同时快速检测大肠杆菌O157:H7(E.coli O157:H7)和鼠伤寒沙门氏菌(Salmonella typhimurium)的可视化抗体阵列技术。【方法】将免疫学技术与蛋白芯片技术相结合,基于双抗体夹心法检测原理利用蛋白质芯片技术的高通量,结合可视化结果判定技术,用一份样品,同步检测大肠杆菌O157:H7和鼠伤寒沙门氏菌两种病原。【结果】检测结果肉眼可见,检测周期短至90 min,纯菌液检测灵敏度达105 CFU/mL,模拟带菌检测灵敏度为106 CFU/mL,与常规的ELISA灵敏度等同且具有良好的特异性和重复性。【结论】该可视化抗体阵列检测结果肉眼可见,检测通量高,无需大型设备,操作简便,检测成本低廉,同时为快速检测致病菌提供一种新途径。