Keeping labs proficient in the detection of Shiga Toxin-producing Escherichia coli
On September 5, 2023 Alberta Health Services declared a Shiga toxin-producing Escherichia coli (STEC) (link), outbreak originating from six locations of a Calgary daycare and five additional sites that shared a central kitchen.
By September 11 there were 231 lab-confirmed cases connected to this outbreak with 26 patients receiving care in hospital. 21 patients have been confirmed as having severe illness/hemolytic uremic syndrome (HUS).
This outbreak clearly demonstrates the importance of laboratories being proficient in the detection of these organisms, have the right methods of detection, and to promptly report the cases to Public Health.
Below is an extract from a Proficiency Testing (PT) case critique provided by CMPT. CMPT has a specific PT program for the detection of STEC (Shiga Toxin | cmpt)
Isolation and Identification
Stool specimens submitted for bacterial enteric culture should be screened for Shiga toxin producing E. coli (STEC). The Sorbitol MacConkey (SMAC) plate is the most commonly used culture screening medium. Most isolates fail to ferment sorbitol and will be recognized as colourless colonies on SMAC.
There are other organisms that can appear as non-sorbitol fermenters, so further testing is required to confirm identification. A sampling of the non-sorbitol fermenting colonies can be tested directly using a latex agglutination test for the presence of the somatic O157 antigen. Species other that E. coli O157 may cross react so all positive latex tests must be confirmed to be E. coli using biochemical tests 1, 2, 3 .
All presumptive E. coli O157 should be tested for the presence of the flagellar (H7) antigen. This test is usually performed at a reference laboratory. Isolates negative for the H7 antigen, or that are non-motile should be tested for the presence of the Shiga toxin or Shiga toxin gene sequences.
Some strains of E. coli O157 ferment sorbitol and will not be detected if the SMAC agar is used. Non O157 STEC are also responsible for outbreaks and may not be isolated unless other media or methods for toxin testing are used 1,4. An alternative may be to perform non-culture Shiga toxin testing either on site or through referral to a reference laboratory.
STEC harbors and expresses the genes for Shiga toxins type 1 (Stx 1) and type 2 (Stx2), the virulence factors that lead to Hemolytic Uremic Syndrome (HUS). Single STEC may express either Stx1, Stx2, or both. Non-culture methods are aimed at the detection of the toxins or toxin genes. They have the advantage of detecting any of the STEC serotypes.
Toxins are detected by cytotoxicity or immunoassays while genes are detected by nucleic acid amplification tests.
Cell Cytotoxicity assays
These assays use Vero or HeLa cell lines to detect the presence of biologically active Shiga toxins in stools. These cell lines are very sensitive to Shiga toxin because they have high concentrations of receptors for the toxin 2.
Sterile fecal or enrichment broth filtrates are inoculated onto the cell monolayer and observed for typical cytopathic effect. Confirmation that the cytopathic effect is caused by Shiga toxin is performed by neutralization using anti-Stx 1 and anti-Stx 2 antibodies 5,6.
Shiga Toxin Immunoassays
There are few immunoassays commercially available for the detection of Shiga toxin. Most assays recommend the use of enrichment broth cultures rather than direct testing of stool specimens because of the low amount of free toxin in stools. Some assays can differentiate between Stx1 and Stx2 6.
Nucleic Acid Amplification Tests (NAAT)
Most NAAT assays are designed and validated for testing isolated colonies taken from plated media while some assays have been validated for testing on stool specimens after incubation in an enrichment broth 2. Depending on the primers used, these assays can distinguish between stx1 and stx2 genes 6.
The natural reservoir is the gastrointestinal tract of cattle. STEC transmission occurs through the consumption of a wide variety of contaminated foods, raw milk, and raw produce, through contact with animals or their environment, and directly from person to person1,7, 8.
Shiga toxin producing strains are capable of causing mild non bloody diarrhea, severe bloody diarrhea and HUS. Symptoms are mediated by toxins which are biochemically and genetically similar to the Shiga toxin produced by Shigella dysenteriae type 1.
Patients infected with STEC present at first with abdominal cramps and watery diarrhea; this may progress within 1 or 2 days to hemorrhagic colitis and about 5 to 15% of patients typically develop HUS 5, 9.
HUS is characterized by thrombocytopenia, hemolytic anemia and kidney injury. Some patients present neurological symptoms including severe headache, and encephalopathy 5.
Bacteremia is rare as STEC are not invasive but they secrete ribosome inactivating toxins which are responsible for the organ damage 10.
Shiga toxins are compound toxins composed of a catalytic A subunit and a multimeric B subunit (AB5) which binds to the cell surface of the target cells 11. Once bound, the toxin is incorporated into the cells by endocytosis and reaches the endoplasmic reticulum by retrograde transport via the Golgi apparatus.
The A subunit is then enzymatically activated and released; the active A subunit has RNA N-glycosidase activity it cleaves a specific N-glycosidic bond in the 28S rRNA, inhibiting protein synthesis and ultimately causing cell death12.
Although O157:H7 is the serotype most frequently isolated from humans, a recent CDC surveillance report showed that O157 serotypes comprised 41.1% of all the STEC isolated. Non-O157:H7 STECs have been emerging in Canada and are common in Australia, Germany, and Austria 4,13.
In 2011 a large outbreak of gastroenteritis with bloody diarrhea and HUS was reported in Germany. The strain involved in the outbreak was an enteroaggregative STEC O104:H4. This strain was notable for its high HUS rate of (22% of cases — 88% of those were adults), and because it expressed an extended-spectrum β-lactamase (ESBL) 14.
Reporting susceptibility test results is not recommended. Treatment of STEC infection is supportive. Generally, patients with HUS are managed for symptoms of renal failure, anemia, bleeding and intestinal injury.
The use of antibiotics is contraindicated as it has been shown to be neither useful nor safe. Some studies have shown that antibiotic usage may induce the development and release of the Shiga toxins. Other studies have shown that antibiotic usage in children with O157 STEC infections can increase the risk of HUS.
Susceptibility testing may be done for epidemiological purposes. Once susceptible to most antimicrobial agents, E coli O157 and other STEC are showing increasing resistance 15, 16.
Main Educational Points
- Some strains of Enterohemorrhagic E. coli O157 and non O157 may not be detected if using Sorbitol MacConkey (SMAC) agar.
- Chromogenic agars have been developed which allow for detection of sorbitol and non-sorbitol fermenting isolates.
- Non O157, verotoxin producing strains, have been involved in outbreaks.
- Non culture Shiga toxin testing may be a useful alternative to culture methods.
- Antimicrobial therapy is not recommended and has been linked to an increased risk of HUS in children.
- CMPT M081. 2008
- Gould L.H, Bopp C, Strockbine N, et al. Recommendations for Diagnosis of Shiga Toxin-Producing Escherichia coli Infections by Clinical Laboratories. MMWR Morbidity and mortality weekly report. 2009;58:1.
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- Ling H, Boodhoo A, Hazes B, et al. Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb3. Biochemistry (N Y ). 1998;37:1777-1788.
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- CDC National Enteric Disease . Shiga toxin-producing Escherichia coli (STEC) Annual Report, 2011 http://www.cdc.gov/ncezid/dfwed/PDFs/national-stec-surv-summ-2011-508c.pdf
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