Evaluation of 5'-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools

5′-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx₁ and stx₂ genes when challenged with reference strains of E. coli (n = 40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx_1c, stx_1d, and stx_2f), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log₁₀ and 3.42 to 4.63 log₁₀ CFU/g of stool for 5′-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation of ≤ 5% for both inter- and intra-assay variation. Clinical performance was identical with a panel of archived positive specimens (n = 19) and a prospective panel of stools associated with bloody diarrhea (n = 115). In conclusion, both assays proved to be sensitive and reproducible.     

Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods

Aims:  To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods.
Methods and Results:  A competitive IAC was constructed and included in an stx -specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx -positive, giving 98·3% and 93·75% concordance, respectively, with the PCR-ELISA reference method.
Conclusions:  A highly sensitive stx -specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors.
Significance and Impact of the Study:  Combined with automated DNA extraction, the stx -IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.     

Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes

AIMS: To develop and evaluate a multiplex PCR (mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) and their main virulence marker genes. METHODS AND RESULTS: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2 (stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin (ehlyA). Moreover, two additional genes (rfb O157 and fliC H7), providing the genotypic identification of the O157:H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157:H7 serotype (six isolates) and O157:NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. CONCLUSIONS: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157:H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.     

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.     

Development of a 5'-nuclease PCR assay for detecting Shiga toxin-producing Escherichia coli O145 based on the identification of an 'O-island 29' homologue

AIMS: A DNA sequence, from Escherichia coli STEC O145, homologous to O-island 29 from STEC O157 is described, together with a real-time PCR assay for detecting it. METHODS AND RESULTS: PCR and sequencing were used to identify the 'O-island 29' homologous DNA sequence from STEC O145 (strain VTH34). The sequence divergence between the STEC O145 and O157 'O-island 29' allowed a STEC O145 5'-nuclease PCR assay to be developed. CONCLUSIONS: The characterization of a novel locus in STEC O145 has allowed a specific O145 serogroup 5'-nuclease PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of serogroup PCR assays available as alternatives to classical O-serotyping of E. coli.     

Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay

AIM: To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. METHODS AND RESULTS: DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg microl(-1) chromosomal DNA, 0.2 CFU g(-1) pork and 0.2 CFU ml(-1) water. The total time required from beginning to end of the procedure was within 16 h. CONCLUSION: The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens.     

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.     

Screening of food raw materials for the presence of Shiga toxin-producing Escherichia coli O91:H21

Aims:  To provide information on the prevalence and detection, in foods, of Shiga toxin-producing Escherichia coli (STEC) O91:H21.
Methods and Results:  Seven hundred fifteen minced beef meats and 205 raw milk samples were analysed by stx -specific PCR-ELISA. Samples positive for stx were subsequently tested for the presence of wzy -O91, fliC -H21 and the adhesin-encoding gene saa . For minced meat, 16 (2·2%) and 11 (1·5%) samples were found positive for ( stx , wzy -O91, fliC -H21) and ( stx , wzy -O91, fliC -H21, saa ) combinations, respectively. For raw milk, seven (3·4%) samples were found positive for the ( stx , wzy -O91, fliC -H21) combination but none of these contained saa . Two STEC O91:H21 saa -positive strains and three STEC O91 H21– and saa -negative strains were isolated by colony hybridization.
Conclusions:  A low prevalence of potentially pathogenic STEC O91:H21 in food products was found using a combination of PCR assays targeting stx , wzy -O91, fliC -H21 and saa .
Significance and Impact of the Study:  The PCR-based approach described here represents a valuable method for rapid screening of food samples contaminated by STEC O91:H21.     

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use

AIMS: To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. METHODS AND RESULTS: First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. CONCLUSIONS: Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. SIGNIFICANCE AND IMPACT OF THE STUDY: We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.     

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.     

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources

AIMS: To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. METHODS AND RESULTS: The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. CONCLUSIONS: The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.     

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.     

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.     

江苏某地健康绵羊群产志贺毒素大肠杆菌体内分离株的分子流行病学及致病力

【目的】探讨江苏某羊场健康绵羊体内产志贺毒素大肠杆菌的带菌和流行情况,同时就分离株的致病力和对Vero细胞的毒性作用作了研究。【方法】基于本实验室已经建立的EHEC O157:H7 EDL933W株的stx1、stx2、eaeA、hlyA四个基因的多重PCR检测并配合选择性增菌、平板筛选等方法对STEC进行分离鉴定。【结果】在为期6个月的连续跟踪调查中,共分离到STEC菌株107株,分离率为19.4%(107/550)。分离株属于41种O血清型、62种O:H血清型,未定型(ONT)有22株,粗糙型(OR)1株。其中属于绵羊STEC的优势血清型有O5(2株)、O91(1株)、O103(1株)。本文检测到的优势血清型为O93,stx2阳性菌株的分离率较stx1阳性菌株的分离率高,LD50测定结果表明分离株对小鼠致病力不高,受试的3个分离株均不能致小鼠死亡。对107株stx阳性分离株噬菌斑试验表明,71株阳性菌株携带噬菌体(66.3%,71/109)。受试分离株进行Vero细胞毒性试验,其中有一个菌株stx基因阳性但不能使Vero细胞产生病变。【结论】绵羊是STEC的天然宿主,可健康带菌。虽然STEC分离株对小鼠的致病力较弱,但不能排除其对人类安全的威胁。STEC携带志贺毒素基因并不意味着一定表达志贺毒素,需对志贺毒素的表达及调控机理做进一步的研究。     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

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.