Related Experiment Video
Updated: Jun 25, 2025
Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis
Published on: May 23, 2021
A challenging STEC strain isolation from patients' stools: an O166:H15 STEC strain with the stx2 gene
Surangi H Thilakarathna1, Vincent Li2, Linda Chui1,2
1Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada.
Abstract:
Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7.
Importance:
Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.
Insights
Shiga toxin-producing Escherichia coli (STEC) O166:H15 strains, carrying the stx2 gene, were challenging to culture but successfully isolated using immunomagnetic separation (IMS). This study highlights IMS as a novel approach for recovering difficult-to-culture STEC from clinical samples.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Biology
Background:
- Shiga toxin-producing Escherichia coli (STEC) infections pose significant health risks, particularly from non-O157:H7 serotypes carrying the stx2 gene.
- The O166:H15 serotype, previously identified as non-STEC, was recovered with the stx2 gene in this study, presenting isolation challenges.
- Severe complications like hemolytic uremic syndrome (HUS) are associated with STEC infections, especially in vulnerable populations.
Purpose of the Study:
- To investigate the recovery of Shiga toxin-producing Escherichia coli (STEC) strains, specifically non-O157:H7 serotypes, from clinical samples.
- To evaluate the efficacy of immunomagnetic separation (IMS) as a novel method for isolating challenging STEC strains.
- To characterize the virulence genes and colony morphology of recovered STEC isolates.
Main Methods:
- Polymerase chain reaction (PCR) was used to detect STEC and the Shiga toxin 2 gene (stx2).
- Immunomagnetic separation (IMS) targeting non-O157:H7 serotypes was employed for STEC isolate recovery from clinical stools.
- Whole-genome sequencing was performed for serotyping, and isolates were cultured on CHROMagar STEC and sheep blood agar (BAP) to observe colony morphology.
Main Results:
- Two patients with acute gastroenteritis were positive for STEC carrying the stx2 gene, identified as O166:H15.
- Routine culture methods failed to recover the STEC isolates, whereas IMS, after two runs, successfully isolated the strains.
- Distinct colony morphologies, including concentric rings on CHROMagar STEC and "fried egg"-like structures on BAP, were observed for the STEC isolates over 15 days.
Conclusions:
- Immunomagnetic separation (IMS) is an effective novel approach for isolating difficult-to-culture STEC strains, including non-O157:H7 serotypes like O166:H15.
- The study identified O166:H15 STEC carrying the stx2 gene, highlighting the increasing importance of non-O157:H7 serotypes in STEC infections.
- Observed cross-reactivity of IMS magnetic beads with other E. coli serotypes warrants further investigation for improved specificity.
