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 (Escherichiacryptic 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.

A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform.

Differentiating enteric Escherichia coli from environmental bacteria through the putative glucosyltransferase gene (ycjM).

This study is to tackle the challenge posed by the "naturalized" Escherichia coli population against the worldwide practice of E. coli-based water quality monitoring. In the literature, the putative glucosyltransferase gene (ycjM) of E. coli has been identified in silico to be one of the 114 genes specific to enteric E. coli. Based on the sequence of E. coli K-12 MG1655, a PCR assay (ycjPCR) targeting ycjM was developed in this study. As demonstrated by the ycjPCR assay using 367 E. coli strains isolated from animal feces, 97.2% of the isolates carried the ycjM with variations from 93.9% to 100% among nine different host sources, but none of the 17 strains of non-E. coli bacteria and only 23.0% of the environment-isolated cryptic Escherichia strains contained the ycjM. These data experimentally confirmed ycjM to be enteric specific. Our study also showed that the ycjPCR assay was superior to the commonly used tuf- or uidA-based PCR methods in differentiating enteric E. coli from ß-D-glucuronidase-positive environmental bacteria. Furthermore, study on 190 E. coli isolates from water samples, using EPA Method 1603 followed by bacterial identification with Biolog MicroStation™ and ycjPCR assay, indicated that the prevalence of ycjM in the E. coli water isolates had a significant (p < 0.05, odds ratio ) spatial variation from 69.6% to 93.8%. These data suggest that E. coli profile using EPA Method 1603 or other ß-D-glucuronidase-activity-based methods may need further analysis using the ycjM profile to accurately determinate fecal pollution in water.