Rapid Concentration and Molecular Enrichment Approach for Sensitive Detection of Escherichia coli and Shigella Species in Potable Water Samples

Abstract

In this work, we used a rapid, simple, and efficient concentration-and-recovery procedure combined with a DNA enrichment method (dubbed CRENAME [ c oncentration and r ecovery of microbial particles, e xtraction of n ucleic a cids, and m olecular e nrichment]), that we coupled to an Escherichia coli / Shigella -specific real-time PCR (rtPCR) assay targeting the tuf gene, to sensitively detect E. coli / Shigella in water. This integrated method was compared to U.S. Environmental Protection Agency (EPA) culture-based Method 1604 on MI agar in terms of analytical specificity, ubiquity, detection limit, and rapidity. None of the 179 non- E. coli / Shigella strains tested was detected by both methods, with the exception of Escherichia fergusonii , which was detected by the CRENAME procedure combined with the E. coli / Shigella -specific rtPCR assay (CRENAME + E. coli rtPCR). DNA from all 90 E. coli / Shigella strains tested was amplified by the CRENAME + E. coli rtPCR, whereas the MI agar method had limited ubiquity and detected only 65 (72.2%) of the 90 strains tested. In less than 5 h, the CRENAME + E. coli rtPCR method detected 1.8 E. coli / Shigella CFU whereas the MI agar method detected 1.2 CFU/100 ml of water in 24 h (95% confidence). Consequently, the CRENAME method provides an easy and efficient approach to detect as little as one Gram-negative E. coli / Shigella cell present in a 100-ml potable water sample. Coupled with an E. coli / Shigella -specific rtPCR assay, the entire molecular procedure is comparable to U.S. EPA Method 1604 on MI agar in terms of analytical specificity and detection limit but provides significant advantages in terms of speed and ubiquity.