Bacteroidales as Indicators and Source Trackers of Fecal Contamination in Tomatoes and Strawberries

Most methods that investigate fecal contamination of vegetables do not address the origin of contamination. Because host-specific sequences are conserved in their genomes, bacteria of the order Bacteroidales are regarded as alternative indicators for tracking sources of contamination of produce. The objective of this study was to determine the efficacy of host-specific Bacteroidales markers to identify sources of fecal contamination and to determine whether detection of Bacteroidales markers correlated with traditional fecal indicator bacteria (FIB) in strawberries and tomatoes. Tomato and strawberry samples were artificially contaminated with human and animal feces, which contained 6 to 7 log CFU Bacteroidales per 100 mL and 3 to 6 log CFU/100 mL of the bacterial indicators Escherichia coli, total coliforms, and Enterococcus. FIB were enumerated by standard procedures. Universal and host-specific Bacteroidales markers were detected and quantified by quantitative PCR, and the detection range was 1.35 to 10.35 logarithmic gene copies, which corresponds to a limit of detection of two Bacteroidales cells. Few correlations between levels of Bacteroidales and levels of FIB were observed. For most of the contaminated tomato and strawberry samples, Bacteroidales levels were higher than FIB levels, and detection of FIB was highly variable. Detection of Bacteroidales markers was similar to total coliforms when ≥0.1 mg of feces was inoculated. These indicators were better than E. coli and Enterococcus for detection of fecal contamination in produce. The host-associated Bacteroidales markers were detected at an inoculum of 1 mg of feces per produce item (except those from bovine feces in strawberry). All of the host-associated Bacteroidales markers were detected at an inoculum of 10 mg of feces per produce item. Thus, Bacteroidales markers are promising tools to identify sources of fecal contamination; however, more research is required for their potential use to reduce the risks of contamination of produce.