通过培养和分子方法检测和计数水样中的大肠杆菌 O157:H7。

Detection and enumeration of E. coli O157:H7 in water samples by culture and molecular methods.

机构信息

Department of Food Science, University of Guelph, Guelph, Ontario, Canada.

出版信息

J Microbiol Methods. 2013 Feb 15;92(2):164-72. doi: 10.1016/j.mimet.2012.11.018. Epub 2012 Dec 5.

DOI:10.1016/j.mimet.2012.11.018

PMID:23220187

Abstract

The performances of three chromogenic agars were evaluated for the recovery of Escherichia coli O157:H7 from spiked dechlorinated tap, ground and surface water, and treated drinking water samples. The chromogenic agars: ChromAgar O157 (CHROM), Rainbow Agar O157 (RB) and HiCrome EC O157 (HC) were compared to cefixime-tellurite Sorbitol MacConkey (CT-SMAC), commonly used for the isolation of E. coli O157:H7. Confirmation of suspect E. coli O157:H7 colonies were performed by colony real-time PCR (C-RTi-PCR) based on the presence of Shiga-toxin genes (stx1 and stx2). Recovery of inoculated E. coli O157:H7 from dechlorinated tap water indicated that RB and CHROM agars demonstrated improved recovery when compared to HC or CT-SMAC. There was a significant drop in recovery on all agars tested after 120h (day 5). Twenty dechlorinated tap and/or treated drinking water samples were inoculated with a pure culture of E. coli O157:H7 (ATCC 43894), and a mixed culture of E. coli O157:H7 (ATCC 43894), E. coli strain K-12, and Enterococcus faecalis (ATCC 063589). After a 48-hour holding time, the recovery using CHROM (99%) and HC (12%) from samples contaminated with the pure culture were found to be significantly different (p<0.05). Recovery results using CHROM (39%) and CT-SMAC (32%) from samples contaminated with the mixed culture after a 48-hour holding time were not significantly different (p>0.05). Analysis by C-RTi-PCR of forty five environmental water samples (surface, sewage, and final effluents) which were negative for E. coli O157:H7 showed an incidence of false suspect positive colonies of 38% (CHROM), 53% (RB), 58% (HC), and 91% (CT-SMAC). Further analysis of eight of the environmental samples inoculated with E. coli (ATCC 43894) showed 100% recovery when utilizing CHROM, 50% when using RB and 40% when using HC. In addition, the C-RTi-PCR positive confirmation rate was 100% for CHROM and HC and 65% for RB. CHROM demonstrated improved recovery of E. coli O157:H7 over RB, HC, and CT-SMAC in terms of sensitivity and specificity.

摘要

三种显色琼脂在检测脱氯自来水、地面和地表水以及处理饮用水样本中大肠杆菌 O157:H7 的回收性能方面进行了评估。显色琼脂：ChromAgar O157（CHROM）、Rainbow Agar O157（RB）和 HiCrome EC O157（HC）与常用于分离大肠杆菌 O157:H7 的头孢克肟-碲酸盐山梨醇麦康凯（CT-SMAC）进行了比较。疑似大肠杆菌 O157:H7 菌落的确认通过基于志贺毒素基因（stx1 和 stx2）存在的菌落实时 PCR（C-RTi-PCR）进行。从脱氯自来水中接种的大肠杆菌 O157:H7 的回收表明，与 HC 或 CT-SMAC 相比，RB 和 CHROM 琼脂的回收率有所提高。在所有测试的琼脂上，在 120 小时（第 5 天）后回收率显著下降。将大肠杆菌 O157:H7 的纯培养物（ATCC 43894）和大肠杆菌 O157:H7（ATCC 43894）、大肠杆菌 K-12 和粪肠球菌（ATCC 063589）的混合培养物接种到二十份脱氯自来水和/或处理饮用水样本中。经过 48 小时的孵育时间后，发现从纯培养物污染的样本中使用 CHROM（99%）和 HC（12%）的回收率有显著差异（p<0.05）。从混合培养物污染的样本中经过 48 小时的孵育时间后，使用 CHROM（39%）和 CT-SMAC（32%）的回收率没有显著差异（p>0.05）。对 45 份环境水样（地表水、污水和最终流出物）进行 C-RTi-PCR 分析，这些水样均未检出大肠杆菌 O157:H7，结果显示 CHROM（38%）、RB（53%）、HC（58%）和 CT-SMAC（91%）的假可疑阳性菌落发生率分别为 38%、53%、58%和 91%。对用大肠杆菌（ATCC 43894）接种的八个环境样本进行进一步分析，结果表明，使用 CHROM 时的回收率为 100%，使用 RB 时的回收率为 50%，使用 HC 时的回收率为 40%。此外，CHROM 和 HC 的 C-RTi-PCR 阳性确认率为 100%，RB 为 65%。CHROM 在灵敏度和特异性方面优于 RB、HC 和 CT-SMAC，可提高大肠杆菌 O157:H7 的回收率。