Identification Of Shiga Toxin-producing Escherichia Coli Research Articles

Real-time PCR primers and probes for the detection of Shiga toxin genes, including novel subtypes

Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes.A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence.The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.

Multiplex Real-Time PCR for the Detection of Shiga Toxin-Producing Escherichia coli in Foods.

Shiga toxin-producing Escherichia coli (STEC) is a group of human foodborne pathogens transmitted to humans through the consumption of different types of food. Their detection is mainly performed by targeting specific serogroups by classical microbiological methods and, later, by molecular typing with different techniques. The application of multiplex real-time PCR (qPCR) can significantly improve the turnaround time of the existing methodologies as in one single run it is possible to detect and characterize specific microorganisms. In the present chapter, a pentaplex qPCR assay is described for the identification of STEC which may also be applied for the rapid screening of these pathogens in different types of foods. The assay targets the most important virulence factors of these microorganisms, the genes stx1, stx2, and eae, along with the rfbE gene which encodes for the "O157" antigen as this is the most prevalent serogroup among all STEC, as well as an internal amplification control to rule out false-negative results due to qPCR inhibition.

Atypical Presentation of Shiga Toxin Haemolytic Uremic Syndrome (STEC-HUS)

Shiga-toxin producing Escherichia coli (STEC) infection is the most common cause of haemolytic uremic syndrome (HUS) in children. It should be noted that 5% of STEC-HUS patients have no prodromal diarrhoea, while it may be present in up to 30% of atypical HUS (aHUS) cases. In patients at risk for aHUS, STEC infection may act as a trigger rather than playing a causative role. Presently, eculizumab is the first-line therapy for children presenting with aHUS, whereas treatment of children with STEC-HUS is mainly supportive. We report the case of a child with STEC-HUS who had a severe presentation, requiring renal replacement therapy in the acute phase and surgical treatment for a colonic stricture that arose as a late extra-renal complication of the disease. We aim to review the diagnostic workup of children presenting with HUS, stressing the resources available in our setting. In Portugal, O157 E. coli isolation medium is widely available in clinical laboratories. Non-O157 STEC identification requires specific testing, which is increasingly relevant as non-O157 STEC is becoming a more common cause of STEC-HUS than serogroup O157. The National Health Institute Doutor Ricardo Jorge (INSA) has the capacity to identify verotoxin genes and to proceed to verotxin-producing E. coli (VTEC) pathogenicity assessment using multiplex polymerase chain reaction. Fresh stool samples must be obtained early in the disease course to be sent for culture in O157 E. coli isolation medium and for non-O157 E. coli identification assays.

Droplet Digital PCR (ddPCR) Analysis for Detecting Shiga-Toxin-Producing Escherichia coli (STEC)

Verocytotoxin-producing Escherichia coli, also referred to as Shiga-toxin-producing Escherichia coli (STEC), can be transmitted to humans through person-to-person contact, consumption of contaminated food or water, or by direct contact with animals. Its clinical and economic consequences have prompted the development of alternative approaches to the official method of analysis “UNI CEN ISO/TS 13136: 2012”, which describes the identification of STEC through the detection of its main virulence genes. Recently, droplet digital PCR (ddPCR) has been proposed as a technique for the sequence-specific detection and direct quantification of nucleic acids. The present study aimed to investigate if ddPCR could be able to detect STEC in less time than that required by the official method. This study consisted of the ddPCR of slices of beef contaminated with STEC and of the sponges used for beef official control at the slaughter stage. The results showed the ability of ddPCR to detect STEC in slices of beef already after sample incubation for 7 h at 37 °C while, in the case of sponges used for official controls, 9 h at 37 °C was needed. In this way, the ddPCR could represent an efficient method for detecting STEC and providing results in less time than the official method.

Shiga toxin-producing Escherichia coli O26:H11 associated with a cluster of haemolytic uraemic syndrome cases in South Africa, 2017.

Introduction Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that may cause diarrhoeal outbreaks and occasionally are associated with haemolytic-uraemic syndrome (HUS). We report on STEC O26:H11 associated with a cluster of four HUS cases in South Africa in 2017.Methodology All case-patients were female and aged 5 years and under. Standard microbiological tests were performed for culture and identification of STEC from specimens (human stool and food samples). Further analysis of genomic DNA extracted from bacterial cultures and specimens included PCR for specific virulence genes, whole-genome sequencing and shotgun metagenomic sequencing.Results For 2/4 cases, stool specimens revealed STEC O26:H11 containing eae, stx2a and stx2b virulence genes. All food samples were found to be negative for STEC. No epidemiological links could be established between the HUS cases. Dried meat products were the leading food item suspected to be the vehicle of transmission for these cases, as 3/4 case-patients reported they had eaten this. However, testing of dried meat products could not confirm this.Conclusion Since STEC infection does not always lead to severe symptoms, it is possible that many more cases were associated with this cluster and largely went unrecognized.

Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices.

Aim:The objective of the study was to detect Shiga toxin-producing Escherichia coli (STEC) and develop a quantitative polymerase chain reaction (qPCR) assay to quantify the bacterial DNA present in different food matrices.Materials and Methods:A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135), pasteurized milk (100), beef (132), buffalo meat (130), chevon (104), beef kheema (115), and beef sausage (42). All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against stx1 gene of STEC by the construction of standard curve using SYBR green chemistry.Results:The overall occurrence of STEC in raw milk (n=135), beef (n=132), buffalo meat (n=130), chevon (n=104), and beef kheema (n=115) samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of stx 1 and stx 2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried stx 1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R2 of linear regression curves were −3.10, 34.24, and 0.99, respectively.Conclusion:The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food safety measures to combat the potential pathogenic effects of harmful bacteria throughout the production chain from production to consumption.

Comparative Evaluation of a Novel Phage Protein Ligand Assay and Immunomagnetic Separation Method To Isolate the Seven Top Serogroups of Escherichia coli (O157, O26, O103, O145, O111, O45, and O121) in Foods at Risk

The presence of Shiga toxin-producing Escherichia coli (STEC) in food is a major concern for food safety authorities and industries. Methods for detecting these pathogenic bacteria are crucial. Enrichment of foods for STEC identification has been optimized, but selective concentration of bacteria before isolation still needs to be improved. In the present study, we tested the performance of the VIDAS ESPT detection method against that of the immunomagnetic separation (IMS) method. A preenrichment inoculation was performed to provide a realistic scenario of the contamination that occurs in foods, and the methods were then compared. Results obtained were then confirmed in naturally contaminated foods. Preenrichment inoculation assays revealed that the novel concentration method using phage recombinant proteins or the selective capture of the target top seven STEC serogroups is as specific and sensitive as IMS. Subsequent evaluation of naturally contaminated samples confirmed that the novel concentration method and IMS are equivalent in performance under the conditions tested.

Improved traceability of Shiga-toxin-producing Escherichia coli using CRISPRs for detection and typing.

Among strains of Shiga-toxin-producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are frequently associated with severe clinical illness in humans. The development of methods for their reliable detection from complex samples such as food has been challenging thus far, and is currently based on the PCR detection of the major virulence genes stx1, stx2, and eae, and O-serogroup-specific genes. However, this approach lacks resolution. Moreover, new STEC serotypes are continuously emerging worldwide. For example, in May 2011, strains belonging to the hitherto rarely detected STEC serotype O104:H4 were identified as causative agents of one of the world's largest outbreak of disease with a high incidence of hemorrhagic colitis and hemolytic uremic syndrome in the infected patients. Discriminant typing of pathogens is crucial for epidemiological surveillance and investigations of outbreaks, and especially for tracking and tracing in case of accidental and deliberate contamination of food and water samples. Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of short, highly conserved DNA repeats separated by unique sequences of similar length. This distinctive sequence signature of CRISPRs can be used for strain typing in several bacterial species including STEC. This review discusses how CRISPRs have recently been used for STEC identification and typing.

Successful detection of pathogenic Shiga-toxin-producing Escherichia coli in shellfish, environmental waters and sediment using the ISO/TS-13136 method.

The presence of highly pathogenic Shiga-toxin-producing Escherichia coli (STEC) in shellfish, upstream waters and sediment from coastal shellfish sites was evaluated using the ISO/TS-13136 method. Shellfish (oysters, mussels and cockles), water and sediment samples were collected monthly over a period of 1 year. The method used real-time PCR detection of stx1, stx2 and eae genes and genetic markers corresponding to the five major serogroups (O157, O26, O103, O111 and O145) on enrichment broths and the identification of STEC when these genes and markers were detected. stx genes were detected in the broth of 33% of shellfish batches (n = 126), 91% of water samples (n = 117) and 28% of sediment (n = 39). One stx1(+), eae(+) O26:H11 strain was isolated from a shellfish batch, and O26:H11, O145:H28 and O103:H2 strains without the stx gene (n = 9) were isolated from shellfish and waters. In conclusion, this study shows the suitability of the ISO/TS-13136 method to assess the presence of highly pathogenic E. coli strains in shellfish farming areas. It also highlights a low prevalence of STEC and consequently suggests a reduced corresponding human health risk. (STEC) infections have been reported following ingestion of contaminated food or water or after bathing in contaminated waters. However, to date, few studies concerning their detection in coastal environment and shellfish have been reported. The aim of this work was to assess the presence of STEC in three shellfish-harvesting areas by the ISO/TS-13136 method, which has recently been used for STEC detection in food.

Rapid Identification and Differentiation of Non-O157 Shiga Toxin–ProducingEscherichia coliUsing Polymerase Chain Reaction Coupled to Electrospray Ionization Mass Spectrometry

A polymerase chain reaction (PCR)-mass spectroscopy assay was developed to identify non-O157 Shiga toxin-producing Escherichia coli (STEC) with Plex-ID biosensor system, a platform identifying short PCR amplicons by specific base compositions. This assay simultaneously amplifies five fragments of two housekeeping genes, two subunits of stx2 gene, and four other virulence genes of STEC. A total of 164 well-characterized STEC isolates were examined with the assay to build a DNA base composition database. Another panel of 108 diverse STEC isolates was tested with the established database to evaluate the assay's identification capability. Among the 108 isolates, the assay specificity was 100% for three (stx1, eae, and aggA) out of five tested virulence genes, but 99% for stx2 and 96% for hlyA, respectively. Main stx1/stx2 subtypes and multiple alleles of stx1/stx2 could be differentiated. The assay successfully identified several clinically significant serotypes, including O91:H14, O103:H25, O145:H28/NM, O113:H21, and O104:H4. Meanwhile, it was able to group isolates with different levels of pathogenic potential. The results suggest that this high-throughput method may be useful in clinical and regulatory laboratories for STEC identification, particularly strains with increased pathogenic potential.

Rapid Identification of Shiga Toxin-producing Escherichia coli (STEC) Using Electric Biochips

Shiga toxin-producing Escherichia coli (STEC) strains are pathogens that cause severe infections, especially in children. Although early data suggested that the STEC phenotype was linked to the O157 serotype, subsequent studies indicated no such strict phenotype-serotype correlation. Thus, it seems that only a test detecting stx1 and stx2 genes (responsible for Shiga toxin production) can provide unambiguous results in STEC identification. Here, we present a system for the rapid detection of the presence of o157, stx1, and stx2 genes, on the basis of an asymmetric polymerase chain reaction and subsequent detection of the amplification products on electric biochips. The whole test lasts less than 100 minutes, and allows for the accurate identification of the stx1 and stx2 genes and the presence of pO157 plasmid in a bacterial colony or in a liquid bacterial culture. The procedure is fully automated, and the only required manual activity is a chip exchange.

Detection of hemolysin variants of Shiga toxin-producing Escherichia coli by PCR and culture on vancomycin-cefixime-cefsulodin blood agar.

The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H-, O103:H2, O103:H-, O26:H11, and O26:H-, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H- strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H- STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 micrograms/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.