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Environmental Risk Factors in the Human Pathogen Transmission Pathways between Animal Operations and Produce Crops
Abstract
Once zoonotic pathogens leave their animal hosts, how they move through the environment and are deposited on unharvested produce remains a persistent industry challenge and research question related to produce safety. The proximity of animals to production areas, animal types and densities, an animal operation’s management practices, and weather conditions are some of the areas explored by researchers to better understand how pathogens contaminate unharvested crops. Water, inputs, airborne particulates, wildlife, and insects may serve as vectors linking pathogens from their animal hosts to produce production areas. Studies have shown a positive correlation between rainfall and pathogen concentrations in agricultural water downstream from animal operations. Bacteria attached to airborne particulates can be deposited onto crops or open water sources. Wildlife and insects share habitat with domesticated animals in rangelands, pasture settings, pens, and feedlots. Plant conditions (injuries, disease) and characteristics (surface topography, genetic traits, age, native microbiota) and environmental conditions (relative humidity, moisture, temperature) play a major role in determining pathogen survival on unharvested produce. This article explores recent research findings elucidating human pathogen dispersion and deposition, subsequent transfer from animals to crops, and the various environmental risk factors along the way that play a role.
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Leafy greens contaminated with Shiga toxin–producing Escherichia coli have continued to cause foodborne illness outbreaks in recent years and present a threat to public health. An important component of foodborne illness outbreak investigations is determining the source of the outbreak vehicle through traceback investigations. The U.S. Food and Drug Administration is home to traceback investigation experts who use a standardized process to initiate, execute, and interpret the results of traceback investigations in collaboration with the Centers for Disease Control and Prevention and state and local partners. Traceback investigations of three outbreaks of Shiga toxin–producing E. coli infections linked to romaine lettuce in 2018 and 2019 were examined to demonstrate challenges, limitations, and opportunities for improvement. The three outbreaks resulted in a total of 474 illnesses, 215 hospitalizations, and 5 deaths. These illnesses were linked to the consumption of romaine lettuce from three distinct growing regions in Arizona and California. Some of the challenges encountered included the time it took to initiate a traceback, limited product-identifying information throughout the supply chain, lack of interoperability in record-keeping systems, and comingling of product from multiple suppliers. These challenges led to time delays in the identification of the farm source of the leafy greens and the inability to identify the root cause of contamination. Implementation of technology-enabled traceability systems, testing of these systems, and future regulations to incentivize adoption of traceability systems are some of the initiatives that will help address these challenges by improving traceback investigations and ultimately preventing foodborne illnesses and future outbreaks from occurring.
HIGHLIGHTS
This study examined the biological and food safety relevance of leaf lesions for potential invasion of food pathogens into the plant tissue (internalization). This was done by determining the role of artificial leaf damage in terms of damaged leaf area on proliferation of E. coli O157:H7 gfp+. In a two-factorial experiment, unwashed fresh baby leaf spinach (Spinacia oleracea L.) was subjected to four damage levels (undamaged, low, moderate, high damage; factor 1) and three incubation intervals (0, 1, 2 days post-inoculation; factor 2). Individual leaves were immersed for 15 s in a suspension loaded with E. coli O157:H7 gfp+ (10⁶ CFU × mL–1). The leaves were analyzed individually using image analysis tools to quantify leaf area and number and size of lesions, and using confocal laser scanning and scanning electron microscopy to visualize leaf lesions and presence of the introduced E. coli strain on and within the leaf tissue. Prevalence of E. coli O157:H7 gfp+ was assessed using a culture-dependent technique. The results showed that size of individual lesions and damaged leaf area affected depth of invasion into plant tissue, dispersal to adjacent areas, and number of culturable E. coli O157:H7 gfp+ directly after inoculation. Differences in numbers of the inoculant retrieved from leaf macerate evened out from 2 days post-inoculation, indicating rapid proliferation during the first day post-inoculation. Leaf weight was a crucial factor, as lighter spinach leaves (most likely younger leaves) were more prone to harbor E. coli O157:H7 gfp+, irrespective of damage level. At the high inoculum density used, the risk of consumers’ infection was almost 100%, irrespective of incubation duration or damage level. Even macroscopically intact leaves showed a high risk for infection. These results suggest that the risk to consumers is correlated with how early in the food chain the leaves are contaminated, and the degree of leaf damage. These findings should be taken into account in different steps of leafy green processing. Further attention should be paid to the fate of viable, but non-culturable, shiga-toxigenic E. coli on and in ready-to-eat leafy vegetables.
The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.
Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains (syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb3syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.
Fugitive dust is an important source of particulate matters (PM) emission in the air. Vegetation barriers (VBs) can be an effective way to mitigate PM from fugitive dust sources. It is meaningful to choose appropriate plants to establish VBs that can efficiently capture PM from various sources. This study was conducted to establish comparable and repeatable conditions to evaluate the capability of different VB species in mitigating PM emission from certain fugitive dust source. The airflow around two VBs and their PM interception mechanism was studied in a wind tunnel with simulated PM emission source of animal feeding operations. The species used for the two VBs were conifers represented by Pinus Sylvestris var. mongolica Litv. (PS) and the broad-leaved species represented by Syringa Oblate Lindl. (SOL). The results showed that the interception efficiency of the PS vegetation barrier was only slightly lower than that of SOL vegetation, while the PS had a lower effect on the wind speed at the similar leaf surface area. On the other hand, there were a large number of disordered “ridged” stripes on the microscopic structure of the hydrophilic leaves of SOL and PS, and a large amount of fine particles deposited on the leaves were observed, indicating that the microscopic geometric surface structure increased the deposition efficiency of the particles. These findings help to better understand the potential of tree species to reduce PM in environments.
This is the first study to quantify the dependence on wind velocity of airborne bacterial emission fluxes from soil. It demonstrates that manure bacteria get aerosolized from fertilized soil more easily than soil bacteria, and it applies bacterial genomic sequencing for the first time to trace environmental faecal contamination back to its source in the chicken barn. We report quantitative, airborne emission fluxes of bacteria during and following the fertilization of agricultural soil with manure from broiler chickens. During the fertilization process, the concentration of airborne bacteria culturable on blood agar medium increased more than 600 000‐fold, and 1 m³ of air carried 2.9 × 10⁵ viable enterococci, i.e. indicators of faecal contamination which had been undetectable in background air samples. Trajectory modelling suggested that atmospheric residence times and dispersion pathways were dependent on the time of day at which fertilization was performed. Measurements in a wind tunnel indicated that airborne bacterial emission fluxes from freshly fertilized soil under local climatic conditions on average were 100‐fold higher than a previous estimate of average emissions from land. Faecal bacteria collected from soil and dust up to seven weeks after fertilization could be traced to their origins in the poultry barn by genomic sequencing. Comparative analyses of 16S rRNA gene sequences from manure, soil and dust showed that manure bacteria got aerosolized preferably, likely due to their attachment to low‐density manure particles. Our data show that fertilization with manure may cause substantial increases of bacterial emissions from agricultural land. After mechanical incorporation of manure into soil, however, the associated risk of airborne infection is low.
The attachment of foodborne pathogens to leaf surfaces is a complex process that involves multiple physical, chemical, and biological factors. Here, we report the results from a study designed to specifically determine the contribution of spinach leaf surface topography as it relates to leaf axis (abaxial and adaxial) and leaf age (15, 45, and 75 days old) to the ability of Escherichia coli to resist removal by surface wash, to avoid inactivation by chlorine, and to disperse through splash impact. We used fresh spinach leaves, as well as so-called “replicasts” of spinach leaf surfaces in the elastomer polydimethylsiloxane to show that leaf vein density correlated positively with the failure to recover E. coli from surfaces, not only using a simple water wash and rinse, but also a more stringent wash protocol involving a detergent. Such failure was more pronounced when E. coli was surface-incubated at 24°C compared to 4°C, and in the presence, rather than absence, of nutrients. Leaf venation also contributed to the ability of E. coli to survive a 50 ppm available chlorine wash and to laterally disperse by splash impact. Our findings suggest that the topographical properties of the leafy green surface, which vary by leaf age and axis, may need to be taken into consideration when developing prevention or intervention strategies to enhance the microbial safety of leafy greens.
Over a 2-year period, drag swabs of orchard soil surface, and air, soil, and almond leaf samples were collected in an almond orchard adjacent to (35 m from first row of trees) and downwind from a poultry operation, and in two almond orchards (controls) that were surrounded by other orchards. Samples were evaluated for aerobic plate count, generic Escherichia coli , other coliforms, presence of Salmonella , bacterial community analysis through sequencing of the 16s rRNA gene, and amounts of dry solids (dust) on leaf surfaces on trees 0, 60, and 120 m into each orchard. E. coli was isolated from 41 of 206 (20%) and one of 207 (0.48%) air samples in the almond-poultry and control orchards, respectively. Salmonella was not isolated from any of the 529 samples evaluated. On average, the amount of dry solids on leaves collected from trees closest to the poultry operation was more than two-fold greater than from trees 120 m into the orchard or from any of the trees in the control orchards. Members of the family Staphylococcaceae —often associated with poultry—were, on average, significantly ( p < 0.001) more abundant in the phyllosphere of trees closest to the poultry operation (10% of relative abundance) than in trees 120 m into the orchard (1.7% of relative abundance) or from any of the trees in control orchards (0.41% of relative abundance). Poultry-associated microorganisms from a commercial operation transferred a short distance into an adjacent downwind almond orchard.
IMPORTANCE The movement of microorganisms, including foodborne pathogens, from animal operations into adjacent plant crop–growing environments is not well characterized. This study provides evidence that dust and bioaerosols moved from a commercial poultry operation a short distance downwind into an almond orchard and altered the microbiome recovered from the leaves. These data provide growers with information they can use to assess food safety risks on their property.
The log-linear die-off rate proposed by FSMA is not always appropriate, as the die-off rates of foodborne bacterial pathogens and specified agricultural water quality indicator organisms appear to commonly follow a biphasic pattern with an initial rapid decline followed by a period of tailing. While we observed substantial variation in the net culturable population levels of Salmonella and E. coli at each time point, die-off rate and FSMA compliance (i.e., at least a 2 log 10 die-off over 4 days) appear to be impacted by produce type, bacteria, and weather; die-off on lettuce tended to be faster than that on spinach, die-off of E. coli tended to be faster than that of attenuated Salmonella , and die-off tended to become faster as relative humidity decreased. Thus, the use of a single die-off rate for estimating time-to-harvest intervals across different weather conditions, produce types, and bacteria should be revised.
This research study was conducted to determine if bird depredation in feedlots is associated with the prevalence of ciprofloxacin-resistant Escherichia coli in cattle and to determine if removal of invasive bird species could be an effective management strategy to help reduce ciprofloxacin-resistant E. coli in cattle within the United States. European starlings (Sturnus vulgaris) were collected from feedlots within multiple geographic regions within the United States and European starlings within all regions tested positive for ciprofloxacin-resistant E. coli, but prevalence differed by region. Total number of birds on feedlots were positively associated with increased cattle fecal shedding of ciprofloxacin-resistant E. coli. Targeted control of invasive European starlings reduced bird numbers on feedlots by 70.4%, but decreasing populations of European starlings was not associated with corresponding reductions in bovine fecal prevalence of ciprofloxacin-resistant E. coli. These data provide evidence for the role of wild bird depredation in feedlots contributing to fecal shedding of ciprofloxacin-resistant E. coli, but a single month of European starling control in feedlots was not sufficient to impact the fecal carriage of this organism in cattle.
Purpose of review:
Fecal contamination of water is a major public health concern. This review summarizes recent developments and advancements in water quality indicators of fecal contamination.
Recent findings:
This review highlights a number of trends. First, fecal indicators continue to be a valuable tool to assess water quality and have expanded to include indicators able to detect sources of fecal contamination in water. Second, molecular methods, particularly PCR-based methods, have advanced considerably in their selected targets and rigor, but have added complexity that may prohibit adoption for routine monitoring activities at this time. Third, risk modeling is beginning to better connect indicators and human health risks, with the accuracy of assessments currently tied to the timing and conditions where risk is measured. Research has advanced although challenges remain for the effective use of both traditional and alternative fecal indicators for risk characterization, source attribution and apportionment, and impact evaluation.
Shiga toxin-producing Escherichia coli (STECs) contamination of produce, as a result of contact with ruminant fecal material, has been associated with serious foodborne illness. Bacteriophages (phages) that infect STECs have primarily been reported to be of cattle origin. However, they likely exist in other environments or in animals that share habitats with cattle, such as goats. To explore the presence and diversity of phages specific to STEC O157 and the top six non-O157 STECs in goat-associated environments, environmental samples consisting of feces (goat and cattle) and soil samples were collected monthly for six months from an organic produce farm. A variety of phages belonging to the Myoviridae, Siphoviridae, and Podoviridae families were isolated from all goat fecal and half of the soil samples. The most commonly isolated phages belonged to Myoviridae and were lytic against STEC O103. The isolated phages had different host ranges, but collectively, showed lytic activity against O157 and the top six non-O157 STEC strains excluding O121. Two non-O157 STECs (O174: H21 and O-antigen-negative: H18) were isolated from soil and cattle feces, respectively. Although prior studies have reported that goats shed STEC into the environment, the findings of the current study suggest that goat feces may also contain lytic STEC-specific phages. The phages of goat origin have the capacity to infect STECs implicated in causing foodborne outbreaks, making them potential candidates for biocontrol pending additional characterization steps. Further work is needed to determine if the addition of goats to the farm environment could potentially reduce the presence of STECs.
Fresh vegetables including baby greens, microgreens, and sprouts can host human pathogens without exhibiting any visible signs of spoilage. It is clear that the vast majority of foodborne disease outbreaks associated with vegetable produce are not simply a result of an oversight by a producer, as it was shown that zoonotic pathogens from Enterobacteriaceae can contaminate produce through various routes throughout the entire production cycle. In this context, phenotypic and genotypic signatures have been used since early ages in agriculture to obtain better produce, and can be used today as a strategy to reduce the risk of outbreaks through plant breeding. In this mini-review, we provide an updated view and perspectives on to what extent the selection of biological markers can be used to select safer cultivars of vegetable crops such as tomato (the most studied), leafy greens and cabbage. Once this knowledge will be better consolidated, these approaches should be integrated into the development of comprehensive farm-to-fork produce safety programs.
An increasing global population demands a continuous supply of nutritious and safe food. Edible products can be contaminated with biological (e.g., bacteria, virus, protozoa), chemical (e.g., heavy metals, mycotoxins), and physical hazards during production, storage, transport, processing, and/or meal preparation. The substantial impact of foodborne disease outbreaks on public health and the economy has led to multidisciplinary research aimed to understand the biology underlying the different contamination processes and how to mitigate food hazards. Here we review the knowledge, opportunities, and challenges of plant breeding as a tool to enhance the food safety of plant-based food products. First, we discuss the significant effect of plant genotypic and phenotypic variation in the contamination of plants by heavy metals, mycotoxin-producing fungi, and human pathogenic bacteria. In addition, we discuss the various factors (i.e., temperature, relative humidity, soil, microbiota, cultural practices, and plant developmental stage) that can influence the interaction between plant genetic diversity and contaminant. This exposes the necessity of a multidisciplinary approach to understand plant genotype × environment × microbe × management interactions. Moreover, we show that the numerous possibilities of crop/hazard combinations make the definition and identification of high-risk pairs, such as Salmonella-tomato and Escherichia coli-lettuce, imperative for breeding programs geared toward improving microbial safety of produce. Finally, we discuss research on developing effective assays and approaches for selecting desirable breeding germplasm. Overall, it is recognized that although breeding programs for some human pathogen/toxin systems are ongoing (e.g., Fusarium in wheat), it would be premature to start breeding when targets and testing systems are not well defined. Nevertheless, current research is paving the way toward this goal and this review highlights advances in the field and critical points for the success of this initiative that were discussed during the Breeding Crops for Enhanced Food Safety workshop held 5–6 June 2019 at University of California, Davis.
There is a need for science-based tools to (i) help manage microbial produce safety hazards associated with preharvest surface water use, and (ii) facilitate comanagement of agroecosystems for competing stakeholder aims. To develop these tools an improved understanding of foodborne pathogen ecology in freshwater systems is needed. The purpose of this study was to identify (i) sources of potential food safety hazards, and (ii) combinations of factors associated with an increased likelihood of pathogen contamination of agricultural water. Sixty-eight streams were sampled between April and October 2018 (196 samples). At each sampling event separate 10-L grab samples (GS) were collected and tested for Listeria, Salmonella, and the stx and eaeA genes. A 1-L GS was also collected and used for Escherichia coli enumeration and detection of four host-associated fecal source-tracking markers (FST). Regression analysis was used to identify individual factors that were significantly associated with pathogen detection. We found that eaeA-stx codetection [Odds Ratio (OR) = 4.2; 95% Confidence Interval (CI) = 1.3, 13.4] and Salmonella isolation (OR = 1.8; CI = 0.9, 3.5) were strongly associated with detection of ruminant and human FST markers, respectively, while Listeria spp. (excluding Listeria monocytogenes) was negatively associated with log10 E. coli levels (OR = 0.50; CI = 0.26, 0.96). L. monocytogenes isolation was not associated with the detection of any fecal indicators. This observation supports the current understanding that, unlike enteric pathogens, Listeria is not fecally-associated and instead originates from other environmental sources. Separately, conditional inference trees were used to identify scenarios associated with an elevated or reduced risk of pathogen contamination. Interestingly, while the likelihood of isolating L. monocytogenes appears to be driven by complex interactions between environmental factors, the likelihood of Salmonella isolation and eaeA-stx codetection were driven by physicochemical water quality (e.g., dissolved oxygen) and temperature, respectively. Overall, these models identify environmental conditions associated with an enhanced risk of pathogen presence in agricultural water (e.g., rain events were associated with L. monocytogenes isolation from samples collected downstream of dairy farms; P = 0.002). The information presented here will enable growers to comanage their operations to mitigate the produce safety risks associated with preharvest surface water use.
Fresh produce contaminated with human pathogens may result in foodborne disease outbreaks that cause a significant number of illnesses, hospitalizations, and death episodes affecting both public health and the agribusiness every year. The ability of these pathogens to survive throughout the food production chain is remarkable. Using a genetic approach, we observed that leaf colonization by Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium 14028s) and Escherichia coli O157:H7 was significantly affected by genetic diversity of lettuce (Lactuca sativa L. and L. serriola L.). In particular, there was a significant variation among 11 lettuce genotypes in bacterial attachment, internalization, and apoplastic persistence after surface- and syringe-inoculation methods. We observed a significant correlation of the bacterial leaf internalization rate with stomatal pore traits (width and area). Moreover, bacterial apoplastic populations significantly decreased in 9 out of 11 lettuce genotypes after 10 days of surface inoculation. However, after syringe infiltration, populations of E. coli O157:H7 and S. Typhimurium 14028s showed positive, neutral, or negative net growth in a 10-day experimental period among seedlings of different lettuce types. The relative ability of the bacteria to persist in the apoplast of lettuce genotypes after syringe inoculation was minimally altered when assessed during a longer period (20 days) using 3.5- to 4-week-old plants. Interestingly, contrasting bacterial persistence in the lettuce genotypes Red Tide and Lollo Rossa was positively correlated with significant differences in the level of reactive oxygen species burst and callose deposition against S. Typhimurium 14028s and E. coli O157:H7 which are related to plant defense responses. Overall, we characterized the genetic diversity in the interaction between lettuce genotypes and enterobacteria S. Typhimurium 14028s and E. coli O157:H7 and discovered that this genetic diversity is linked to variations in plant immune responses towards these bacteria. These results provide opportunities to capitalize on plant genetics to reduce pathogen contamination of leaves.
The shedding dynamics of foodborne pathogens by wild birds on farmland are not well characterized. This yearlong study sampled wild birds for foodborne pathogens within agricultural lands in northern California. There was a low prevalence of Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga-toxin producing E. coli (prevalence, 0.34% to 0.50%) identified in bird populations in this study. However, pathogens of public health importance (such as Salmonella Newport, E. coli O157:H7, and STEC O103 and O26) were identified in fecal samples, and two birds carried STEC on their feet or feathers. Identical pathogen strains were shared episodically among birds and between wild geese and free-range cattle. This result suggests a common source of contamination in the environment and potential transmission between species. These findings can be used to assess the risk posed by bird intrusions in produce fields and enhance policy decisions toward the comanagement of food safety and farmland habitat conservation.
Water quality standards for drinking water and recreational waters have long been based on the enumeration of fecal coliforms in the various water supplies, with 0 CFU Escherichia coli/100 ml for drinking water and <126 CFU generic E. coli/100ml for recreational waters. Irrigation water will soon undergo the same scrutiny in the U.S. For over 50 years the Most Probable Number (MPN) method has been used by laboratories to estimate the level of viable bacteria in a sample, but this method is labor intensive and slow, especially if large numbers of samples need to be tested. In this review we describe some recent innovations in methods to enumerate enteric pathogens in water. These methods are based on different reasoning schemes that can be categorized as biosensors and nucleic acid‐based methods. All the methods described here used natural water sources. Several were also used to survey the bacterial levels in naturally contaminated samples. The different methods vary in their Limits of Detection (LOD), ease of use, and potential portability. Some combine very good limits of detection with the ability to overcome technical challenges; however, there is considerable room for improvement, as none of the methods are without shortcomings. This article is protected by copyright. All rights reserved.
The impact of plant development, environmental conditions at the time of inoculation, and inoculum concentration on survival of attenuated BSL1 Escherichia coli O157:H7 strain ATCC 700728 on field-grown romaine lettuce was evaluated over 3 years. E. coli 700728 was inoculated onto 4- and 6-week-old romaine lettuce plants in the Salinas Valley, CA, at night or the next morning with either low (5 log) or high (7 log) cell numbers per plant to simulate a single aqueous contamination event. At night, when leaf wetness and humidity levels were high, E. coli cell numbers declined by 0.5 log CFU/plant over the first 8-10 h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2 h. However, similar numbers of E. coli were retrieved from lettuce plants at 2 and 7 days. E. coli cell numbers per plant were significantly lower (P < 0.05) 7 days after application onto 4-week-old compared to 6-week-old plants. E. coli 700728 could be recovered by plating or enrichment from a greater proportion of plants for longer times when inoculated at high compared with low initial concentrations and after inoculation of 6-week-old plants compared with 4-week-old plants, even at the low initial inoculum. A contamination event near harvest or when leaf wetness and humidity levels are high may enhance survivability, even when low numbers of E. coli are introduced.
Microbial air pollution from livestock farms has raised concerns regarding public health. Little is known about airborne livestock-related microbial levels in residential areas. We aimed to increase insights into this issue. Air measurements were performed in 2014 and 2015 at 61 residential sites in The Netherlands. Quantitative-PCR was used to assess DNA concentrations of selected bacteria (commensals: Escherichia coli and Staphylococcus spp.; a zoonotic pathogen: Campylobacter jejuni) and antimicrobial resistance (AMR) genes ( tetW, mecA) in airborne dust. Mixed models were used to explore spatial associations (temporal adjusted) with livestock-related characteristics of the surroundings. DNA from commensals and AMR genes was detectable even at sites furthest away from farms (1200 m), albeit at lower levels. Concentrations, distinctly different between sites, were strongly associated with the density of farms in the surroundings especially with poultry and pigs. C. jejuni DNA was less prevalent (42% of samples positive). Presence of C. jejuni was solely associated with poultry (OR: 4.7 (95% CI: 1.7-14), high versus low poultry density). Residential exposure to livestock-related bacteria and AMR genes was demonstrated. Identified associations suggest contribution of livestock farms to microbial air pollution in general and attribution differences between farm types. This supports the plausibility of recent studies showing health effects in relation to residential proximity to farms.
Shiga toxin-producing Escherichia coli O157:H7 (O157) can cause mild to severe gastrointestinal disease in humans. Cattle are the primary reservoir for O157, which colonizes the intestinal tract without inducing any overt clinical symptoms. Parenteral vaccination can reduce O157 shedding in cattle after challenge and limit zoonotic transmission to humans, although the impact of vaccination and vaccine formulation on cellular and mucosal immune responses are undetermined. To better characterize the cattle immune response to O157 vaccination, cattle were vaccinated with either water-in-oil-adjuvanted, formalin-inactivated hha deletion mutant of Shiga toxin 2 negative (stx2−) O157 (Adj-Vac); non-adjuvanted (NoAdj-Vac); or non-vaccinated (NoAdj-NoVac) and peripheral T cell and mucosal antibody responses assessed. Cattle in Adj-Vac group had a higher percentage of O157-specific IFNγ producing CD4+ and γδ+ T cells in recall assays compared to the NoAdj-Vac group. Furthermore, O157-specific IgA levels detected in feces of the Adj-Vac group were significantly lower in NoAdj-Vac group. Extracts prepared only from Adj-Vac group feces blocked O157 adherence to epithelial cells. Taken together, these data suggest parenteral administration of adjuvanted, inactivated whole-cell vaccines for O157 can induce O157-specific cellular and mucosal immune responses that may be an important consideration for a successful vaccination scheme.
Field trials were conducted in July–August and October 2012 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, California. One-half milliliter of rabbit, chicken, or pig fecal slurry, containing an average of 4.05 × 10 ⁷ CFU E. coli O157:H7 (C 0 ), was inoculated onto the upper (adaxial) surface of a lower leaf on 288 heads of lettuce per trial immediately following a 2.5 h irrigation event. To estimate the bacterial inactivation rate as a function of time, fecal matrix, irrigation and seasonal climate effects, sets of lettuce heads ( n = 28) were sampled each day over 10 days and the concentration of E. coli O157:H7 (C t ) determined. E. coli O157:H7 was detected on 100% of heads during the 10-day duration, with concentrations ranging from ≤340 MPN/head (∼5-log reduction) to >3.45 × 10 ¹² MPN/head (∼5-log growth). Relative to C 0 , on day 10 (C t = 12 ) we observed an overall 2.6-log and 3.2-log mean reduction of E. coli O157:H7 in July and October, respectively. However, we observed relative maximum concentrations due to bacterial growth on day 6 (maximum C t = 8 ) apparently stimulated by foliar irrigation on day 5. From this maximum there was a mean 5.3-log and 5.1-log reduction by day 10 (C t = 12 ) for the July and October trials, respectively. This study provides insight into the inactivation and growth kinetics of E. coli O157:H7 on romaine lettuce leaves under natural field conditions. This study provides evidence that harvesting within 24 h post irrigation has the potential to increase the concentration of E. coli O157:H7 contamination, if present on heads of romaine lettuce; foliar irrigation can temporarily stimulate substantial regrowth of E. coli O157:H7.
The interaction of wind with aquatic and terrestrial surfaces is known to control the creation of microbial aerosols allowing for their entrainment into air masses that can be transported regionally and globally. Near surface interactions between urban waterways and urban air are understudied but some level of interaction among these bacterial communities would be expected and may be relevant to understanding both urban air and water quality. To address this gap related to patterns of local air-water microbial exchange, we utilized next-generation sequencing of 16S rRNA genes from paired air and water samples collected from 3 urban waterfront sites and evaluated their relative bacterial diversity. Aerosol samples at all sites were significantly more diverse than water samples. Only 17–22% of each site’s bacterial aerosol OTUs were present at every site. These shared aerosol OTUs included taxa associated with terrestrial systems (e.g., Bacillus), aquatic systems (e.g., Planktomarina) and sewage (e.g., Enterococcus). In fact, sewage-associated genera were detected in both aerosol and water samples, (e.g., Bifidobacterium, Blautia, and Faecalibacterium), demonstrating the widespread influence of similar pollution sources across these urban environments. However, the majority (50–61%) of the aerosol OTUs at each site were unique to that site, suggesting that local sources are an important influence on bioaerosols. According to indicator species analysis, each site’s aerosols harbored the highest percentage of bacterial OTUs statistically determined to uniquely represent that site’s aquatic bacterial community, further demonstrating a local connection between water quality and air quality in the urban environment.
Natural aquatic environments such as oceans, lakes, and rivers are home to a tremendous diversity of microorganisms. Some may cross the air-water interface within droplets and become airborne, with the potential to impact the Earth’s radiation budget, precipitation processes, and spread of disease. Larger droplets are likely to return to the water or adjacent land, but smaller droplets may be suspended in the atmosphere for transport over long distances. Here, we report on a series of controlled laboratory experiments to quantify wind-driven droplet production from a freshwater source for low wind speeds. The rate of droplet production increased quadratically with wind speed above a critical value (10-m equivalent 5.7 m/s) where droplet production initiated. Droplet diameter and ejection speeds were fit by a gamma distribution. The droplet mass flux and momentum flux increased with wind speed. Two mechanisms of droplet production, bubble bursting and fragmentation, yielded different distributions for diameter, speed, and angle. At a wind speed of about 3.5 m/s, aqueous suspensions of the ice-nucleating bacterium Pseudomonas syringae were collected at rates of 283 cells m−2 s−1 at 5 cm above the water surface, and at 14 cells m−2 s−1 at 10 cm above the water surface. At a wind speed of about 4.0 m/s, aqueous suspensions of P. syringae were collected at rates of 509 cells m−2 s−1 at 5 cm above the water surface, and at 81 cells m−2 s−1 at 10 cm above the water surface. The potential for microbial flux into the atmosphere from aquatic environments was calculated using known concentrations of bacteria in natural freshwater systems. Up to 3.1 × 104 cells m−2 s−1 of water surface were estimated to leave the water in potentially suspended droplets (diameters
Several produce-borne outbreaks have been associated with the use of contaminated water during preharvest applications. Salmonella has been implicated in a number of these outbreaks. The purpose of this study was to evaluate the microbial quality of agricultural surface water used in preharvest production on the Eastern Shore of Virginia in accordance with the Food Safety Modernization Act's Produce Safety Rule water standards. The study also examined the prevalence, concentration, and diversity of Salmonella in those water sources. Water samples (1 L) from 20 agricultural ponds were collected during the 2015 and 2016 growing seasons ( n = 400). Total aerobic bacteria, total coliforms, and Escherichia coli were enumerated for each sample. Population levels of each microorganism were calculated per 100-mL sample and log transformed, when necessary. Samples (250 mL) were also enriched for Salmonella. Presumptive Salmonella isolates were confirmed by PCR ( invA gene) and were serotyped. In 2016, the concentration of Salmonella in each sample was also estimated by most probable number (MPN). Indicator bacteria and environmental and meteorological factors were analyzed for their association with the detection of a Salmonella-positive water sample by using logistic regression analysis. Seventeen of the 20 ponds met the Food Safety Modernization Act's Produce Safety Rule standards for production agricultural water. Three ponds did not meet the standards because the statistical threshold value exceeded the limit. Salmonella was detected in 19% of water samples in each year (38 of 200 in 2015 and 38 of 200 in 2016). Of the 118 Salmonella isolates serotyped, 14 serotypes were identified with the most prevalent being Salmonella Newport. E. coli concentration, farm, and total aerobic bacteria concentration were significantly associated with the likelihood of detecting a Salmonella-positive sample The average concentration of Salmonella in all samples was 4.44 MPN/100 mL, with the limit of detection being 3.00 MPN/100 mL. The highest concentration of Salmonella was 93.0 MPN/100 mL. These data will assist in a better understanding of the risks that production water poses to produce contamination events.
Escherichia coli O157:H7 fecal shedding in feedlot cattle is common and is a public health concern due to the risk of foodborne transmission that can result in severe, or even fatal, disease in people. Despite a large body of research, few practical and cost-effective farm-level interventions have been identified. In this study, a randomized controlled trial was conducted to assess the effect of reducing the level of water in automatically refilling water-troughs on fecal shedding of E. coli O157:H7 in feedlot cattle. Pens in a feedlot in the Texas Panhandle were randomly allocated as control (total number: 17) or intervention (total number: 18) pens. Fecal samples (2,759 in total) were collected both at baseline and three weeks after the intervention, and tested for the presence of E. coli O157:H7 using immunomagnetic bead separation and selective culture. There was a strong statistical association between sampling date and the likelihood of a fecal sample testing positive for E. coli O157:H7. Pen was also a strong predictor of fecal prevalence. Despite accounting for this high level of clustering, a statistically significant association between reduced water levels in the trough and increased prevalence of E. coli O157:H7 in the feces was observed (Odds Ratio = 1.6; 95% Confidence Interval: 1.2–2.0; Likelihood Ratio Test: p = 0.02). This is the first time that such an association has been reported, and suggests that increasing water-trough levels may be effective in reducing shedding of E. coli O157:H7 in cattle feces, although further work would be needed to test this hypothesis. Controlling E. coli O157:H7 fecal shedding at the pre-harvest level may lead to a reduced burden of human foodborne illness attributed to this pathogen in beef.
Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the area—around or within city—with predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology between free STEC-specific phages and STEC bacteria for further pre-harvest food safety management in produce environment.
Plant injury is inherent to the production and processing of fruit and vegetables. The opportunistic colonization of damaged plant tissue by human enteric pathogens may contribute to the occurrence of outbreaks of foodborne illness linked to produce. Escherichia coli O157:H7 (EcO157) responds to physicochemical stresses in cut lettuce and lettuce lysates by upregulation of several stress response pathways. We investigated the tolerance of EcO157 to osmotic stress imposed by the leakage of osmolytes from injured lettuce leaf tissue. LC-MS analysis of bacterial osmoprotectants in lettuce leaf lysates and wound washes indicated an abundant natural pool of choline, but sparse quantities of glycine betaine and proline. Glycine betaine was a more effective osmoprotectant than choline in EcO157 under osmotic stress conditions in vitro. An EcO157 mutant with a deletion of the betTIBA genes, which are required for biosynthesis of glycine betaine from imported choline, achieved population sizes twofold lower than those of the parental strain (P < 0.05) over the first hour of colonization of cut lettuce in modified atmosphere packaging (MAP). The cell concentrations of the betTIBA mutant also were 12-fold lower than those of the parental strain (P < 0.01) when grown in hypertonic lettuce lysate, indicating that lettuce leaf cellular contents provide choline for osmoprotection of EcO157. To demonstrate the utilization of available choline by EcO157 for osmoadaptation in injured leaf tissue, deuterated (D-9) choline was introduced to wound sites in MAP lettuce; LC-MS analysis revealed the conversion of D9-choline to D-9 glycine betaine in the parental strain, but no significant amounts were observed in the betTIBA mutant. The EcO157 ΔbetTIBA-ΔotsBA double mutant, which is additionally deficient in de novo synthesis of the compatible solute trehalose, was significantly less fit than the parental strain after their co-inoculation onto injured lettuce leaves and MAP cut lettuce. However, its competitive fitness followed a different time-dependent trend in MAP lettuce, likely due to differences in O2 content, which modulates betTIBA expression. Our study demonstrates that damaged lettuce leaf tissue does not merely supply EcO157 with substrates for proliferation, but also provides the pathogen with choline for its survival to osmotic stress experienced at the site of injury.
Whole genome sequencing (WGS) of important foodborne pathogens is a technology under development, but is already employed in routine surveillance by public health agencies and is being increasingly exploited in tracing transmission routes and identifying contamination events (source tracking) that take place in the farm-to-fork continuum. Furthermore, data generated from WGS, complemented by other -omics data, have the potential to be integrated into and strengthen microbiological risk assessment. In this paper, we discuss the contribution of WGS in diverse areas important to food safety and public health. Additionally, an outlook of future WGS applications, which should contribute to our understanding of the ecology and physiology of foodborne microorganisms, is presented.
Produce has become a major source of foodborne illness, and may become contaminated through surface water irrigation. The objectives of this study were to (i) determine the frequency of verotoxigenic E. coli (VTEC), Listeria monocytogenes, and Salmonella in surface waters used for irrigation in the Lower Mainland of British Columbia, (ii) assess the suitability of fecal coliforms and generic E. coli as hygiene indicators, and (iii) investigate the correlations of environmental factors with pathogen occurrence. Water samples were collected semi-monthly for 18 months from seven irrigation ditches across the Serpentine and Sumas watersheds. VTEC colonies on water filters were detected using a verotoxin colony immunoblot, and the presence of virulence genes vt1 and vt2 was ascertained via multiplex PCR. Detection of L. monocytogenes and Salmonella was completed using standard, Health Canada Compendium of Analytical Methods. Fecal coliforms and generic E. coli were enumerated by 3M™ Petrifilm™ and filtration methods, and meteorological and geographic data were collected from government records. VTEC, L. monocytogenes, and Salmonella were detected in 4.93%, 10.3%, and 2.69% of 223 samples, respectively. L. monocytogenes occurrence was greatest in the Serpentine watershed (χ²; p < 0.05), and was most common during the winter and fall (Fisher exact test; p < 0.05). Site dependence of VTEC and Salmonella occurrence was observed within watersheds (Fisher’s exact test; p < 0.10). Pathogen occurrence correlated with fecal coliform counts (r = 0.448), while VTEC occurrence also correlated with precipitation over the five days before sampling (r = 0.239). The density of upstream livestock correlated with VTEC (rs = 0.812), and L. monocytogenes (rs = 0.841) detection. These data show that foodborne pathogens are present in the waters used for irrigation in the Lower Mainland of British Columbia, but their frequency may depend on spatial and temporal factors.
Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.
IMPORTANCE
This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.
Aerosolized microorganisms may play an important role in climate change, disease transmission, water and soil contaminants, and geographic migration of microbes. While it is known that bioaerosols are generated when bubbles break on the surface of water containing microbes, it is largely unclear how viable soil-based microbes are transferred to the atmosphere. Here we report a previously unknown mechanism by which rain disperses soil bacteria into the air. Bubbles, tens of micrometres in size, formed inside the raindrops disperse micro-droplets containing soil bacteria during raindrop impingement. A single raindrop can transfer 0.01% of bacteria on the soil surface and the bacteria can survive more than one hour after the aerosol generation process. This work further reveals that bacteria transfer by rain is highly dependent on the regional soil profile and climate conditions.
Lack of access to clean water and adequate sanitation continues to be a major brake on development. Here we present the results of a 12-month investigation into the dynamics of Escherichia coli, a commonly used indicator of faecal contamination in water supplies, in three small, rural catchments in Laos, Thailand and Vietnam. We show that land use and hydrology are major controlling factors of E. coli concentrations in streamwater and that the relative importance of these two factors varies between the dry and wet seasons. In all three catchments, the highest concentrations were observed during the wet season when storm events and overland flow were highest. However, smaller peaks of E. coli concentration were also observed during the dry season. These latter correspond to periods of intense farming activities and small, episodic rain events. Furthermore, vegetation type, through land use and soil surface crusting, combined with mammalian presence play an important role in determining E. coli loads in the streams. Finally, sampling during stormflow revealed the importance of having appropriate sampling protocols if information on maximum contamination levels is required as grab sampling at a fixed time step may miss important peaks in E. coli numbers.
Concerns about the microbiological safety of fresh produce have attracted attention in the past three decades due to multiple foodborne outbreaks. Animal manure contaminated with enteric pathogens has been identified as an important preharvest pathogen source. This study investigated the survival of Salmonella enterica in dust particles of dehydrated turkey manure and how association with manure dust may enhance the survival of salmonellae on leafy greens in the field. The survival of a cocktail of multiple Salmonella serotypes in the dried fecal material of various particle sizes (125 to 500 lm) was examined at varying moisture contents (5, 10, and 15%). Survival times of the pathogen were inversely related to moisture content and particle size of manure dust, with viable Salmonella still detectable for up to 291 days in the smallest particle size (125 lm) with 5% moisture. Association with manure dust particles increased the survival of Salmonella when subjected to UV light both under laboratory conditions and on the surface of spinach leaves in a greenhouse setting. The results of this study suggest that aerosolized manure particles could be a potential vehicle for Salmonella dispersal to leafy greens if the microorganism is present in the dry manure.
Significance
Fresh produce has become the primary cause of foodborne illness in the United States. A widespread concern that wildlife vector foodborne pathogens onto fresh produce fields has led to strong pressure on farmers to clear noncrop vegetation surrounding their farm fields. We combined three large datasets to demonstrate that pathogen prevalence in fresh produce is rapidly increasing, that pathogens are more common on farms closer to land suitable for livestock grazing, and that vegetation clearing is associated with increased pathogen prevalence over time. These findings contradict widespread food safety reforms that champion vegetation clearing as a pathogen mitigation strategy. More generally, our work indicates that achieving food safety and nature conservation goals in produce-growing landscapes is possible.
The safety of fresh produce is an important concern in the United States, especially in the wake of recent national foodborne illness outbreaks. The agricultural industry has implemented steps to enhance food safety along the entire farm-to-fork supply chain. This includes on-farm measures to exclude wildlife and to remove its habitat in and around fields. Farmers and others from across the United States have expressed concern about the ecological consequences and uncertain food safety benefits of such practices. This article reviews the scientific rationale behind management of wildlife and its habitat as part of good agriculture practices for enhancing food safety. The review concludes that, although pathogen prevalence has been documented in wildlife at overall low levels, the potential role that wildlife and its habitat play in pathogenic contamination remains unclear and is interwoven with pathogenic risk from human and domesticated animal sources. The characterization and disruption of potential links between livestock and wildlife is highlighted as a research priority. The findings underscore the importance of appropriate wildlife research and management in the context of food safety and to human-wildlife interactions in general, and they have implications wherever fresh produce is grown in the United States.
Vaccination of Cattle against Escherichia coli O157:H7, Page 1 of 2
Abstract
Human infection with Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is relatively rare, but the consequences can be serious, especially in the very young and the elderly. Efforts to control the flow of STEC O157 during beef processing have meaningfully reduced the incidence of human STEC O157 infection, particularly prior to 2005. Unfortunately, despite early progress, the incidence of STEC O157 infection has not changed meaningfully or statistically in recent years, suggesting that additional actions, for example, targeting the cattle reservoir, are necessary to further reduce STEC O157 illness. Ideally, preharvest interventions against STEC O157 should reduce the likelihood that cattle carry the organism, have practical application within the beef production system, and add sufficient value to the cattle to offset the cost of the intervention. A number of STEC O157 antigens are being investigated as potential vaccine targets. Some vaccine products have demonstrated efficacy to reduce the prevalence of cattle carrying STEC O157 by making the gut unfavorable to colonization. However, in conditions of natural exposure, efficacy afforded by vaccination depends on how the products are used to control environmental transmission within groups of cattle and throughout the production system. Although cattle vaccines against STEC O157 have gained either full or preliminary regulatory approval in Canada and the United States, widespread use by cattle feeders is unlikely until there is an economic signal to indicate that cattle vaccinated against STEC O157 are valued over other cattle.
Raw vegetables are a key food for a healthy diet, but their increased consumption brings a higher risk for foodborne disease. Contamination of salad greens with Shiga toxin-producing Escherichia coli (STEC) O157:H7 has caused severe disease and important economic losses almost yearly in the United States over the last 10 years. To curb the risk of infections from contaminated produce, approaches based on bacterial virus – commonly known as bacteriophage or phage – have recently started to draw interest among other antimicrobial strategies. Phages enter bacterial cells to reproduce and cause cellular lysis to release their phage progeny at the end of their infection cycle. This lytic effect is caused by lysins, phage-encoded enzymes that have evolved to degrade the bacterial cell wall resulting in hypotonic lysis. When applied externally in their purified form, such enzymes are able to kill sensitive bacteria on contact in a similar way. Their unique bactericidal properties have made lysins effective antimicrobial agents in a variety of applications, from treating multidrug-resistant infections in humans to controlling bacterial contamination in several areas, including microbiological food safety. Here we describe a novel lysin, namely PlyEc2, with potent bactericidal activity against key gram-negative pathogens including E. coli, Salmonella, Shigella, Acinetobacter and Pseudomonas. PlyEc2 displayed high bactericidal activity against STEC to a concentration of 12.5 μg/ml under different pH conditions. This lysin was also able to reduce the bacterial titer of several pathogenic bacterial strains in vitro by more than 5 logarithmic units, resulting in complete sterilization. Importantly, PlyEc2 proved to be a powerful produce decontamination agent in its ability to clear 99.7% of contaminating STEC O157:H7 in our Romaine lettuce leaf model. PlyEc2 was also able to eradicate 99.8% of the bacteria contaminating the washing solution, drastically reducing the risk of cross-contamination during the washing process. A sensory evaluation panel found that treatment with PlyEc2 did not alter the visual and tactile quality of lettuce leaves compared to the untreated leaves. Our study is the first to describe a highly effective lysin treatment to control gram-negative pathogenic contamination on fresh lettuce without the addition of membrane destabilizing agents.
Background:
Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. CDC queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation.
Methods:
A case was defined as an infection between March 13 and August 22, 2018 with one of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole genome sequencing. We conducted epidemiologic and traceback investigations to identify illness sub-clusters and common sources. An FDA-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination.
Results:
We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and five died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty sub-clusters were identified. Product traceback from sub-cluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157.
Conclusion:
We report on the largest multistate leafy green-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.
Recent pathogenic Escherichia coli contamination of fresh vegetables that originated from irrigation water has increased awareness and importance of identifying sources of E. coli entering agroecosystems. However, inadequate methods for accurately predicting E. coli occurrence and sources in waterways continue to limit the identification of appropriate and effective prevention and treatment practices. Therefore, the primary objectives of this study were to: (1) Determine the concentration of E. coli during storm events in a hydrologic controlled stream situated in a livestock research operation that is located in the Central Flyway for avian migration in the United States. Great Plains; and (2) Identify trends between E. coli concentrations, grazing rotations, and avian migration patterns. The study sampled five rainfall events (three summer and two fall) to measure E. coli concentrations throughout storm events. A combination of cattle density and waterfowl migration patterns were found to significantly impact E. coli concentrations in the stream. Cattle density had a significant impact during the summer season (p < .0001), while waterfowl density had a significant impact on E. coli concentrations during the fall (p = .0422). The downstream reservoir had exceedance probabilities above the Environmental Protection Agency freshwater criteria > 85% of the growing season following rainfall events. Based on these findings, implementation of best management practices for reducing E. coli concentrations during the growing season and testing of irrigation water prior to application are recommended.
Nearly one-half of foodborne illnesses in the United States can be attributed to fresh produce consumption. The preharvest stage of production presents a critical opportunity to prevent produce contamination in the field from contaminating postharvest operations and exposing consumers to foodborne pathogens. One produce-contamination route that is not often explored is the transfer of pathogens in the soil to edible portions of crops via splash water. We report here on the results from multiple field and microcosm experiments examining the potential for Salmonella contamination of produce crops via splash water, and the effect of soil moisture content on Salmonella survival in soil and concentration in splash water. In field and microcosm experiments, we detected Salmonella for up to 8 to 10 days after inoculation in soil and on produce. Salmonella and suspended solids were detected in splash water at heights of up to 80 cm from the soil surface. Soil-moisture conditions before the splash event influenced the detection of Salmonella on crops after the splash events—Salmonella concentrations on produce after rainfall were significantly higher in wet plots than in dry plots (geometric mean difference = 0.43 CFU/g; P = 0.03). Similarly, concentrations of Salmonella in splash water in wet plots trended higher than concentrations from dry plots (geometric mean difference = 0.67 CFU/100 mL; P = 0.04). These results indicate that splash transfer of Salmonella from soil onto crops can occur and that antecedent soil-moisture content may mediate the efficiency of microbial transfer. Splash transfer of Salmonella may, therefore, pose a hazard to produce safety. The potential for the risk of splash should be further explored in agricultural regions in which Salmonella and other pathogens are present in soil. These results will help inform the assessment of produce safety risk and the development of management practices for the mitigation of produce contamination.
HIGHLIGHTS
Leafy greens are leading vehicles for Escherichia coli O157:H7 foodborne illness. Pest flies can harbor this pathogen and may disseminate it to produce. We determined the occurrence of E. coli O157:H7-positive flies in leafy greens planted up to 180 m from a cattle feedlot and assessed their relative risk to transmit this pathogen to leafy greens. The primary fly groups captured on sticky traps at the feedlot and leafy greens plots included house flies (Musca domestica L.), face flies (Musca autumnalis L.), stable flies (Stomoxys calcitrans L.), flesh flies (family Sarcophagidae), and blow flies (family Calliphoridae). E. coli O157:H7 carriage rates of house, face, flesh, and blow flies were similar (P > 0.05), ranging from 22.3 to 29.0 flies per 1,000 flies. In contrast, the carriage rate of stable flies was lower at 1.1 flies per 1,000 flies (P < 0.05). Differences in carriage rates are likely due to the uses of fresh bovine feces and manure by these different pest fly groups. E. coli O157:H7 carriage rates of total flies did not differ (P > 0.05) by distance (ranging from 0 to 180 m) from the feedlot. Most fly isolates were the same predominant pulsed-field gel electrophoresis types found in feedlot surface manure and leafy greens, suggesting a possible role for flies in transmitting E. coli O157:H7 to the leafy greens. However, further research is needed to clarify this role and to determine set-back distances between cattle production facilities and produce crops that will reduce the risk for pathogen contamination by challenging mechanisms like flies.
Results of previous studies revealed that (i) splash can transfer microbes from in-field feces to preharvest produce and (ii) wildlife can be vectors for the introduction of foodborne pathogens into produce fields. However, few peer-reviewed studies have been conducted to examine pathogen transfer from wildlife feces to in-field produce via splash during irrigation. Although two previous studies found a significant relationship between distance and Escherichia coli transfer via splash, the studies sampled produce <1 m from the feces. The present study was conducted to refine our understanding of the impact of distance on E. coli splash. Two trials were conducted 1 month apart. For each trial, fecal pellets inoculated with a three-strain E. coli cocktail were placed in a lettuce field 2.5 h before irrigation. After irrigation, E. coli levels on lettuce heads 0 to 6 m from the pellets were determined. Although E. coli was not detected in any of the heads ≥2 m from the fecal pellets (n = 39), 39% of heads (13 of 33) <2 m from the pellets tested positive for E. coli. According to logistic regression, the odds of harvesting a head that tested positive for E. coli decreased by a factor of 50 (odds ratio, 0.02; 95% confidence interval, <0.01, 0.28; P = 0.004) for each meter increase in the distance between the lettuce and the feces. Thus, the likelihood of E. coli transfer from feces to produce should be minimal at a given distance from the feces. Our model can be used to predict the probability of harvesting a microbially contaminated lettuce head following implementation of a no-harvest buffer around in-field feces. For example, our model suggests that the probability of harvesting a contaminated head was 0.1% at 3 m from the feces. Although the approaches utilized in this study provide a conceptual framework that can be used to help define appropriate no-harvest buffers, delineation of appropriate buffer zones requires additional information (e.g., acceptable risk and regional data).
HIGHLIGHTS
Plant genotype has been advocated to have an important role in the fate of enteric pathogens residing in lettuce foliage. This study was therefore undertaken under the premise that different pathogen responses could occur in lettuce cultivars with cultivar selection being one of several hurdles in an overall strategy for controlling foodborne pathogens on field-grown produce. Up to eight lettuce cultivars ('Gabriella', 'Green Star', 'Muir', 'New Red Fire', 'Coastal Star', 'Starfighter', 'Tropicana', and 'Two Star') were examined in these experiments in which the plants were subjected to spray contamination of their foliage with pathogens. In an experiment that addressed internalization of Salmonella, cultivar was determined to be a significant variable (P < 0.05) with 'Gabriella' and 'Muir' being the least and most likely to exhibit internalization of this pathogen, respectively. Furthermore, antimicrobials (total phenols and antioxidant capacity chemicals) could be part of the plant's defenses to resist internalization as there was an inverse relationship between the prevalence of internalization at 1 h and the levels of these antimicrobials (r = -0.75 to -0.80, P = 0.0312 to 0.0165). Internalized cells appeared to be transient residents in that across all cultivars, plants sampled 1 h after being sprayed were 3.5 times more likely to be positive for Salmonella than plants analyzed 24 h after spraying (95% CI from 1.5 to 8.2, P = 0.0035). The fate of surface-resident Salmonella and Escherichia coli O157:H7 was addressed in subsequent growth chamber and field experiments. In the growth chamber study, no effect of cultivar was manifested on the fate of either pathogen when plants were sampled up to 12 days after spray contamination of their foliage. However, in the field study, five days after spraying the plants, Salmonella contamination was significantly affected by cultivar (P < 0.05) and the following order of prevalence of contamination was observed: 'Muir' < 'Gabriella' < 'Green Star' = 'New Red Fire' < 'Coastal Star'. Nine days after spray contamination of plants in the field, no effect of cultivar was exhibited due primarily to the low prevalence of contamination observed for Salmonella (8 of 300 plant samples positive by enrichment culture) and E. coli O157 (4 of 300 plant samples positive by enrichment culture). Given the narrow window of time during which cultivar differences were documented.
Dust, fine particulate matter suspended in air, represents an understudied vehicle for microbial dispersal in agricultural environments and fruit contamination by microorganisms pathogenic to humans. Dust not only affects biological processes in plants, such as stomatal gas exchange, but also the plant surface microbiome. While the risk of growing fruits and vegetables in proximity of livestock operations is well recognized, a full understanding of the mechanisms by which fresh produce become contaminated remains incomplete without the consideration of dust. Currently no recommendations on the microbiological quality of air in produce production environments exist. This review explores the association between carpoplane (fruit surface) contamination by enteric bacterial pathogens and the ability of these pathogens to survive and disperse with aerosolized dust.
Filth flies have been implicated in the dispersal of human disease pathogens; however, the epidemiological parameters of the transmission of human pathogens fromflies to plants are largely undescribed. The capacity of the black blow fly, Phormia regina Meigen, to acquire and subsequently deposit bacteria onto baby lettuce leaves was compared with that of the house fly, Musca domestica (L.). Adult P. regina and M. domestica were exposed to green fluorescent protein-tagged Escherichia coli O157:H7-or Salmonella enterica-inoculated manure and then allowed access to the lettuce plants. Bacteria on the plants and flies were assessed by plating and polymerase chain reaction. Although blowflies acquired significantlymore E. coli O157:H7 than house flies, there was no significant difference between the deposition of bacteria on lettuce by the two fly species. In contrast, therewas no significant difference in the acquisition of S. enterica by the two fly species. However, blow flies deposited more S. enterica onto lettuce than house flies. Tomore accurately assess transmission parameters, flies were given timed exposure and inoculation periods of 10 and 30 s. Blowflies acquiredmore E. coli O157:H7 than S. enterica in the both time periods. Flies exposed to manure for 30 s were then tested for deposition by forcing the flies to walk on lettuce leaves. Blow flies deposited comparable amounts of E. coli O157:H7 and S. enterica. Although house flies have historically been implicated in the transmission of human pathogens to food, the data presented suggest that blow flies aremore efficient vectors of E. coli O157:H7 and S. enterica to leafy greens than house flies. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved.
Studies have shown that irrigation water can be a vector for pathogenic bacteria. Due to this, the Food Safety Modernization Act's (FSMA) produce safety rule requires that agricultural water directly applied to produce be safe and of adequate sanitary quality for use, which may pose a challenge for some farmers. The purpose of this research was to assess the presence and concentration of Salmonella and generic Escherichia coli in irrigation water from distribution systems in a mixed produce production region of southern Georgia. Water samples were collected during three growing seasons at three farms irrigating crops with surface water (Pond 1, Pond 2) or groundwater (Well) during 2012-2013. Salmonella and generic E. coli populations were monitored by culture and Most Probable Number (MPN). Confirmed isolates were characterized by pulsed-field gel electrophoresis and serotyping. In Pond 1, Salmonella was detected in 2/21 surface, 5/26 subsurface, 10/50 center pivot, and 0/16 solid set sprinkler head water samples. In Pond 2, Salmonella was detected in 2/18 surface, 1/18 subsurface, 6/36 drip line start, and 8/36 drip line end water samples. Twenty-six well pumps and 64 associated drip line water samples were negative. The overall mean Salmonella concentration for positive water samples was 0.03 MPN/100 mL (range <0.0011-1.8 MPN/100 mL). Nine Salmonella serovars comprising 22 pulsotypes were identified. Identical serovars and subtypes were found three times on the same day and location: Pond 1-Pivot-Cantaloupe (serovar Rubislaw), Pond 1-Pivot-Peanut (serovar Saintpaul), and Pond 2-Drip Line Start-Drip Line End-Yellow Squash (serovar III_16z10:e,n,x,z15). Generic E. coli was detected in water from both farm ponds and irrigation distribution systems, but the concentrations met FSMA microbial water quality criteria. The results from this study will allow producers in southern Georgia to better understand how potential pathogens move through irrigation distribution systems.
Production of both livestock and food crops are central priorities of agriculture; however, food safety concerns arise where these practices intersect. In this study, we investigated the public health risks associated with potential bioaerosol deposition to crops grown in the vicinity of manure application sites. A field sampling campaign at dairy manure application sites supported the emission, transport, and deposition modeling of bioaerosols emitted from these lands following application activities. Results were coupled with a quantitative microbial risk assessment model to estimate the infection risk due to consumption of leafy green vegetable crops grown at various distances downwind from the application area. Inactivation of pathogens ( spp., spp., and O157:H7) on both the manure-amended field and on crops was considered to determine the maximum loading of pathogens to plants with time following application. Overall median one-time infection risks at the time of maximum loading decreased from 1:1300 at 0 m directly downwind from the field to 1:6700 at 100 m and 1:92,000 at 1000 m; peak risks (95th percentiles) were considerably greater (1:18, 1:89, and 1:1200, respectively). Median risk was below 1:10,000 at >160 m downwind. As such, it is recommended that a 160-m setback distance is provided between manure application and nearby leafy green crop production. Additional distance or delay before harvest will provide further protection of public health.
