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.

Contamination of Fresh Produce by Microbial Indicators on Farms and in Packing Facilities: Elucidation of Environmental Routes.

To improve food safety on farms, it is critical to quantify the impact of environmental microbial contamination sources on fresh produce. However, studies are hampered by difficulties achieving study designs with powered sample sizes to elucidate relationships between environmental and produce contamination. Our goal was to quantify, in the agricultural production environment, the relationship between microbial contamination on hands, soil, and water and contamination on fresh produce. In 11 farms and packing facilities in northern Mexico, we applied a matched study design: composite samples (n = 636, equivalent to 11,046 units) of produce rinses were matched to water, soil, and worker hand rinses during two growing seasons. Microbial indicators (coliforms, Escherichia coli, Enterococcus spp., and somatic coliphage) were quantified from composite samples. Statistical measures of association and correlations were calculated through Spearman's correlation, linear regression, and logistic regression models. The concentrations of all microbial indicators were positively correlated between produce and hands (ρ range, 0.41 to 0.75; P < 0.01). When E. coli was present on hands, the handled produce was nine times more likely to contain E. coli (P < 0.05). Similarly, when coliphage was present on hands, the handled produce was eight times more likely to contain coliphage (P < 0.05). There were relatively low concentrations of indicators in soil and water samples, and a few sporadic significant associations were observed between contamination of soil and water and contamination of produce. This methodology provides a foundation for future field studies, and results highlight the need for interventions surrounding farmworker hygiene and sanitation to reduce microbial contamination of farmworkers' hands.IMPORTANCE This study of the relationships between microbes on produce and in the farm environment can be used to support the design of targeted interventions to prevent or reduce microbial contamination of fresh produce with associated reductions in foodborne illness.

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.

Elucidating the role of the phenylacetic acid metabolic complex in the pathogenic activity of Rhizoctonia solani anastomosis group 3.

The soil fungus Rhizoctonia solani produces phytotoxic phenylacetic acid (PAA) and hydroxy (OH-) and methoxy (MeO-) derivatives of PAA. However, limited information is available on the specific role that these compounds play in the development of Rhizoctonia disease symptoms and concentration(s) required to induce a host response. Reports that PAA inhibits the growth of R. solani conflict with the established ability of the fungus to produce and metabolize PAA. Experiments were conducted to clarify the role of the PAA metabolic complex in Rhizoctonia disease. In this study the concentration of PAA and derivatives required to induce tomato root necrosis and stem canker, in the absence of the fungus, and the concentration that inhibits mycelial growth of R. solani were determined. The effect of exogenous PAA and derivatives of PAA on tomato seedling growth also was investigated. Growth of tomato seedlings in medium containing 0.1-7.5 mM PAA and derivatives induced necrosis of up to 85% of root system. Canker development resulted from injection of tomato seedling stems with 7.5 mM PAA, 3-OH-PAA, or 3-MeO-PAA. PAA in the growth medium reduced R. solani biomass, with 50% reduction observed at 7.5 mM. PAA, and derivatives were quantified from the culture medium of 14 isolates of R. solani belonging to three distinct anastomosis groups by GC-MS. The quantities ranged from below the limit of detection to 678 nM, below the concentrations experimentally determined to be phytotoxic. Correlation analyses revealed that isolates of R. solani that produced high PAA and derivatives in vitro also caused high mortality on tomato seedlings. The results of this investigation add to the body of evidence that the PAA metabolic complex is involved in Rhizoctonia disease development but do not indicate that production of these compounds is the primary or the only determinant of pathogenicity.

An In Planta Method for Assessing the Role of Basidiospores in Rhizoctonia Foliar Disease of Tomato.

A tomato (Solanum lycopersicum) foliar blight disease of unknown etiology was observed in North Carolina (NC) during 2005 to 2006. Symptoms included necrotic lesions and blighted leaves, with signs of white mycelial growth on abaxial leaf surfaces. The morphology of isolates from symptomatic leaves was consistent with that of Rhizoctonia solani. Because the pattern of symptom expression suggested that basidiospores were the primary inoculum source, Koch's postulates were fulfilled using a method to generate basidiospores in planta. Isolates were characterized by morphology, DNA sequence analysis, hyphal anastomosis, and somatic hyphal interactions. Phylogenetic analyses and hyphal anastomosis criteria support placement of the isolates in R. solani anastomosis group 3 (AG-3). Tomato foliar blight isolates from NC form a single phylogenetic group with tomato isolates of R. solani AG-3 from Japan and are more closely related to R. solani AG-3 isolates from potato than tobacco. Isolates exhibited both compatible and incompatible hyphal interactions when paired in vitro. To our knowledge, this is the first detailed report of tomato foliar blight caused by R. solani AG-3 in North America. A comprehensive description of the technique employed for producing basidiospores is presented with potential utility for understanding foliar disease etiology in other Rhizoctonia pathosystems.

Low Prevalence of Human Pathogens on Fresh Produce on Farms and in Packing Facilities: A Systematic Review.

Foodborne illness burdens individuals around the world and may be caused by consuming fresh produce contaminated with bacterial, parasite, and viral pathogens. Pathogen contamination on produce may originate at the farm and packing facility. This research aimed to determine the prevalence of human pathogens (bacteria, parasites, and viruses) on fresh produce (fruits, herbs, and vegetables) on farms and in packing facilities worldwide through a systematic review of 38 peer-reviewed articles. The median and range of the prevalence was calculated, and Kruskal-Wallis tests and logistic regression were performed to compare prevalence among pooled samples of produce groups, pathogen types, and sampling locations. Results indicated a low median percentage of fresh produce contaminated with pathogens (0%). Both viruses (p-value = 0.017) and parasites (p-value = 0.033), on fresh produce, exhibited higher prevalence than bacteria. No significant differences between fresh produce types or between farm and packing facility were observed. These results may help to better quantify produce contamination in the production environment and inform strategies to prevent future foodborne illness.

Microbial Load of Fresh Produce and Paired Equipment Surfaces in Packing Facilities Near the U.S. and Mexico Border.

Several produce-associated outbreaks have been linked to the packing facility. Equipment surfaces may be an important source of contamination. The goal was to assess whether the microbial load of packing facility surfaces is associated with the microbial load of produce. From November 2000 to December 2003, 487 matched produce (14 types) and equipment surfaces (six production steps) were sampled from eight packing facilities in the United States near the border with Mexico and enumerated for aerobic plate counts (APC), Escherichia coli , Enterococcus, and coliforms. Bivariate correlations were assessed by Spearman's ρ, and adjusted associations were assessed by multilevel mixed linear regression models. In general, the microbial load both increased and decreased on produce (0.2 to 1.0 log CFU/g) and equipment surfaces (0.5 to 3.0 log CFU/cm2) across production steps. Equipment surface and produce microbial loads were correlated, but correlations varied from none to high depending on the equipment surface. For example, significant correlations (P < 0.01) included APC (ρ = 0.386) and Enterococcus (ρ = 0.562) with the harvest bin, E. coli (ρ = 0.372) and Enterococcus (ρ = 0.355) with the merry-go-round, Enterococcus (ρ = 0.679) with rinse cycle equipment, APC (ρ = 0.542) with the conveyer belt, and for all indicators with the packing box (ρ = 0.310 to 0.657). After controlling for crop type, sample replicate group, and sample location, there were significant positive associations between the log concentration of Enterococcus on produce and the harvest bin (ß = 0.259, P < 0.01) and the rinse cycle (ß = 0.010, P = 0.01), and between the log concentration of all indicators on produce and the packing box (ß = 0.155 to 0.500, all P < 0.01). These statistically significant associations between microbial loads on packing facility surfaces and fresh produce confirm the importance of packing facility sanitation to protect produce quality and safety.

Somatic Coliphage Profiles of Produce and Environmental Samples from Farms in Northern México.

Somatic coliphages were quantified in 459 produce and environmental samples from 11 farms in Northern Mexico to compare amounts of somatic coliphages among different types of fresh produce and environmental samples across the production steps on farms. Rinsates from cantaloupe melons, jalapeño peppers, tomatoes, and the hands of workers, soil, and water were collected during 2011-2012 at four successive steps on each farm, from the field before harvest through the packing facility, and assayed by FastPhage MPN Quanti-tray method. Cantaloupe farm samples contained more coliphages than jalapeño or tomato (p range <0.01-0.03). Across production steps, jalapeños had higher coliphage percentages before harvest than during packing (p = 0.03), while tomatoes had higher coliphage concentrations at packing than all preceding production steps (p range <0.01-0.02). These findings support the use of targeted produce-specific interventions at multiple points in the process of growing and packing produce to reduce the risk of enteric virus contamination and improve food safety during fruit and vegetable production.

Microbial Indicator Profiling of Fresh Produce and Environmental Samples from Farms and Packing Facilities in Northern Mexico.

To compare microbiological indicator and pathogen contamination among different types of fresh produce and environmental samples along the production chain, 636 samples of produce (rinsates from cantaloupe melons, jalapeño peppers, and tomatoes) and environmental samples (rinsates from hands of workers, soil, and water) were collected at four successive steps in the production process (from the field before harvest through the packing facility) on 11 farms in northern Mexico during 2011 and 2012. Samples were assayed for enteric pathogens (Escherichia coli O157:H7, other Shiga toxigenic E. coli, Salmonella, and Listeria monocytogenes) and microbial indicators (coliforms, other E. coli strains, and Enterococcus spp.). Salmonella was the only pathogen detected; it was found in one preharvest jalapeño sample (detection limits: 0.0033 CFU/ml in produce and hand samples, 0.0013 CFU/ml in water, and 0.04 CFU/g in soil). Microbial indicator profiles for produce, worker hands, and soil from jalapeño and tomato farms were similar, but cantaloupe farm samples had higher indicator levels (P < 0.05 for all comparisons) on fruit (6.5, 2.8, and 7.2 log CFU per fruit) and hands (6.6, 3.1, and 7.1 log CFU per hand) for coliforms, E. coli, and Enterococcus, respectively, and lower E. coli levels in soil (<1 CFU/g). In water from tomato farms, E. coli indicators were significantly more prevalent (70 to 89% of samples were positive; P = 0.01 to 0.02), and geometric mean levels were higher (0.3 to 0.6 log CFU/100 ml) than those in cantaloupe farm water (32 to 38% of samples were positive, geometric mean <1 CFU/100 ml). Microbial indicators were present during all production steps, but prevalence and levels were generally highest at the final on-farm production step (the packing facility) (P < 0.03 for significant comparisons). The finding that microbial contamination on produce farms is influenced by produce type and production step can inform the design of effective approaches to mitigate microbial contamination.

Validation of a novel rinse and filtration method for efficient processing of fresh produce samples for microbiological indicator enumeration.

Several methods have been described to prepare fresh produce samples for microbiological analysis, each with its own advantages and disadvantages. The aim of this study was to compare the performance of a novel combined rinse and membrane filtration method to two alternative sample preparation methods for the quantification of indicator microorganisms from fresh produce. Decontaminated cantaloupe melons and jalapeño peppers were surface inoculated with a cocktail containing 10(6) CFU/ml Escherichia coli, Salmonella Typhimurium, and Enterococcus faecalis. Samples were processed using a rinse and filtration method, homogenization by stomacher, or a sponge-rubbing method, followed by quantification of bacterial load using culture methods. Recovery efficiencies of the three methods were compared. On inoculated cantaloupes, the rinse and filtration method had higher recovery of coliforms (0.95 log CFU/ml higher recovery, P = 0.0193) than the sponge-rubbing method. Similarly, on inoculated jalapeños, the rinse and filtration method had higher recovery for coliforms (0.84 log CFU/ml higher, P = 0.0130) and E. coli (1.46 log CFU/ml higher, P < 0.0001) than the sponge-rubbing method. For jalapeños, the rinse and filtration method outperformed the homogenization method for all three indicators (0.79 to 1.71 log CFU/ml higher, P values ranging from 0.0075 to 0.0002). The precision of the three methods was also compared. The precision of the rinse and filtration method was similar to that of the other methods for recovery of two of three indicators from cantaloupe (E. coli P = 0.7685, E. faecalis P = 0.1545) and was more precise for recovery of two of three indicators from jalapeño (coliforms P = 0.0026, E. coli P = 0.0243). Overall, the rinse and filtration method performed equivalent to, and sometimes better than, either of the compared methods. The rinse and filtration method may have logistical advantages when processing large numbers of samples, improving sampling efficiency and facilitating microbial detection.

Ability of Hand Hygiene Interventions Using Alcohol-Based Hand Sanitizers and Soap To Reduce Microbial Load on Farmworker Hands Soiled during Harvest.

Effective hand hygiene is essential to prevent the spread of pathogens on produce farms and reduce foodborne illness. The U.S. Food and Drug Administration Food Safety Modernization Act Proposed Rule for Produce Safety recommends the use of soap and running water for hand hygiene of produce handlers. The use of alcohol-based hand sanitizer (ABHS) may be an effective alternative hygiene intervention where access to water is limited. There are no published data on the efficacy of either soap or ABHS-based interventions to reduce microbial contamination in agricultural settings. The goal of this study was to assess the ability of two soap-based (traditional or pumice) and two ABHS-based (label-use or two-step) hygiene interventions to reduce microbes (coliforms, Escherichia coli, and Enterococcus spp.) and soil (absorbance of hand rinsate at 600 nm [A600]) on farmworker hands after harvesting produce, compared with the results for a no-hand-hygiene control. With no hand hygiene, farmworker hands were soiled (median A600, 0.48) and had high concentrations of coliforms (geometric mean, 3.4 log CFU per hand) and Enterococcus spp. (geometric mean, 5.3 log CFU per hand) after 1 to 2 h of harvesting tomatoes. Differences in microbial loads in comparison to the loads in the control group varied by indicator organism and hygiene intervention (0 to 2.3 log CFU per hand). All interventions yielded lower concentrations of Enterococcus spp. and E. coli (P < 0.05), but not of coliforms, than were found in the control group. The two-step ABHS intervention led to significantly lower concentrations of coliforms and Enterococcus spp. than the pumice soap and label-use ABHS interventions (P < 0.05) and was the only intervention to yield significantly fewer samples with E. coli than were found in the control group (P < 0.05). All interventions removed soil from hands (P < 0.05), soap-based interventions more so than ABHS-based interventions (P < 0.05). ABHS-based interventions were equally as effective as hand washing with soap at reducing indicator organisms on farmworker hands. Based on these results, ABHS is an efficacious hand hygiene solution for produce handlers, even on soiled hands.

Modulation of the phenylacetic acid metabolic complex by quinic acid alters the disease-causing activity of Rhizoctonia solani on tomato.

The metabolic control of plant growth regulator production by the plant pathogenic fungus Rhizoctonia solani Kühn (teleomorph=Thanatephorus cucumeris (A.B. Frank) Donk) and consequences associated with the parasitic and saprobic activity of the fungus were investigated. Fourteen genetically distinct isolates of the fungus belonging to anastomosis groups (AG) AG-3, AG-4, and AG-1-IA were grown on Vogel's minimal medium N with and without the addition of a 25 mM quinic acid (QA) source of carbon. The effect of QA on fungal biomass was determined by measuring the dry wt of mycelia produced under each growth condition. QA stimulated growth of 13 of 14 isolates of R. solani examined. The production of phenylacetic acid (PAA) and the chemically related derivatives 2-hydroxy-PAA, 3-hydroxy-PAA, 4-hydroxy-PAA, and 3-methoxy-PAA on the two different media was compared by gas chromatography coupled with mass spectrometry (GC-MS). The presence of QA in the growth medium of R. solani altered the PAA production profile, limiting the conversion of PAA to derivative forms. The effect of QA on the ability of R. solani to cause disease was examined by inoculating tomato (Solanum lycopersicum L.) plants with 11 isolates of R. solani AG-3 grown on media with and without the addition of 25 mM QA. Mean percent survival of tomato plants inoculated with R. solani was significantly higher when the fungal inoculum was generated on growth medium containing QA. The results of this study support the hypotheses that utilization of QA by R. solani leads to reduced production of the plant growth regulators belonging to the PAA metabolic complex which can suppress plant disease development.