A challenging STEC strain isolation from patients’ stools: an O166:H15 STEC strain with the stx2 gene

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.

led to severe disease outcomes (6,7).Therefore, it is crucial to study the new emerging STEC strains, especially when they carry stx2 (2).
Serotyping and cluster analysis are crucial for public health surveillance and gastroenteric outbreak detection.The recovery of STEC isolates from patients' stools is essential for these purposes as well as for further characterizations.Recovering the isolates through culture is labor-intensive and mostly challenging as not all non-O157 STEC would grow on selective media when directly inoculated from stools (8)(9)(10).Therefore, it is crucial to investigate alternate approaches to recover these pathogens from patients' stools.This is a short report on the successful isolation and characteriza tion of an uncommon serotype of O166:H15 STEC from two cases from Alberta, Canada, which were particularly challenging to isolate through culture alone.
Two patients' stool specimens: P-1 and P-2 from Alberta, Canada, submitted due to acute gastroenteritis, tested positive only for STEC by polymerase chain reaction (PCR) using the BD Max enteric bacterial panel targeting STEC, Salmonella spp., Shigella spp./ enteroinvasive E. coli (EIEC), and Campylobacter spp.These specimens were sent to Alberta Precision Laboratory-Provincial Laboratory for Public Health (ProvLab) for pathogen isolation and identification.ProvLab reported both stools were PCR-positive for stx2 but failed to isolate the strains through routine culture.Consequently, these samples were referred to ProvLab Chui research laboratory for further investigations.Upon receipt, 10 µL of stools was enriched in trypticase soy broth (TSB) overnight at 37°C.Stools and the enriched TSB cultures were inoculated onto CHROMagar STEC and MacConkey agar (MAC) plates (Dalynn Biologicals, Calgary, AB, Canada) and were incubated overnight at 37°C.Both samples showed growth only on MAC plates, and colony sweeps tested PCR-positive for stx2.The colonies were small and light pink in color and were embedded within off-white-colored commensal bacterial colonies (PCR-negative for stx).The nature of the colony growth and pinpoint size complicated STEC isolation through culture.Multiple attempts were made to recover pure colonies through TSB, MAC, and onto CHROMagar STEC culture, but failed.
Considering these STEC strains carrying stx2 showed no growth on CHROMagar STEC and proved challenging to isolate, there was a possibility that these were not O157:H7 strains.Consequently, they were presumed to be in the top six non-O157:H7 category.As isolation through culture was difficult, the immunomagnetic separation (IMS) method was explored as an alternative approach for the recovery.In IMS, a pool of immunomagnetic beads (RapidCheck CONFIRM STEC IMS, Romer Lab Division Holdings GmbH, Getzersdorf, Austria) that target O26, O45, O103, O111, O121, and O145 STEC are used to separate these STEC from the other background bacteria in a sample.A common application of IMS is to isolate the top six STEC in contaminated food products (11), but its usage to recover STEC from stools has not been previously documented.The flow chart (Fig. 1) illustrates the STEC isolation process that was followed.
IMS was applied on enriched stool TSB according to the manufacturer's instructions with slight modifications.Pea-sized amounts of stools from P-1 and P-2 were separately inoculated in 3.5 mL TSB and were incubated at 37°C overnight.A 1-mL aliquot from each of these enriched TSB were treated with 51 µL of pooled immunomagnetic beads, washed three times with Tween-PBS, and then resuspended in 100 µL of Tween-PBS (a protocol used for immunocapture in routine food laboratory in ProvLab).A 50-µL aliquot was subjected to lysis to obtain the DNA for PCR analysis (12).The remaining 50-µL aliquot was inoculated onto MAC plates.STEC-captured beads from P-1 and P-2 tested PCR-positive for stx2.The colony morphologies from both samples were similar on MAC plates, and sweeps from all four quadrants tested positive for stx2.However, a mixed culture of small pink and large off-white colonies was observed, indicating the crossreactivity of the top six non-O157 immunocapture beads.The small pink colonies tested positive for stx2 but not the larger ones.Consequently, the recovery of pure STEC colonies was not possible at this stage.Therefore, a second IMS was carried out using a sweep of colonies from the first IMS-MAC agar.P-1 and P-2 MAC colony sweeps were added separately to 1.5 mL of TSB, and 1 mL of the mixture was retrieved for the IMS protocol as described previously.After the separation, the beads were spread on MAC plates and incubated overnight (~20 h).On the following day, although large off-white colonies were still present, numerous light pink isolated colonies were also found and were confirmed stx2-positive by PCR.
To observe the colony growth over time, colonies from P-1 and P-2 were inoculated onto CHROMagar STEC and BAP.Plates were incubated at 37°C overnight and then left at room temperature until day 15.The small P-2 colonies grew fine colorless colonies that were barely visible on day 1 on both types of media (Fig. 2A).These colonies became visible after day 4 at room temperature.The large colonies from P-1 and P-2 showed similar morphologies on each type of media on day 1.By day 8, both large and small colonies on BAP resembled "fried egg"-like structures with a raised circular center and a flat surrounding (Fig. 2A and B), and ridges were radiating from the circular center.On CHROMagar STEC, the small colonies remained smaller with a pinpoint center (Fig. 2A) and the large colonies further expanded in size and exhibited concentric ring structures with a darker center (Fig. 2B).The colonies on both BAP and CHROMagar STEC developed diffused outer layers during days 8 to 15, and the colony morphology did not significantly change on each media over this period.In parallel, a STEC O157:H7 was inoculated onto CHROMagar, MAC, and BAP, and the colony morphologies were not comparable to those of O166:H15 under the same conditions and time duration (data not shown).
In this investigation, we aimed to report this uncommon case of E. coli O166:H15 STEC and to emphasize the challenges faced in the recovery of these isolates from stools.We tested IMS as an alternative method to using culture alone, which was a novel approach to recover STEC from clinical stools.Although the immunomagnetic beads targeted the top six serotypes, the cross-reactivity allowed us to recover this O166:H15 serotype, which is not from the top six serotypes.A gastroenteritis outbreak pertaining to E. coli O166:H15 was previously reported in Osaka, Japan, in 1996 (18).However, this particular O166:H15 strain was not considered an STEC as it did not carry any stx genes.Since the O166:H15 STEC we isolated carried stx2, it has a great potential to cause complications such as HUS in patients.Unfortunately, the patient chart reviews were not available, so disease outcomes could not be discussed here.

FIG 2
FIG 2 Colony morphology of O166:H15.The morphology of the (A) small and (B) large O166:H15 colonies on sheep blood agar and CHROMagar STEC at day 1 and day 8.The same large colonies on CHROMagar STEC are displayed under different contrasts to emphasize on the concentric ring structures.

TABLE 1
Quality statistics of WGS analysis

TABLE 2
The virulence factors identified in the O166:H15 strains a a The listed virulence factors were present in both small and large colonies.