Vidovic Sinisa, Germida Jim J, Korber Darren R
Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Sask, Canada S7N 5A8.
Food Microbiol. 2007 Sep;24(6):633-9. doi: 10.1016/j.fm.2006.12.001. Epub 2006 Dec 30.
The aim of this study was to investigate the sensitivity and reliability of two techniques commonly used for the isolation of Escherichia coli O157: (i) buffered peptone water (BPW) containing vancomycin, cefsulodin and cefixime followed by immunomagnetic separation (IMS-VCC) and (ii) modified E. coli (EC) broth supplemented with novobiocin (m ECn), both followed by culturing on cefixime tellurite sorbitol McConkey (ctSMAC) agar plates. Over a 2-year period, 24 feedlots located over a large geographical area (approximately 600 x 450 km) were screened for the presence of E. coli O157. A total of 194 E. coli O157 isolates were identified; 151 (77.4%) using IMS-VCC and 108 (55.4%) using m ECn. The recovery rates of IMS-VCC varied from 100% to 47%, whereas for m ECn ranged from 100% to 16%. All isolates were grouped, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), into 3 major clusters that comprised 39 different subtypes, providing evidence of considerable genetic heterogeneity. The results from this study revealed false negatives in IMS-VCC technique, most probably due to the high genetic diversity of environmental E. coli O157 isolates and antibiotic sensitivity. Using only IMS-VCC as a method for detection may result in significant underestimation of the pathogen. Performing two different enrichment steps in parallel can lead to markedly improved recovery rates of E. coli O157 isolates from naturally infected samples.
本研究的目的是调查两种常用于分离大肠杆菌O157的技术的敏感性和可靠性：（i）含有万古霉素、头孢磺啶和头孢克肟的缓冲蛋白胨水（BPW），随后进行免疫磁珠分离（IMS-VCC）；（ii）添加新生霉素的改良大肠杆菌（EC）肉汤（m ECn），两者随后均在头孢克肟亚碲酸盐山梨醇麦康凯（ctSMAC）琼脂平板上培养。在两年时间里，对位于大片地理区域（约600×450公里）的24个饲养场进行了大肠杆菌O157检测。共鉴定出194株大肠杆菌O157分离株；使用IMS-VCC鉴定出151株（77.4%），使用m ECn鉴定出108株（55.4%）。IMS-VCC的回收率在100%至47%之间，而m ECn的回收率在100%至16%之间。所有分离株通过随机扩增多态性DNA聚合酶链反应（RAPD-PCR）分为3个主要簇，包含39种不同亚型，这证明了其存在相当大的遗传异质性。本研究结果揭示了IMS-VCC技术存在假阴性，最可能的原因是环境中大肠杆菌O157分离株的高度遗传多样性和抗生素敏感性。仅使用IMS-VCC作为检测方法可能会导致对病原体的严重低估。并行执行两个不同的富集步骤可显著提高从自然感染样本中分离大肠杆菌O157的回收率。
