Gilmour Matthew W, Chui Linda, Chiu Theodore, Tracz Dobryan M, Hagedorn Kathryn, Tschetter Lorelee, Tabor Helen, Ng Lai King, Louie Marie
National Microbiology Laboratory, Winnipeg, MN, Canada.
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada.
J Med Microbiol. 2009 Jul;58(Pt 7):905-911. doi: 10.1099/jmm.0.007732-0. Epub 2009 Jun 5.
The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.
从临床粪便样本中分离出除O157血清群之外的产志贺毒素大肠杆菌（STEC）存在问题，因为它与非致病性大肠杆菌缺乏可区分的表型特征。能够识别毒素和血清群特异性遗传决定因素的分子试剂的开发，有望对粪便样本进行更全面的表征并分离出STEC菌株。在本研究中，使用细胞毒性测定、商业免疫测定和针对志贺毒素决定因素的常规PCR，对876份来自患有肠胃炎的儿科患者的粪便样本进行了STEC筛查。此外，还采用了分离O157 STEC的常规培养方法。筛查试验确定了45份粪便样本可能含有STEC，使用非鉴别培养技术共分离出20株O157菌株和22株非O157菌株。这些菌株包括STEC血清型O157 : H7、O26 : H11、O121 : H19、O26 : NM、O103 : H2、O111 : NM、O115 : H18、O121 : NM、O145 : NM、O177 : NM和O5 : NM。值得注意的是，从两份临床粪便样本中分离出了多种STEC血清型（分别得到O157 : H7和O26 : H11，或O157 : H7和O103 : H2分离株）。将这些数据与使用针对O157菌毛基因lpfA的LUX实时PCR测定、针对espZ等位基因变体的微球悬浮阵列以及基于gnd的分子O抗原血清群分型方法直接从粪便富集培养物中确定的分子血清群谱进行了比较。单个粪便培养物的基因谱表明，espZ微球阵列和lpfA实时PCR测定能够准确预测临床样本中存在的STEC菌株的存在并提供初步分型。基于gnd的分子血清群分型方法为血清群身份提供了额外的确证证据。这个分子方法工具箱为临床粪便样本中的STEC提供了强大的检测能力，包括多个血清群的共感染情况。
