Weagant S D, Bound A J
Pacific Regional Laboratory Northwest, US Food and Drug Administration, Bothell, WA 98021, USA.
Int J Food Microbiol. 2001 Dec 4;71(1):87-92. doi: 10.1016/s0168-1605(01)00558-x.
Because sprouted seed products are kept wet during and after production, have high levels of nutrients, and a neutral pH, they are subject to the outgrowth of pathogens such as Escherichia coli O157:H7. For these same reasons, these products also contain high levels of heterotrophic organisms and in particular coliform bacteria. Recent outbreaks have focused attention on the need to improve methodology for isolating this pathogen from sprouts. When 40 E. coli O157:H7 strains were grown in pure culture in enterohemorrhagic E. coli enrichment broth (EEB) as prescribed in the U.S. FDA-Bacteriological Analytical Manual (FDA-BAM) and in EEB modified by varying the cefixime concentration, outgrowth for all strains in EEB was inhibited at 0.05 mg/l but for only 2 of 40 strains when the cefixime level was adjusted to 0.0125 mg/l. These two enrichment formulae were compared to modified E. coli broth (mEC), modified Tryptic Soy Broth with 20 mg/l novobiocin (mTSB + N), modified Buffered Peptone Water (mBPW), and mBPW with added 10 mg/l acriflavin, 10 mg/l cefsulodin, and 8 mg/l vancomycin (mBPW + ACV) for isolation of E. coli O157:H7 from sprouts. These comparisons were performed using low-level (0.12 to 0.42 cfu/g) artificially contaminated alfalfa and mixed salad sprouts. After enrichment, two isolation methods were compared for recovery; direct plating to Tellurite-Cefixime Sorbitol MacConkey agar (TCSMAC) and immunomagnetic separation (IMS) (Dynabeads anti-E. coli O157, Dynal, Oslo, Norway) followed by plating to TCSMAC. In addition, an immunoprecipitin detection kit, VIP (BioControl, Bellevue, WA), was evaluated for detection after enrichment. We found that five of the six enrichments were equivalent for detection or recovery while one enrichment (mTSB + N without agitation) was less productive. Incubation for 24 h was more effective in recovering E. coli O157:H7 from sprouts than 6 h for all enrichment broths. Plating after IMS was more productive than direct plating at these low levels of contamination, yielding recovery in 70 of 90 trials compared to 37 of 90 trials without IMS for six enrichments. The sensitivity of VIP for detection of E. coli O157:H7 varied depending on the enrichment broth. Because of the rapid rate of growth of E. coli O157:H7 in mBPW, the high productivity of mBPW + ACV after 24-h enrichment and its compatibility with both IMS and detection with immunoprecipitin tests, mBPW + ACV at 42 degrees C with agitation was found to be the most promising enrichment protocol for testing sprouts.
由于发芽种子产品在生产期间及之后保持湿润，营养成分含量高，且pH值呈中性，因此易受诸如大肠杆菌O157:H7等病原体滋生的影响。出于同样的原因，这些产品中还含有大量的异养生物，尤其是大肠菌群。近期的疫情爆发使得人们开始关注改进从豆芽中分离这种病原体的方法的必要性。当按照美国食品药品监督管理局细菌学分析手册（FDA - BAM）的规定，将40株大肠杆菌O157:H7菌株在肠出血性大肠杆菌增菌肉汤（EEB）中进行纯培养，并在通过改变头孢克肟浓度进行改良的EEB中培养时，在EEB中所有菌株的生长在头孢克肟浓度为0.05mg/L时受到抑制，但当头孢克肟水平调整为0.0125mg/L时，40株菌株中只有2株受到抑制。将这两种增菌配方与改良大肠杆菌肉汤（mEC）、添加20mg/L新生霉素的改良胰蛋白胨大豆肉汤（mTSB + N）、改良缓冲蛋白胨水（mBPW）以及添加10mg/L吖啶黄素、10mg/L头孢磺啶和8mg/L万古霉素的mBPW（mBPW + ACV）进行比较，以从豆芽中分离大肠杆菌O157:H7。这些比较是使用低水平（0.12至0.42cfu/g）人工污染的苜蓿芽和混合沙拉芽进行的。增菌后，比较了两种分离方法的回收率；直接接种到亚碲酸盐 - 头孢克肟山梨醇麦康凯琼脂（TCSMAC）平板上，以及免疫磁珠分离（IMS）（抗大肠杆菌O157 Dynabeads，Dynal，挪威奥斯陆）后再接种到TCSMAC平板上。此外，还评估了一种免疫沉淀检测试剂盒VIP（BioControl，华盛顿州贝尔维尤）在增菌后的检测效果。我们发现六种增菌方法中有五种在检测或回收率方面相当，而一种增菌方法（未搅拌的mTSB + N）效率较低。对于所有增菌肉汤，培养24小时从豆芽中回收大肠杆菌O157:H7比培养6小时更有效。在这些低污染水平下，IMS后接种比直接接种更有效，在90次试验中，六种增菌方法中有70次通过IMS后接种获得回收率，而未使用IMS的情况下90次试验中只有37次获得回收率。VIP检测大肠杆菌O157:H7的灵敏度因增菌肉汤而异。由于大肠杆菌O157:H7在mBPW中生长迅速，mBPW + ACV在24小时增菌后效率高，且与IMS以及免疫沉淀检测兼容，因此发现42摄氏度搅拌条件下的mBPW + ACV是检测豆芽最有前景的增菌方案。
