Karch H, Janetzki-Mittmann C, Aleksic S, Datz M
Institut fur Hygiene und Mikrobiologie der Universitat Wurzburg, Germany.
J Clin Microbiol. 1996 Mar;34(3):516-9. doi: 10.1128/jcm.34.3.516-519.1996.
We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.
我们检查了30例典型溶血尿毒综合征（HUS）患儿的粪便样本中是否存在肠出血性大肠杆菌（EHEC）菌株，并测定了这些患者急性期血清样本中对O157脂多糖的特异性免疫反应。从18例（60%）患者的粪便样本中分离出EHEC O157菌株，从5例（17%）患者中分离出非O157 EHEC菌株。为了从粪便中分离O157菌株，我们引入了一个选择性富集步骤，使用附着在顺磁性颗粒上的O157特异性抗体（免疫磁珠分离[IMS]法）。该方法能够在存在10⁷大肠菌群背景菌群的情况下，检测到每克粪便中10²CFU的O157菌株。通过使用IMS，然后接种到山梨醇麦康凯（SMAC）琼脂和头孢克肟-亚碲酸盐SMAC（CT-SMAC）琼脂上，在18份样本中检测到O157菌株，而菌落杂交检测到12份阳性样本的一个子集，直接接种到CT-SMAC或SMAC琼脂上仅检测到7份。18份O157阳性粪便中有3份通过粪便滤液进行的细胞毒性试验以及从粪便中提取DNA进行的直接PCR检测为阴性。IMS技术从20例有O157感染血清学证据的患者中的18例分离出了O157菌株。与直接培养相比，除了提高了O157检测的灵敏度外，IMS技术还具有比分子方法劳动强度小、耗时少的优点。因此，IMS可被认为是一种在临床微生物实验室中广泛用于检测O157菌株的有效方法。然而，由于大量HUS病例归因于非O157 EHEC血清群，在HUS的细菌学诊断中除了IMS外还需要使用其他方法。
