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

Authors: MIRE, MARGO - LINCOLN UNIVERSITY OF MISSOURI; KIM, CHYER - VIRGINIA STATE UNIVERSITY; Baffaut, Claire; LIU, FENGJING - MICHIGAN TECHNOLOGICAL UNIVERSITY; WULIJI, TUMEN - LINCOLN UNIVERSITY OF MISSOURI; ZHENG, GUOLU - LINCOLN UNIVERSITY OF MISSOURI

Submitted to: Science of the Total Environment
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2022
Publication Date: 7/30/2022
Citation: Mire, M., Kim, C., Baffaut, C., Liu, F., Wuliji, T., Zheng, G. 2022. Escherichia cryptic clade II through clade VIII: Rapid detection and prevalence in feces and surface water. Science of the Total Environment. 848. Article 157741. https://doi.org/10.1016/j.scitotenv.2022.157741.
DOI: https://doi.org/10.1016/j.scitotenv.2022.157741

Interpretive Summary: Escherichia coli (E. coli) bacteria are a common fecal indicator used to manage water quality. Recent studies have identified groups of organisms (clades) that derive from E. coli but are genetically distinguishable. However, current standard methods of enumerating E. coli are unable to differentiate these clades from the original E. coli. For that reason, they were given the name of cryptic E. coli. In the past decade, eight clades of cryptic E. coli, namely clade I through clade VIII (C-I through C-VIII), have been reported. Recent studies suggest that cryptic E. coli may be living in water environments, except for clade I. Unfortunately, the existence of cryptic E. coli in aquatic environment may cause false alarms of fecal pollution because environmental-living bacteria do not indicate the presence of harmful fecal material. This study was performed to develop a rapid method for the detection of cryptic E. coli and to investigate their potential impact on water quality monitoring. One gene named cec (cryptic E. coli) was identified to be unique to C-II through C-VIII and a method was developed by targeting this gene. This method provides 100% sensitivity and 100% specificity in detecting C-II through C-VIII among strains of known cryptic E. coli. Prevalence of cryptic E. coli among E. coli isolates obtained from the fecal samples of 13 different host species was 0.6 %. Prevalence of cryptic E. coli among isolates from surface water samples taken at four locations of a watershed in mid-Missouri was 1.6%. In addition to the low prevalence, clades C-II through C-VIII in stream water samples were found at a single sampling site, but not at a nearby downstream site. The results suggest that the cryptic E. coli identified at that site may not be environmentally living but from hosts that have not been well studied. If confirmed at other sites, this study indicates that regulators and water managers may not need to account for cryptic E. coli in their water quality assessment.

Technical Abstract: Bacteria of the cryptic lineage of genus Escherichia, cryptic E. coli, are phenotypically distinguishable from E. coli using standard biochemical tests. Except for clade I (C-I), cryptic E. coli are commonly believed to be environmentally living. If so, they would hinder the interpretation of current E. coli-based water quality (fecal pollution) monitoring in the United States (US) because environmental-living bacteria do not indicate the presence of harmful fecal material. This study was performed to develop a rapid method for the detection of cryptic E. coli and to investigate their potential impact on water quality monitoring. By whole-genome comparison, one gene named cec (cryptic E. coli), was identified to be unique to C-II through C-VIII. An end-point polymerase chain reaction (PCR) method, cecPCR, was developed by targeting the cec. The results of in-silico and wet tests demonstrated 100% sensitivity and specificity for cecPCR to detect C-II through C-VIII. Based on the EPA Method 1603, 596 presumptive E. coli isolates were obtained from the fecal samples of 13 different host species and 196 isolates from surface water samples taken at four locations of a watershed in mid-Missouri. As indicated by the cecPCR 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. Furthermore, clades C-II through C-VIII in stream water samples were found at a single sampling site, but not five kilometers downstream of the site. The data suggests that the cryptic E. coli may not be environmentally living but from hosts that have not been well studied.