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.     

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater

AIMS: Developing and evaluating a rapid real-time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre-enrichment step before DNA extraction. METHODS AND RESULTS: Wastewater samples were collected from a dairy farm producing mozzarella cheese and located in Puglia (south of Italy). Plate count and other microbial assays were performed 1 h after sampling. Wastewater samples were artificially inoculated with 10(4), 10(7) and 10(8) cells ml(-1) of E. coli O157:H7, strain EDL933. PCR protocols for stx1, stx2 and eae genes were first tested on pure DNA extracted from type strains, in order to optimize the amplification conditions and reagent concentration before real-time PCR experiments. Three specific fragments of ca 106, 150 and 200 bp corresponding to genes eae, stx1 and stx2, respectively, were obtained. Real-time PCR experiments were performed with DNA extracted from dairy and manure wastewater samples inoculated with 10(4), 10(7) and 10(8) colony-forming units (CFU) ml(-1) of E. coli O157:H7 strain EDL 933. The sensitivity limit of the assay was 10(-1) pg microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-1) for stx1 gene respectively. CONCLUSIONS: A real-time PCR protocol has been developed and used in order to identify potential pathogens in dairy wastewater, in which previous methods (including standard PCR) failed to work. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle and dairy wastewater samples produced from mozzarella cheese factories may harbour verocytotoxin-producing E. coli. The availability of rapid and sensitive molecular methods may be useful to monitor the persistence of verocytotoxin-producing E. coli in general and to assess the effectiveness of wastewater treatments.     

A carboxy-terminal domain of Tir from enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) required for efficient type III secretion

The type III secreted protein Tir from Enterohemorrhagic Escherichia coli (EHEC O157:H7) plays a central role in adherence and pedestal formation during infection. Little is known about how Tir domains outside of the amino-terminus contribute to efficient Tir secretion and translocation. We found a 6 amino acid (519-524) carboxy-terminal region which was required for efficient Tir secretion and translocation. Interestingly, EHEC O157:H7 Tir(Delta)519-524 was efficiently secreted when expressed in the related pathogen enteropathogenic E. coli. These data suggest that this region may play a role in maintaining EHEC O157:H7 Tir in a secretion-competent conformation.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

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.     

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.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

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.     

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12–34% in mussels, 48–69.5% in oysters and 30–65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 °C. Increasing enrichment temperatures from 37 to 42 °C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.     

肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

Production of an autoinducer of growth by norepinephrine cultured Escherichia coli O157:H7

Abstract Escherichia coli O157:H7 were cultured in the presence or absence of norepinephrine to generate conditioned media. The presence of a growth-inducing factoKs) in the conditioned media was examined by measurement of the ability of conditioned media to support the growth of fresh cultures of E. coli O157:H7. Supplementation of fresh cultures with as little as 0.024% (v/v) norepinephrine conditioned medium resulted in increased growth as compared to controls, thereby indicating the presence of an autoinducer of growth. Analysis of the production kinetics for the autoinducer during the generation of conditioned media indicates that it differs from other more well characterized autoinducers. It is proposed that the neurohumoral environment of the host may contribute to the production of bacterial growth factors.     

Thermal death of Escherichia coli O157:H7 in cattle feeds

AIMS: To determine if the temperatures used in feed manufacture are likely to destroy Escherichia coli O157. METHODS AND RESULTS: Two commercial feeds were ground and inoculated with E. coli O157 cells. The feeds were heated to 50, 55, 60, 65 or 70 degrees C. Heating produced quadratic survivor curves, with rapid initial decreases. The survival characteristics of E. coli O157 differed in the two feeds. The reductions observed in one feed may not have been due to heat alone. There was evidence that indigenous anti-E. coli O157 factor(s) in one feed acted with the heat and contributed to the observed rates of bacterial death. Heating at 70 degrees C for 20 or 120 s resulted in approx. 1.3 and 2.2 log reductions in E. coli O157 numbers respectively. Lesser reductions were observed at lower temperatures. CONCLUSIONS: The time/temperature combinations used in commercial pelleting processes would not effectively kill high numbers of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to look at the survival of E. coli O157 strains after heat treatment within concentrated animal feed. The study provides information on the likely risk of E. coli O157 surviving the animal feed manufacturing process.     

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.     

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.