Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada.
Alberta Precision Laboratories - Public Health Laboratory (ProvLab), Edmonton, Canada.
Microbiol Spectr. 2024 Jul 2;12(7):e0009824. doi: 10.1128/spectrum.00098-24. Epub 2024 May 30.
Two patients with acute gastroenteritis tested positive for Shiga toxin-producing (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 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other 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.
Shiga toxin-producing (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 (), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. 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 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.
通过聚合酶链反应 (PCR)，两名急性肠胃炎患者的检测结果呈产志贺毒素大肠杆菌（STEC）阳性，且两种菌株均携带志贺毒素 2 编码基因。由于常规使用 CHROMagar STEC 进行培养未能回收这些分离株，因此使用针对前六种非-O157:H7 血清型的免疫磁分离（IMS）进行分离株回收。在进行了两次后续的 IMS 运行后，分别从胰蛋白酶大豆肉汤中回收了在运行 1 和 2 中进行过夜富集的 STEC 菌株。基于全基因组测序的血清型鉴定显示，两名患者均携带 O166:H15 STEC 菌株，该菌株带有 基因。因此，IMS 中使用的磁珠似乎与其他 血清型具有交叉反应性。当从两份粪便样本中培养 STEC 分离株并分别接种在 CHROMagar STEC 和绵羊血琼脂（BAP）平板上时，经过过夜孵育后可明显观察到两种不同大小的菌落。挑取小和大的菌落，分别在两种培养基上进行单独培养，在 37°C 下初始过夜孵育后，在室温下观察菌落生长 2 周。第 1 周后，CHROMagar STEC 平板上的菌落呈现出同心环结构，中央较暗，周围较浅；BAP 平板上的菌落呈现出“煎蛋”状结构，中央凸起，周围平坦。在 15 天的时间里，两种类型的菌落都在 CHROMagar STEC 平板上保持形态学差异。然而，在 BAP 平板上，第 7 天时它们的外观相似。
产志贺毒素大肠杆菌（STEC）感染可导致严重并发症，如血性腹泻和溶血性尿毒症综合征（HUS），尤其是在幼儿和老年人中。携带志贺毒素 2 基因（）的菌株，如 O157:H7，与严重疾病结局大多相关。近年来，非-O157:H7 菌株引起的暴发有所增加。O166:H15 先前曾被报道为 1996 年非 STEC 菌株引起的肠胃炎暴发，但我们分离到的 O166:H15 血清型携带 基因。通过培养从粪便中分离该菌株极具挑战性。因此，我们测试了免疫磁分离法用于 STEC 的回收，这是一种对临床粪便的新方法。为了对这些分离株进行表征，我们还检测了毒力基因。
