Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture

Karch, Helge; Janetzki-Mittmann, C; Aleksić, S.; Datz, M

doi:10.1128/jcm.34.3.516-519.1996

Cited by 111 publications

(35 citation statements)

References 20 publications

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“…With the exception of an Enterococcus faecalis isolate, these strains grew well in mTSB + a. 0157 VTEC, but some sorbitol-fermenting E. coli 0157:NM do not grow on CT-SMAC (Karch et al 1996). Almost all 0157 STEC lack GUD activity and to exploit this characteristic, media that contain the fluorogenic compound, 4-methylumbelliferyl-fJ-D-glucuronide (MUG) (Szabo et al 1986;Okrend et al 1990a) or the chromogenic compound 5-bromo-4-chloro-3-indolyl-fJ-D-glucoronide (BCIG) (Okrend et al 1990b), were developed.…”

Section: Enrichment Mediamentioning

confidence: 97%

Methods for the detection and isolation of Shiga toxin-producingEscherichia coli

Boer

Heuvelink

2000

Journal of Applied Microbiology

119

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SUMMARY Shiga toxin‐producing Escherichia coli (STEC) are an important cause of haemorrhagic colitis and the diarrhoea‐associated form of the haemolytic uraemic syndrome. Of the numerous serotypes of E. coli that have been shown to produce Shiga toxin (Stx), E. coli O157:H7 and E. coli O157:NM (non‐motile) are most frequently implicated in human disease. Early recognition of STEC infections is critical for effective treatment of patients. Furthermore, rapid microbiological diagnosis of individual patients enables the prompt notification of outbreaks and implementation of control measures to prevent more cases. Most human infections caused by STEC have been acquired by the consumption of contaminated foods, especially those of bovine origin such as undercooked ground beef and unpasteurized cows' milk, and by person‐to‐person contacts. To identify the reservoirs of STEC and the routes of transmission to man, sensitive methods are needed as these pathogens may only be present in food, environmental and faecal samples in small numbers. In addition, sensitive and rapid detection methods are necessary for the food industry to ensure a safe supply of foods. Sensitive methods are also needed for surveillance programmes in risk assessment studies, and for studies on survival and growth of STEC strains. Cultural methods for the enrichment, isolation and confirmation of O157 STEC are still evolving. Several selective enrichment media have been described, of which modified tryptone soy broth with novobiocin and modified E. coli broth with novobiocin, seem to be the most appropriate. These media are minimally‐selective broths that give a somewhat limited differential specificity favouring isolation of O157 STEC, as opposed to other Gram‐negative bacteria, in the sample. An incubation temperature of 41–42°C further enhances selectivity. The occurrence of heat‐, freeze‐, acid‐ or salt‐stressed STEC in foods means that it is important to be able to detect cells that are in a stressed state, as STEC generally have a very low infectious dose, and injured cells mostly retain their pathogenic properties. For the isolation of stressed O157 STEC, pre‐enrichment in a non‐selective broth is necessary. The most widely used plating medium for the isolation of typical sorbitol‐non‐fermenting strains of STEC of serogroup O157 is sorbitol MacConkey agar with cefixime and tellurite (CT‐SMAC). As some STEC strains are sensitive for tellurite and/or are sorbitol‐fermenting, the use of a second isolation medium, such as one of the newer chromogenic media, is recommended. Immunomagnetic separation (IMS) following selective enrichment, and subsequent spread‐plating of the concentrated target cells onto CT‐SMAC agar, appears to be the most sensitive and cost‐effective method for the isolation of E. coli O157 from raw foods. IMS increases sensitivity by concentrating E. coli O157 relative to background microflora, which may overgrow or mimic O157 STEC cells on selective agars. While cultural isolation of O157 STEC from foods and faeces is time‐consu...

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Section: Enrichment Mediamentioning

confidence: 97%

Methods for the detection and isolation of Shiga toxin-producingEscherichia coli

Boer

Heuvelink

2000

Journal of Applied Microbiology

119

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“…Bacteria with beads attached are separated by magnetic force and plated onto SMAC agar and on a selective cefixime-tellurite (CT)-SMAC agar. The study performed in our laboratory [45] demonstrated that the IMS technique was able to detect 10 2 of E. coli O157/g of feces in the presence of 10 7 of background coliform flora and significantly increased the isolation rate of E. coli O157 from stool as compared with direct plating.…”

Section: Isolation Of Stec O157:h7mentioning

confidence: 88%

“…However, this is often not possible, especially in HUS patients in whom the number of STEC O157:H7 in stool can be very low when HUS is diagnosed (usually 1 week or later after the onset of prodromal diarrhea). To increase the sensitivity of the isolation of STEC O157 in such cases, selective enrichment using the technique of immunomagnetic separation (IMS) is recommended [45]. In the IMS procedure, fecal samples are enriched in GN Broth Hajna for 4±6 hours and then incubated with magnetic beads coated with anti-O157 antibody.…”

Section: Isolation Of Stec O157:h7mentioning

confidence: 99%

Diagnosis of Shiga Toxin-Producing Escherichia coli Infections/Die Diagnostik von Shiga Toxin-produzierenden Escherichia coli-Infektionen

Friedrich¹,

Bielaszewska²,

Karch³

2002

LaboratoriumsMedizin

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Shiga toxin-producing E. coli (STEC) cause a spectrum of diseases ranging from watery diarrhea through hemorrhagic colitis to hemolytic uremic syndrome (HUS). Most STEC strains cannot be identified using conventional culture procedures. The exception is STEC O157:H7, which grows in colonies of typical morphology on certain selective culture media due to its inability to ferment sorbitol and to produce b-Dglucuronidase. Sorbitol-fermenting E. coli O157:H ± and STEC of the major non-O157 serotypes, of which most ferment sorbitol, cannot be distinguished on such media. Among various virulence factors, the ability to produce Shiga toxins is a common characteristic of all STEC. The E. coli Shiga toxin family includes two major types, Stx1 and Stx2, and several toxin variants termed Stx1c, Stx2c, Stx2d, Stx2e and Stx2 f. The aim of the STEC laboratory diagnosis is the detection of Shiga toxins, which is achieved at three levels. At the first level, STEC screening is performed using culture on comercially available selective media and commercial enzyme immunoassays with enriched stool cultures. The detection of the toxin by an enzyme immunoassay must be followed by the isolation and characterization of the STEC isolate at the second level with the aim to confirm Shiga toxin production. The Shiga toxin colony immunoblot assay represents an effective, serotype independent, commercially available method for the isolation of STEC. In this method, Shiga toxin-positive colonies are identified using monoclonal antibodies. At the third step, subtyping of the STEC isolates is performed for epidemiological purposes, especially for STEC surveillance. This subtyping is usually done by National Reference Centers. In patients with HUS, the bacteriological detection of STEC may fail because the number of STEC in stool may be extremely low. In such cases, selective enrichment using immunomagnetic separation is recommended. In case of negative stool culture, the detection of specific serum antibodies against lipopolysaccharides of the major HUS-associated STEC serogroups, e. g. O157, is recommended.

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“…The sensitivities of PCR techniques to detect E. coli 0157 and other foodborne pathogens are dependent on many factors. Previous procedures differed in the type of DNA recovery technique used (Lindqvist 1997;Venkateswaran et al 1997); the PCR parameters (Chen et al 1998;Stone et al 1995;Wang et al 1997); the type of sample (Karch et al 1992;Stone et al 1995;Witham et al 1996); and the physiological state of the cells (Uyttendaele et al 1998). The PCR-LS-SOB and PCR-7200 procedures differed in the amount of MgCl, used, the thermal cycling conditions, and the plate readers.…”

Section: Discussionmentioning

confidence: 99%

“…Other genes, such as the eaeA gene also have been used as primer targets for the detection of E. coli 0157:H7 (Fratamico et al 1995;Gannon et al 1997;Paton and Paton 1998). Because the srx genes are subject to frequent loss during cultivation (Karch et al 1992), the eaeA gene may be a better predictor of the presence of enterohemorrhagic E. coli 0157:H7 (Gannon et al 1993). Simultaneous detection of multiple genes of this pathogen in a multiplex PCR has been reported (Franck et al 1998;Fratamico et al 1995;Gannon et al 1992Gannon et al , 1997Meng et al 1997;Paton and Paton 1998).…”

Section: Introductionmentioning

confidence: 99%

EVALUATION OF 5’NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

Bohra¹,

Oberst²,

Phebus³

et al. 2001

Rapid Methods Automation Mic

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Two 5’nuclease‐based PCR methods (PCR‐LS‐50B and PCR‐7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR‐7200 method was able to detect E. coli O157:H7 at ± 102 CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR‐7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR‐LS‐50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR‐based detection techniques for E. coli O157:H7, however, the 5’nuclease‐based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.

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Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture

Cited by 111 publications

References 20 publications

Methods for the detection and isolation of Shiga toxin-producingEscherichia coli

Methods for the detection and isolation of Shiga toxin-producingEscherichia coli

Diagnosis of Shiga Toxin-Producing Escherichia coli Infections/Die Diagnostik von Shiga Toxin-produzierenden Escherichia coli-Infektionen

EVALUATION OF 5’NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

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