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

作者：Ngwa G A; Schop R; Weir S; Leon Velarde C G; Odumeru J A^*

来源：Journal of Microbiological Methods, 2013, 92(2): 164-172.

DOI：10.1016/j.mimet.2012.11.018

• Escherichia coli O157:H7
• Chromogenic media
• Membrane filtration
• Multiplex colony real-time PCR (mC-RTi-PCR)
• Shiga-toxin genes

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 120 h (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%26lt;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%26gt;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.

• 出版日期2013-2-15