Escherichia cryptic clade II through clade VIII: Rapid detection and prevalence in feces and surface water

Bacteria of the cryptic lineage of genus Escherichia, or Escherichia cryptic clades (cryptic clades), are phenotypically indistinguishable from Escherichia coli (E. coli) using standard biochemical tests. Except for clade I (C-I), cryptic clades were hypothetically believed to be environmental but not enteric. If so, they would hinder the interpretation of current E. coli-based water quality (fecal pollution) monitoring in the United States because environmental bacteria do not indicate the presence of harmful fecal material. This study was performed to develop a rapid method for the detection of cryptic clades and to investigate their potential impact on water quality monitoring. By whole-genome comparison, one gene, named ecc (Escherichia cryptic clades), was identified to be unique to C-II through C-VIII. An end-point polymerase chain reaction (PCR) method, eccPCR, was developed by targeting the ecc. The results of in-silico and wet tests demonstrated 100 % sensitivity and specificity of the eccPCR to detect C-II through C-VIII. Based on the EPA Method 1603, 519 presumptive E. coli isolates were obtained from the fecal samples of 13 different host species and 192 isolates from surface water samples taken at four locations in a watershed of mid-Missouri. As indicated by the eccPCR amplification, the overall prevalence of C-II through C-VIII in the presumptive E. coli isolates was estimated to be about 0.6 % in the fecal samples and about 1.6 % in the water samples. Therefore, the potential impact of cryptic clades on water quality monitoring may be limited if EPA Method 1603 is used. Furthermore, clades C-II through C-VIII in stream water samples were found repeatedly only at a single sampling site, but neither at the upstream sites nor five kilometers downstream of the site. The data do not support nor reject the environmental hypothesis about cryptic clades. Further study is needed to determine the implication of the observation.