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In recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of foodborne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based on Bacteroidales 16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universal Bacteroidales marker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10 genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10 GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10 GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation between Bacteroidales and generic Escherichia coli across all samples. This study provides evidence that Bacteroidales markers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host. [ABSTRACT FROM AUTHOR]

Abstract: Identification of the source of fecal pollution is becoming a priority for states and territories in the U.S. in order to meet water quality standards and to develop and implement total maximum daily loads. The goal of this research was to relate microbial source tracking (MST) assay concentrations to land use and levels of impervious surfaces in order to gauge how increasing development is associated with human fecal contamination in inland watersheds. The concentrations of two proposed MST markers, targeting nifH of Methanobrevibacter smithii and HuBac of Bacteroides sp., were positively correlated with increasing anthropogenic development and impervious surfaces. Higher concentrations of these MST markers in more urbanized watersheds suggest that increasing development negatively affects water quality. Neither MST marker concentration was correlated with antecedent rainfall levels, and detection of markers did not differ between dry weather and rain events. Water samples were also analyzed for norovirus and enterovirus, but these enteric viruses were rarely detected. These MST results differ from previous studies that have found correlations between traditional fecal indicator bacteria (FIB) and antecedent rainfall. This difference suggests that the MST markers used in this study may be more specific for recent, land-based contamination events as opposed to resuspension of particle-associated organisms in waterways. HuBac was detected in 98% of samples, correlating with fecal coliform and Escherichia coli concentrations. The ubiquity of the HuBac marker in our samples suggests that this marker does not provide sufficiently different or additional information than FIB, and it is likely this marker was amplifying non-human targets. The nifH marker was detected in 30% of samples. Less than half of the nifH-positive samples contained levels of fecal coliforms or E. coli above regulatory thresholds, suggesting that nifH would be more useful when utilized simultaneously with FIB than in a tiered monitoring strategy. The results of this research suggests that land use factors play an important role in characterizing and mitigating fecal contamination in watersheds. [Copyright &y& Elsevier]

Abstract: An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10–50 gene copies or plaques × 50 ml−1) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40–70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent. [Copyright &y& Elsevier]

Abstract: A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study. [Copyright &y& Elsevier]