Both Handwashing and an Alcohol-Based Hand Sanitizer Intervention Reduce Soil and Microbial Contamination on Farmworker Hands during Harvest, but Produce Type Matters.

Hand hygiene interventions are critical for reducing farmworker hand contamination and preventing the spread of produce-associated illness. Hand hygiene effectiveness may be produce-commodity specific, which could influence implementation strategies. This study's goal was to determine if produce commodity influences the ability of handwashing with soap and water or two-step alcohol-based hand sanitizer (ABHS) interventions to reduce soil and bacteria on farmworker hands. Farmworkers (n = 326) harvested produce (cantaloupe, jalapeño, and tomato) for 30 to 90 minutes before engaging in handwashing, two-step ABHS (jalapeño and cantaloupe), or no hand hygiene. Hands were rinsed to measure amounts of soil (absorbance at 600 nm) and indicator bacteria (coliforms, Enterococcus sp., generic Escherichia coli, and Bacteroidales universal [AllBac] and human-specific [BFD] 16S rRNA gene markers). Without hand hygiene, bacterial concentrations (0.88 to 5.1 log10 CFU/hand) on hands significantly differed by the produce commodity harvested. Moderate significant correlations (ρ = -0.41 to 0.56) between soil load and bacterial concentrations were observed. There were significant produce-commodity-specific differences in the ability of handwashing and two-step ABHS interventions to reduce soil (P < 0.0001), coliforms (P = 0.002), and Enterococcus sp. (P = 0.003), but not the Bacteroidales markers AllBac (P = 0.4) or BFD (P = 0.3). Contamination on hands of farmworkers who harvested cantaloupe was more difficult to remove. Overall, we found that a two-step ABHS intervention was similar to handwashing with soap and water at reducing bacteria on farmworker hands. In summary, produce commodity type should be considered when developing hand hygiene interventions on farms.IMPORTANCE This study demonstrated that the type of produce commodity handled influences the ability of handwashing with soap and water or a two-step alcohol-based hand sanitizer (ABHS) intervention to reduce soil and bacterial hand contamination. Handwashing with soap and water, as recommended by the FDA's Produce Safety Rule, when tested in three agricultural environments, does not always reduce bacterial loads. Consistent with past results, we found that the two-step ABHS method performed similarly to handwashing with soap and water but also does not always reduce bacterial loads in these contexts. Given the ease of use of the two-step ABHS method, which may increase compliance, the two-step ABHS method should be further evaluated and possibly considered for implementation in the agricultural environment. Taken together, these results provide important information on hand hygiene effectiveness in three agricultural contexts.

Hepatitis E virus and coliphages in waters proximal to swine concentrated animal feeding operations.

North Carolina is the second leading state in pork production in the United States, with over 10 million swine. Swine manure in NC is typically collected and stored in open-pit lagoons before the liquid waste is sprayed onto agricultural fields for disposal. Components of this waste may be able to impact surface water quality with the potential for human exposure. This study examined viruses of public health concern in creeks adjacent to swine concentrated animal feeding operation (CAFO) spray fields. Surface water samples (n=154) were collected from public access waters in proximity to swine CAFO spray fields for six months and were tested for hepatitis E virus (HEV) and coliphages. HEV was detected in one sample. Somatic coliphages were detected in 98% of samples (geometric mean 24 ± 4.1 PFU per 100 ml), and F+ coliphages were detected in 85% of samples (geometric mean 6.8 ± 5.0 PFU per 100 ml). Only 3% (21) of the F+ coliphage isolates were RNA phage, and all of the F+ RNA coliphages belonged to genogroup I. Although the pervasiveness of swine CAFOs in this area prevented a comparison with samples from un-impacted sites, the near ubiquity of coliphages, as well as the presence of HEV, suggests that current waste management practices may be associated with the dissemination of viruses of public health concern in waters proximal to CAFO spray fields.

Persistence of human norovirus RT-qPCR signals in simulated gastric fluid.

Human noroviruses (HuNoV) are a leading cause of foodborne disease and are known to be environmentally persistent. Foods usually become contaminated by contact with fecal material, both on hands and on surfaces. Emerging evidence suggests that HuNoVs are also shed and potentially aerosolized during projectile vomiting, resulting in another source of contamination. The purpose of this study was to compare the persistence of HuNoV in vomitus-like material (simulated gastric fluid, SGF, pH 2.5) to that in a pH neutral buffer (phosphate buffered saline, PBS, pH 7.4) in suspension and on surfaces. Human fecal suspensions containing two HuNoV strains (GI.1 and GII.4) were suspended in SGF and PBS. Suspension and surface samples were held at room temperature, and subsamples were collected from both samples for a period up to 42 days. Subsamples were subjected to RNA isolation, with and without inclusion of an RNase pre-treatment, followed by RT-qPCR amplification. In suspension assays, the genome copy number of HuNoV GII.4 decreased by ≤1.0-1.3 log10 over 42 days, irrespective of suspension buffer. On stainless steel, there was virtually no reduction in HuNoV GII.4 RT-qPCR signal over the 42-days experimental period, regardless of suspension buffer. Overall, the GI.1 RT-qPCR signal dropped more precipitously. In most cases, there were no statistically significant differences (p > 0.05) between persistence in solution or on surfaces when comparing RT-qPCR assays with and without prior RNase treatment. This study suggests that HuNoV suspended in vomitus-like material can persist for long periods, a likely contributor to foodborne transmission.

Human health and the water environment: using the DPSEEA framework to identify the driving forces of disease.

There is a growing awareness of global forces that threaten human health via the water environment. A better understanding of the dynamic between human health and the water environment would enable prediction of the significant driving forces and effective strategies for coping with or preventing them. This report details the use of the Driving Force-Pressure-State-Exposure-Effect-Action (DPSEEA) framework to explore the linkage between water-related diseases and their significant driving forces. The DPSEEA frameworks indicate that a select group of driving forces, including population growth, agriculture, infrastructure (dams and irrigation), and climate change, is at the root cause of key global disease burdens. Construction of the DPSEEA frameworks also allows for the evaluation of public health interventions. Sanitation was found to be a widely applicable and effective intervention, targeting the driver/pressure linkage of most of the water-related diseases examined. Ultimately, the DPSEEA frameworks offer a platform for constituents in both the health and environmental fields to collaborate and commit to a common goal targeting the same driving forces.

Use of Bacteroidales microbial source tracking to monitor fecal contamination in fresh produce production.

In recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne 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.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

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.

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study.

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.

Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation-emission matrix spectroscopy and PARAFAC.

Assays that utilize PCR offer powerful tools to detect pathogens and other microorganisms in environmental samples. However, PCR inhibitors present in nucleic acid extractions can increase a sample's limit of detection, skew calculated marker concentrations, or cause false-negative results. It would be advantageous to predict which samples contain various types and levels of PCR inhibitors, especially the humic and fulvic acids that are frequently cited as PCR inhibitors in natural water samples. This study investigated the relationships between quantitative PCR (qPCR) inhibition and the humic and fulvic content of dissolved organic matter (DOM), as well as several other measures of DOM quantity and quality, in water samples. QPCR inhibition was also compared to water quality parameters, precipitation levels, and land use adjacent to the sampling location. Results indicate that qPCR inhibition in the tested water samples was correlated to several humic substance-like, DOM components, most notably terrestrially-derived, humic-like DOM and microbially-derived, fulvic-like DOM. No correlation was found between qPCR inhibition and water quality parameters or land use, but a relationship was noted between inhibition and antecedent rainfall. This study suggests that certain fractions of humic substances are responsible for PCR inhibition from temperate, freshwater systems. PARAFAC modeling of excitation-emission matrix spectroscopy provides insight on the components of the DOM pool that impact qPCR success and may be useful in evaluating methods to remove PCR inhibitors present in samples.

Similar concentration and extraction recoveries allow for use of turnip crinkle virus as a process control for enteroviruses in water.

Enteric viruses are etiological agents of waterborne disease that may be detected using molecular techniques such as PCR. However, processing water samples in preparation for PCR typically involves concentration of samples and extraction of nucleic acids, steps that have low and variable recovery efficiencies. This study evaluated a plant virus, turnip crinkle virus (TCV), for its ability to serve as a process control for human enteroviruses during concentration and extraction procedures. Enteroviruses and TCV have similar sizes and morphologies, and both contain single stranded, positive-sense RNA genomes. Results from the study demonstrate that the tested viruses experience similar losses during sample processing. Virus recoveries averaged 0.03% for EV and 0.02% for TCV from DI water, and 0.004% for EV and 0.009% for TCV from a creek sample. Surface water and wastewater samples from around the U.S. were evaluated for the presence of TCV to ensure the virus is not present in environmental samples. All were negative. With similar recovery efficiencies to EV, TCV may be a suitable process control for enteroviruses in environmental water samples in the U.S. Use of process controls as proposed in this study would allow better detection and quantitation methods to be employed in water quality monitoring.

HuBac and nifH source tracking markers display a relationship to land use but not rainfall.

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.