E. coli O157:H7

Test code: B0059 - Ultrasensitive qualitative detection of E. coli strain O157:H7 by real time polymerase chain reaction

Escherichia coli serotype O157:H7 is a gram-negative, rod-shaped bacterium and is one of hundreds of serotypes of the bacterium E. coli. While most strains are harmless and are found normally in the intestines of mammals, strain O157:H7 produces shiga-like toxins, causes severe illness, and is a member of a class of pathogenic E. coli known as enterohemorrhagic Escherichia coli (EHEC). They are sometimes also referred to by their toxin producing capabilities, eg “verocytotoxin producing E. coli” (VTEC) or “shiga-like toxin producing E. coli” (STEC).

E. coli O157:H7 differs from other pathogenic E. coli in that it is not invasive, elaborates no colonization factors (CFA/I nor CFA/II), does not produce heat stable or heat labile toxins and is non-hemolytic. In addition, E. coli O157:H7 is sorbitol negative whereas 93% of all E. coli ferment sorbitol. E. coli O157:H7 also does not hydrolyze 4-methylumbelliferyl-ß-D-glucuronide (MUG), nor does it grow at 45ºC in the presence of 0.15% bile salts. Because of the latter characteristic this serotype cannot be isolated by using standard fecal coliform methods that include incubation at 45ºC.

E. coli O157:H7 can be transmitted in food or drinking water, and outbreaks of E. coli O157:H7 infection have been attributed to the presence of this bacterium in groundwater and surface water (Chalmers et al. 2000; Lee et al. 2002). A likely source of contamination of aquatic systems is cattle manure and agricultural runoff. The intestinal cells of cattle, swine and deer lack the highly specific surface receptors that the toxin requires in order to attach and enter the cell. Therefore these animals do not exhibit disease when infected (Pruimboom-Brees et al. 2000) and can be carriers, shedding the organism in their feces.

E. coli O157:H7 persists in cattle manure (Wang et al. 1996; Bolton et al. 1999; Fukushima et al. 1999; Osek 2002) and manure-amended soil (Jiang et al. 2002) and experiments with models have suggested that it may leach through soil (Gagliardi and Karns 2000). Meat can become contaminated during slaughter, and the organisms can be thoroughly mixed into beef when it is ground. Bacteria present on cows’ udders or on equipment may get into raw milk. Although the number of organisms required to cause disease is not known, it is suspected to be very small.

A major source of human infection is undercooked ground beef; other sources include consumption of unpasteurized milk and juice, raw sprouts, lettuce and salami, as well as contact with infected live animals. Waterborne transmission occurs through swimming in contaminated lakes or pools, or drinking inadequately treated water. The organism is easily transmitted from person to person and has been difficult to control in child daycare centers. Infection often causes severe, acute bloody diarrhea (although nonbloody diarrhea is also possible) and abdominal cramps. Usually little or no fever is present, and the illness resolves in 5 to 10 days. It can also be asymptomatic.

Rapid methods to detect E. coli O157:H7 are important to identify the source of outbreaks. Both molecular and culture-based methods have been used for the detection of E. coli O157:H7. Culture-based methods developed for clinical samples have been applied to environmental samples. These methods rely on enrichment cultures followed by confirmation based on metabolic and antigenic properties. A disadvantage of this approach is the lack of complete correlation of these antigenic and metabolic properties with Shigella toxin (stx) production (Karch and Bielaszewska 2001). Also, culture-based methods for the detection of O157:H7 are slow and labor intensive, so they are not ideal for analysis of the large numbers of samples that must be tested when possible environmental sources of an outbreak are being investigated.

As the infectious dose is very small and the number of cells contaminating environmental samples or food may be low, immunomagnetic capture with anti-O157 antibody has been suggested as a means to concentrate and detect the target cells (Pyle et al. 1999). However, this approach is limited to cells displaying a specific antigen, making it unsuitable for other STEC. Molecular approaches for bacterial detection avoid the need for culture and can be designed to be more specific. Primers targeting stx1, stx2 and other genes specific to E. coli O157:H7 have been used in PCR and real time PCR (Olsvik and Strockbine 1993; Fratamico et al. 2000; Fortin et al. 2001; Li and Drake 2001; Ibekwe et al. 2002; Ibekwe and Grieve 2003). Specific, rapid diagnosis of E. coli O157:H7 is possible using PCR techniques (Al-Ajmi et al., 2006; Holicka et al., 2006).

Utilities:

• Confirm the disease causing agent
• Shorten the time required to confirm a clinical diagnosis of E. coli O157:H7 infection.
• Ensure that horse populations are free of E. coli O157:H7
• Early prevention of spread of this bacterial strain
• Minimize human exposure to this bacterial strain

References:
Al-Ajmi, D., Padmanabha, J., Denman, S.E., Gilbert, R.A., Al Jassim, R.A. and McSweeney, C.S.(2006) Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples. Lett Appl Microbiol. 42:386-391.

Holicka, J., Guy, R.A., Kapoor, A., Shepherd, D. and Horgen, P.A. (2006) A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef. Can J Microbiol. 52:992-8.

Specimen requirements: Rectal swab, 1 ml fecal specimen or bacterial culture, shipped overnight at room temperature.

For specimen types other than those listed here, please call to confirm specimen acceptability and shipping instructions.

For all specimen types, if there will be a delay in shipping, or during very warm weather, refrigerate specimens until shipped and ship with a cold pack unless more stringent shipping requirements are specified. Frozen specimens should be shipped so as to remain frozen in transit. See shipping instructions for more information.

Turnaround time: 2 business days

Methodology: Qualitative real time PCR

Normal range: Nondetected