Publications by authors named "Manan Sharma"

47 Publications

"Zooming" Our Way through Virtual Undergraduate Research Training: A Successful Redesign of the CONSERVE Summer Internship Program.

J Microbiol Biol Educ 2021 31;22(1). Epub 2021 Mar 31.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD 20742.

The COVID-19 pandemic has had an enormous impact on education globally, forcing the teaching community to think outside the box and create innovative educational plans to benefit students at home. Here, we narrate how the undergraduate, laboratory-based Summer Internship Program of our CONSERVE Center of Excellence, which focuses heavily on engaging women and underrepresented minorities in STEM programming, took a turn from an in-person research experience to a fully virtual one. We share our challenges and how we overcame them. Additionally, we provide a description of our virtual internship professional development curriculum, as well as the creative research projects that our seven interns were able to achieve in an 8-week virtual internship, including projects focused on the microbiological water quality of recycled irrigation water; social media promotion, enhancement and marketing of online educational resources focused on water, microbial contamination, and food crop irrigation; decision support systems for using recycled water in agricultural settings; and the effectiveness of zero-valent iron sand filtration in improving agricultural water quality, to name a few. Upon evaluating our internship program, we observed that more than 80% of our interns were either very satisfied or satisfied with the overall virtual internship experience. Through this experience, both the educators and the interns learned that although a virtual laboratory internship cannot completely replace in-person learning, it can still result in a very meaningful educational experience.
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http://dx.doi.org/10.1128/jmbe.v22i1.2625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060145PMC
March 2021

Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters.

Appl Environ Microbiol 2021 Apr 23. Epub 2021 Apr 23.

University of Delaware, Department of Animal and Food Sciences, Newark, DE, United States

Enteric viruses (EV) are the largest contributors to foodborne illness and outbreaks globally. Their ability to persist in the environment, coupled with the challenges experienced in environmental monitoring create a critical aperture through which agricultural crops may become contaminated. This study involved a seventeen-month investigation of select human enteric viruses and viral indicators in non-traditional irrigation water sources, surface and reclaimed waters, in the Mid-Atlantic region of the United States. Real-time quantitative PCR was used for detection of Aichi virus, hepatitis A virus, noroviruses GI and GII. Pepper mild mottle virus (PMMoV), a common viral indicator of human fecal contamination, was also evaluated along with atmospheric (air and water temperature, cloud cover, and precipitation 24 h, 7 d and 14 d prior to sample collection) and physicochemical (dissolved oxygen, pH, salinity and turbidity) data to determine if there were any association between EV and measured parameters. EV were detected more frequently in reclaimed waters (32%, n=22) than in surface waters (4%, n=49) similar to PMMoV detection frequency in surface (33%, n=42) and reclaimed (67%, n=21) water. Our data show a significant correlation between EV and PMMoV (R=0.628; <0.05) detection in reclaimed water samples, but not in surface waters (R=0.476; =0.78). Water salinity significantly affected the detection of both EV and PMMoV (<0.05) as demonstrated by logistic regression analyses. These results provide relevant insights into the extent and degree of association between human enteric (pathogenic) viruses and water quality data in Mid-Atlantic surface and reclaimed waters as potential sources for agricultural irrigation.Microbiological analysis of agricultural waters is fundamental to ensure microbial food safety. The highly variable nature of non-traditional sources of irrigation water are particularly difficult to test for the presence of viruses. Multiple characteristics influence viral persistence in a water source as well as affect the recovery and detection methods which are employed. Testing for a suite of viruses in water samples is often too costly and labor intensive, making identification of suitable indicators for viral pathogen contamination necessary. The results from this study address two critical data gaps: enteric virus prevalence in surface and reclaimed waters of the Mid-Atlantic region of the United States and subsequent evaluation of physicochemical and atmospheric parameters used to inform the potential for use of indicators of viral contamination.
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http://dx.doi.org/10.1128/AEM.00211-21DOI Listing
April 2021

Temporal and Agricultural Factors Influence Escherichia coli Survival in Soil and Transfer to Cucumbers.

Appl Environ Microbiol 2021 03 11;87(7). Epub 2021 Mar 11.

USDA ARS, Northeast Area, Beltsville Agricultural Research Center, Environmental Microbial and Food Safety Laboratory, Beltsville, Maryland, USA

Biological soil amendments of animal origin (BSAAO) increase nutrient levels in soils to support the production of fruits and vegetables. BSAAOs may introduce or extend the survival of bacterial pathogens which can be transferred to fruits and vegetables to cause foodborne illness. survival over 120 days in soil plots (3 m) covered with (mulched) or without plastic mulch (not mulched), amended with either poultry litter, composted poultry litter, heat-treated poultry pellets, or chemical fertilizer, and transfer to cucumbers in 2 years (2018 and 2019) were evaluated. Plots were inoculated with (8.5 log CFU/m) and planted with cucumber seedlings (Supremo). The number of days needed to reduce levels by 4 log CFU (dpi4log) was determined using a sigmoidal decline model. Random forest regression and one-way analysis of variance (ANOVA; < 0.05) identified predictors (soil properties, nutrients, and weather factors) of dpi4log of and transfer to cucumbers. The combination of year, amendment, and mulch (25.0% increase in the mean square error [IncMSE]) and year (9.75% IncMSE) were the most prominent predictors of dpi4log and transfer to cucumbers, respectively. Nitrate levels at 30 days and soil moisture at 40 days were also impactful predictors of dpi4log. Differing rainfall amounts in 2018 (24.9 in.) and 2019 (12.6 in.) affected survival in soils and transfer to cucumbers. spp. were recovered sporadically from various plots but were not recovered from cucumbers in either year. Greater transfer of to cucumbers was also shown to be partially dependent on dpi4log of in plots containing BSAAO. Poultry litter and other biological soil amendments are commonly used fertilizers in fruit and vegetable production and can introduce enteric pathogens such as O157:H7 or previously associated with outbreaks of illness linked to contaminated produce. survival duration in soils covered with plastic mulch or uncovered and containing poultry litter or heat-treated poultry litter pellets were evaluated. Nitrate levels on day 30 and moisture content in soils on day 40 on specific days were good predictors of survival in soils; however, the combination of year, amendment, and mulch type was a better predictor. Different cumulative rainfall totals from year to year most likely affected the transfer of from soils to cucumbers and survival durations in soil. survival in soils can be extended by the addition of several poultry litter-based soil amendments commonly used in organic production of fruits and vegetables and is highly dependent on temporal variation in rainfall.
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http://dx.doi.org/10.1128/AEM.02418-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091612PMC
March 2021

Bacterial Community Dynamics Distinguish Poultry Compost from Dairy Compost and Non-Amended Soils Planted with Spinach.

Microorganisms 2020 Oct 18;8(10). Epub 2020 Oct 18.

Department of Nutrition and Food Sciences, University of Vermont, Marsh Life Science, 109 Carrigan Drive, Burlington, VT 05405, USA.

The aim of this study was to determine whether and how poultry litter compost and dairy manure compost alter the microbial communities within field soils planted with spinach. In three successive years, separate experimental plots on two fields received randomly assigned compost treatments varying in animal origin: dairy manure (DMC), poultry litter (PLC), or neither (NoC). The composition and function of bacterial and fungal communities were characterized by the amplicon sequencing of marker genes and by the ecoenzyme activity, respectively. The temporal autocorrelation within and among years was adjusted by principal response curves (PRC) to analyze the effect of compost on community composition among treatments. Bacteria in the phylum Bacteriodetes, classes Flavobacteriia and Spingobacteriales (, , and ), were two to four times more abundant in soils amended with PLC than DMC or NoC consistently among fields and years. Fungi in the phylum Ascomycota were relatively abundant, but their composition was field-specific and without treatment differences. The ecoenzyme data verify that the effects of PLC and DMC on soil communities are based on their microbial composition and not a response to the C source or nutrient content of the compost.
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http://dx.doi.org/10.3390/microorganisms8101601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603165PMC
October 2020

Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, spp., and spp. in Alternative Irrigation Water Sources: a CONSERVE Study.

Appl Environ Microbiol 2020 10 1;86(20). Epub 2020 Oct 1.

Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland, USA

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (, total coliforms [TC], , and ). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. , TC, enterococci (∼86%, 98%, and 90% positive, respectively;  = 333), and (∼98% positive;  = 133) were widespread in water samples tested. Highest counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (0.001 in all cases) compared to other water types. counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting. Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.
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http://dx.doi.org/10.1128/AEM.00342-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531960PMC
October 2020

A Predictive Model for Survival of Escherichia coli O157:H7 and Generic E. coli in Soil Amended with Untreated Animal Manure.

Risk Anal 2020 07 7;40(7):1367-1382. Epub 2020 May 7.

Center for Food Safety and Applied Nutrition, Food and Drug Administration, Office of Analytics and Outreach, College Park, MD, USA.

This study aimed at developing a predictive model that captures the influences of a variety of agricultural and environmental variables and is able to predict the concentrations of enteric bacteria in soil amended with untreated Biological Soil Amendments of Animal Origin (BSAAO) under dynamic conditions. We developed and validated a Random Forest model using data from a longitudinal field study conducted in mid-Atlantic United States investigating the survival of Escherichia coli O157:H7 and generic E. coli in soils amended with untreated dairy manure, horse manure, or poultry litter. Amendment type, days of rain since the previous sampling day, and soil moisture content were identified as the most influential agricultural and environmental variables impacting concentrations of viable E. coli O157:H7 and generic E. coli recovered from amended soils. Our model results also indicated that E. coli O157:H7 and generic E. coli declined at similar rates in amended soils under dynamic field conditions.The Random Forest model accurately predicted changes in viable E. coli concentrations over time under different agricultural and environmental conditions. Our model also accurately characterized the variability of E. coli concentration in amended soil over time by providing upper and lower prediction bound estimates. Cross-validation results indicated that our model can be potentially generalized to other geographic regions and incorporated into a risk assessment for evaluating the risks associated with application of untreated BSAAO. Our model can be validated for other regions and predictive performance also can be enhanced when data sets from additional geographic regions become available.
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http://dx.doi.org/10.1111/risa.13491DOI Listing
July 2020

Influence of Poultry Litter Amendment Type and Irrigation Events on Survival and Persistence of Salmonella Newport.

J Food Prot 2020 May;83(5):821-828

Department of Animal and Food Sciences, University of Delaware, Newark, Delaware 19716 (ORCID: https://orcid.org/0000-0002-2673-0728 [P.K.L.]); and.

Abstract: Salmonella enterica subsp. enterica serovar Newport is a bacterial foodborne pathogen isolated from several environmental reservoirs on the Delmarva Peninsula and has been associated with several produce-related outbreaks. However, little is known about specific interactions between Salmonella Newport and soil amendments used as fertilizers. The purpose of this study was to determine Salmonella Newport persistence and resuscitation in raw poultry litter (PLR), a common biological soil amendment, and in soils containing poultry litter-based (heat-treated poultry pellets [HTPP]) or chemical fertilizer (urea [U]) amendments to provide equivalent levels of nitrogen to the soil. Inoculated samples were stored in a growth chamber and irrigated regularly over 4 weeks. Soil samples were collected every week for 4 weeks to determine moisture content and surviving Salmonella Newport populations (log CFU per gram dry weight). Data were analyzed by using a one-way analysis of variance and Student's t test. The PLR supported significantly higher (5.07 log CFU/g dry weight [gdw]) populations of Salmonella Newport than HTPP only (1.70 log CFU/gdw). However, PLR-amended (PRLA) soil (2.5 log CFU/gdw) samples had significantly (P < 0.05) lower Salmonella Newport populations compared with HTPP-amended (4.5 log CFU/gdw) and U-amended (4.0 log CFU/gdw) soil samples. The effect of irrigation on Salmonella Newport population levels in PRLA soils was significant, and in a comparative study, the overall increase in the pathogen levels in U-amended soil (mean = 1.12 log CFU/gdw) was significantly greater than that in PLRA soil (mean = 0.54 log CFU/gdw), whereas that in HTPP-amended soil (0.80 log CFU/gdw) was not significantly different from PLRA soil.

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http://dx.doi.org/10.4315/0362-028X.JFP-19-431DOI Listing
May 2020

Prevalence of Salmonella and Listeria monocytogenes in non-traditional irrigation waters in the Mid-Atlantic United States is affected by water type, season, and recovery method.

PLoS One 2020 17;15(3):e0229365. Epub 2020 Mar 17.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, United States of America.

Irrigation water contaminated with Salmonella enterica and Listeria monocytogenes may provide a route of contamination of raw or minimally processed fruits and vegetables. While previous work has surveyed specific and singular types of agricultural irrigation water for bacterial pathogens, few studies have simultaneously surveyed different water sources repeatedly over an extended period of time. This study quantified S. enterica and L. monocytogenes levels (MPN/L) at 6 sites, including river waters: tidal freshwater river (MA04, n = 34), non-tidal freshwater river, (MA05, n = 32), one reclaimed water holding pond (MA06, n = 25), two pond water sites (MA10, n = 35; MA11, n = 34), and one produce wash water site (MA12, n = 10) from September 2016-October 2018. Overall, 50% (84/168) and 31% (53/170) of sampling events recovered S. enterica and L. monocytogenes, respectively. Results showed that river waters supported significantly (p < 0.05) greater levels of S. enterica than pond or reclaimed waters. The non-tidal river water sites (MA05) with the lowest water temperature supported significantly greater level of L. monocytogenes compared to all other sites; L. monocytogenes levels were also lower in winter and spring compared to summer seasons. Filtering 10 L of water through a modified Moore swab (MMS) was 43.5 (Odds ratio, p < 0.001) and 25.5 (p < 0.001) times more likely to recover S. enterica than filtering 1 L and 0.1 L, respectively; filtering 10 L was 4.8 (p < 0.05) and 3.9 (p < 0.05) times more likely to recover L. monocytogenes than 1L and 0.1 L, respectively. Work presented here shows that S. enterica and L. monocytogenes levels are higher in river waters compared to pond or reclaimed waters in the Mid-Atlantic region of the U.S., and quantitatively shows that analyzing 10 L water is more likely recover pathogens than smaller samples of environmental waters.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229365PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077874PMC
June 2020

Pharmaceuticals, herbicides, and disinfectants in agricultural water sources.

Environ Res 2019 07 17;174:1-8. Epub 2019 Apr 17.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD 20742, USA. Electronic address:

Agricultural water withdrawals account for the largest proportion of global freshwater use. Increasing municipal water demands and droughts are straining agricultural water supplies. Therefore, alternative solutions to agricultural water crises are urgently needed, including the use of nontraditional water sources such as advanced treated wastewater or reclaimed water, brackish water, return flows, and effluent from produce processing facilities. However, it is critical to ensure that such usage does not compromise soil, crop, and public health. Here, we characterized five different nontraditional water types (n = 357 samples) for the presence of pharmaceuticals, herbicides, and disinfectants using ultra-high-pressure liquid chromatography tandem mass spectrometry based method (UPLC-MS/MS). We then evaluated whether the levels of these contaminants were influenced by season. The highest level of herbicides (atrazine) was detected in untreated pond water (median concentration 135.9 ng/L). Reclaimed water had the highest levels of antibiotics and stimulants including azithromycin (215 ng/L), sulfamethoxazole (232.1 ng/L), and caffeine (89.4 ng/L). Produce processing plant water also tended to have high levels of atrazine (102.7 ng/L) and ciprofloxacin (80.1 ng/L). In addition, we observed seasonal variability across water types, with the highest atrazine concentrations observed during summer months, while the highest median azithromycin concentrations were observed in reclaimed water during the winter season. Further studies are needed to evaluate if economically feasible on-farm water treatment technologies can effectively remove such contaminants from nontraditional irrigation water sources.
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http://dx.doi.org/10.1016/j.envres.2019.04.011DOI Listing
July 2019

Zero-valent iron sand filtration reduces concentrations of virus-like particles and modifies virome community composition in reclaimed water used for agricultural irrigation.

BMC Res Notes 2019 Apr 11;12(1):223. Epub 2019 Apr 11.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, School of Public Health Building (255), 4200 Valley Drive, Room 2234P, College Park, MD, 20742, USA.

Objective: Zero-valent iron sand filtration can remove multiple contaminants, including some types of pathogenic bacteria, from contaminated water. However, its efficacy at removing complex viral populations, such as those found in reclaimed water used for agricultural irrigation, has not been fully evaluated. Therefore, this study utilized metagenomic sequencing and epifluorescent microscopy to enumerate and characterize viral populations found in reclaimed water and zero-valent iron-sand filtered reclaimed water sampled three times during a larger greenhouse study.

Results: Zero-valent iron-sand filtered reclaimed water samples had significantly less virus-like particles than reclaimed water samples at all collection dates, with the reclaimed water averaging between 10 and 10 and the zero-valent iron-sand filtered reclaimed water averaging between 10 and 10 virus-like particles per mL. In addition, for both sample types, viral metagenomes (viromes) were dominated by bacteriophages of the order Caudovirales, largely Siphoviridae, and genes related to DNA metabolism. However, the proportion of sequences homologous to bacteria, as well as the abundance of genes possibly originating from a bacterial host, was higher in the viromes of zero-valent iron-sand filtered reclaimed water samples. Overall, zero-valent iron-sand filtered reclaimed water had a lower total concentration of virus-like particles and a different virome community composition compared to unfiltered reclaimed water.
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http://dx.doi.org/10.1186/s13104-019-4251-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458639PMC
April 2019

The efficacy of zero valent iron-sand filtration on the reduction of Escherichia coli and Listeria monocytogenes in surface water for use in irrigation.

Environ Res 2019 06 19;173:33-39. Epub 2019 Feb 19.

Department of Animal and Food Science, University of Delaware, Newark, DE, USA. Electronic address:

The use of surface and recycled water sources for irrigation can reduce demand on critical groundwater resources. Treatment or mitigation may be necessary for the use of these alternative water sources in order to reduce risk associated with microbial pathogens present in the water. In this study, the efficacy of a zero-valent iron (ZVI) sand filter was assessed for the reduction of Listeria monocytogenes and Escherichia coli in surface water. Water recovered from an agricultural pond was inoculated with E. coli TVS353 and an environmental L. monocytogenes isolate at 7 Log CFU/mL and horizontally filtered over a six-month period through a PVC pipe filter, filled with 35%:65% (volume:volume) ZVI:sand or sand alone. Filtered water was used to irrigate lettuce and bacterial persistence on lettuce leaves was determined for 7 days post-irrigation. Both ZVI:sand-filtered water and sand-filtered water contained significantly (p < 0.005) lower levels of E. coli and L. monocytogenes compared to initial unfiltered inoculated water. Population reductions of E. coli and L. monocytogenes were comparable after sand filtration. However, ZVI:sand filtration resulted in significantly greater population reductions of L. monocytogenes (P < 0.05) compared to E. coli. Populations of E. coli on leaves of lettuce plants irrigated with ZVI:sand-filtered water were not significantly lower than populations on plants irrigated with sand-filtered irrigation water over the 7-day period. However, populations of L. monocytogenes on lettuce leaves irrigated with ZVI-treated water were significantly lower than counts on plants irrigated with sand-filtered irrigation water on days 3 and 4 post irrigation (p = 0.052 and p = 0.042 for days 3 and 4, respectively. The differences observed in reductions of L. monocytogenes and E. coli by ZVI filtration is due to the differing effect that ZVI disruption has on Gram-positive and Gram-negative cell walls and membranes. ZVI- sand filters show promising results as an inexpensive on-farm technology for the mitigation of enteric foodborne bacterial populations in pond water over a six-month period.
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http://dx.doi.org/10.1016/j.envres.2019.02.028DOI Listing
June 2019

Prevalence of Shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S.

Environ Res 2019 05 13;172:630-636. Epub 2019 Feb 13.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, United States.

The microbial quality of irrigation water has increasingly become a concern as a source of contamination for fruits and vegetables. Non-traditional sources of water are being used by more and more growers in smaller, highly diversified farms in the Mid-Atlantic region of the U.S. Shiga-toxigenic E. coli (STEC) have been responsible for several outbreaks of infections associated with the consumption of leafy greens. Our study evaluated the prevalence of the "big seven" STEC serogroups and the associated enterohemorrhagic E. coli (EHEC) virulence factors (VF) genes in conventional and nontraditional irrigation waters in the Mid-Atlantic region of the U.S. Water samples (n = 510) from 170 sampling events were collected from eight untreated surface water sites, two wastewater reclamation facilities, and one vegetable processing plant, over a 12-month period. Ten liters of water were filtered through Modified Moore swabs (MMS); swabs were then enriched into Universal Pre-enrichment Broth (UPB), followed by enrichment into non-O157 STEC R&F broth and isolation on R & F non-O157 STEC chromogenic plating medium. Isolates (n = 2489) from enriched MMS from water samples were screened for frequently reported STEC serogroups that cause foodborne illness: O26, O45, O103, O111, O121, O145, and O157, along with VF genes stx1, stx2, eae, and ehxA. Through this screening process, STEC isolates were found in 2.35% (12/510) of water samples, while 9.0% (46/510) contained an atypical enteropathogenic E. coli (aEPEC) isolate. The eae gene (n = 88 isolates) was the most frequently detected EHEC VF of the isolates screened. The majority of STEC isolates (stx1 or stx2) genes mainly came from either a pond or reclamation pond water site on two specific dates, potentially indicating that these isolates were not spatially or temporally distributed among the sampling sites. STEC isolates at reclaimed water sites may have been introduced after wastewater treatment. None of the isolates containing eae were determined to be Escherichia albertii. Our work showed that STEC prevalence in Mid-Atlantic untreated surface waters over a 12-month period was lower than the prevalence of atypical EPEC.
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http://dx.doi.org/10.1016/j.envres.2019.02.019DOI Listing
May 2019

in Soils Amended with Heat-Treated Poultry Pellets Survived Longer than Bacteria in Unamended Soils and More Readily Transferred to and Persisted on Spinach.

Appl Environ Microbiol 2019 05 2;85(10). Epub 2019 May 2.

U.S. Department of Agriculture, Agricultural Research Service, Northeast Area, Environmental Microbial Food Safety Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland, USA

Untreated biological soil amendments of animal origin (BSAAO) are commonly used as biological fertilizers but can harbor foodborne pathogens like , leading to potential transfer from soils to fruits and vegetables intended for human consumption. Heat-treated poultry pellets (HTPP) can provide produce growers with a slow-release fertilizer with a minimized risk of pathogen contamination. Little is known about the impact of HTPP-amended soil on the survival of The contributions of RpoS and formation of viable but nonculturable cells to survival in soils are also inadequately understood. We quantified the survival of subsp. serovar Newport wild-type (WT) and -deficient (Δ mutant) strains in HTPP-amended and unamended soil with or without spinach plants over 91 days using culture and quantitative PCR methods with propidium monoazide (PMA-qPCR). Simulated "splash" transfer of Newport from soil to spinach was evaluated at 35 and 63 days postinoculation (dpi). The Newport WT and Δ mutant reached the limit of detection, 1.0 log CFU/g (dry weight), in unamended soil after 35 days, whereas 2 to 4 log CFU/g (dry weight) was observed for both WT and Δ mutant strains at 91 dpi in HTPP-amended soil. Newport levels in soils determined by PMA-qPCR and plate count methods were similar ( > 0.05). HTPP-amended soils supported higher levels of Newport transfer to and survival on spinach leaves for longer periods of time than did unamended soils ( < 0.05). Newport introduced to HTPP-amended soils survived for longer periods and was more likely to transfer to and persist on spinach plants than was Newport introduced to unamended soils. Heat-treated poultry pellets (HTPP) often are used by fruit and vegetable growers as a slow-release fertilizer. However, contamination of soil on farms may occur through contaminated irrigation water or scat from wild animals. Here, we show that the presence of HTPP in soil led to increased Newport survival in soil and to greater likelihood of its transfer to and survival on spinach plants. There were no significant differences in survival durations of WT and Δ mutant isolates of Newport. The statistically similar populations recovered by plate count and estimated by PMA-qPCR for both strains in the amended and unamended soils in this study indicate that all viable populations of Newport in soils were culturable.
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http://dx.doi.org/10.1128/AEM.00334-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498161PMC
May 2019

Zerovalent iron-sand filtration can reduce the concentration of multiple antimicrobials in conventionally treated reclaimed water.

Environ Res 2019 05 10;172:301-309. Epub 2019 Feb 10.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, 4200 Valley Drive, College Park, MD 20742, USA. Electronic address:

Irrigation with reclaimed water is increasing in areas that lack access to, and infrastructure for, high-level treatment and distribution. Antimicrobial residues are known to persist in conventionally treated reclaimed water, necessitating the investigation of reuse site-based mitigation options to further reduce these contaminants. We examined the effectiveness of a 50:50 volume/volume, particle matched, micro-scale zerovalent iron (ZVI)-sand filter in reducing concentrations of mixtures of antimicrobials present in pH-unadjusted conventionally treated reclaimed water. Twelve antimicrobials (azithromycin, ciprofloxacin, erythromycin, linezolid, oxacillin, oxolinic acid, penicillin G, pipemidic acid, sulfamethoxazole, triclocarban, tetracycline and vancomycin) were quantified using high performance-liquid chromatography-tandem mass spectrometry in reclaimed water, and ZVI-sand filtered reclaimed water, in a two-month long greenhouse-based experiment. Data were analyzed using a non-parametric rank-based approach. ZVI-sand filtration significantly reduced concentrations of azithromycin, ciprofloxacin, oxolinic acid, penicillin G, sulfamethoxazole, linezolid, pipemidic acid and vancomycin. Azithromycin, the antimicrobial with the highest median concentration (320 ng/L), was reduced to below the limit of detection after ZVI-sand filtration. Inorganic element (antimony, beryllium, cadmium, chromium, iron, lead, selenium and thallium) and water quality (free and total chlorine, nitrates, nitrites, pH and total dissolved solids) analyses showed that ZVI-sand filtered reclaimed water quality (nitrate, salinity, and inorganic elements) met the recommended guidelines for agricultural irrigation with reclaimed water. Based on our initial results, ZVI-sand filtration may be a promising basis for a point-of-use filtration system for reclaimed water irrigation on small-scale farms.
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http://dx.doi.org/10.1016/j.envres.2019.02.012DOI Listing
May 2019

Survival and Growth of Wild-Type and rpoS-Deficient Salmonella Newport Strains in Soil Extracts Prepared with Heat-Treated Poultry Pellets.

J Food Prot 2019 Mar;82(3):501-506

2 U.S. Department of Agriculture, Agricultural Research Service, Northeast Area, Beltsville Agricultural Research Center, Environmental Microbial Food Safety Laboratory, Beltsville, Maryland 20705 (ORCID: http://orcid.org/000-0002-8585-0308 [M.S.]).

Manure runoff can transfer pathogens to farmlands or to water sources, leading to subsequent contamination of produce. Untreated biological soil amendments, like manure, can be contaminated with foodborne pathogens, such as Salmonella Newport, which may lead to transfer of the pathogen to fruits or vegetables. Studies have reported the occurrence and survival of Salmonella in manure or manure slurries. However, data on the survival and growth of Salmonella Newport is lacking in matrices simulating runoff. We quantified the survival and growth of wild-type (WT) Salmonella Newport and rpoS-deficient (Δ rpoS) strains in sterile and nonsterile soil extracts prepared with (amended) or without (unamended) heat-treated poultry pellets at 25°C. Salmonella Newport WT and Δ rpoS populations reached a maximum cell density of 6 to 8 log CFU/mL in 24 to 30 h in amended and unamended soil extracts and remained in stationary phase for up to 4 days. Salmonella Newport in amended soil extracts exhibited a decreased lag phase (λ , 2.87 ± 1.01 h) and greater maximum cell densities ( N, 6.84 ± 1.25 CFU/mL) compared with λ (20.10 ± 9.53 h) and N (5.22 ± 0.82 CFU/mL) in unamended soil extracts. In amended soil extract, the Δ rpoS strain had no measurable λ , similar growth rates (μ) compared with WT, and a lower N compared with the WT strain. Unamended, nonsterile soil extracts did not support the growth of Salmonella Newport WT and led to a decline in populations for the Δ rpoS strain. Salmonella Newport had lower cell densities in nonsterile soil extracts (5.94 ± 0.95 CFU/mL) than it did in sterile soil extracts (6.66 ± 1.50 CFU/mL), potentially indicating competition for nutrients between indigenous microbes and Salmonella Newport. The most favorable growth conditions were provided by amended sterile and nonsterile soil extracts, followed by sterile, unamended soil extracts for both Salmonella Newport strains. Salmonella Newport may grow to greater densities in amended extracts, providing a route for increased Salmonella levels in the growing environments of produce.
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http://dx.doi.org/10.4315/0362-028X.JFP-18-465DOI Listing
March 2019

Incidence of fecal indicator and pathogenic bacteria in reclaimed and return flow waters in Arizona, USA.

Environ Res 2019 03 1;170:122-127. Epub 2018 Dec 1.

School of Animal and Comparative Biomedical Sciences, University of Arizona, 1117 E. Lowell Street, Tucson, AZ 85721, United States. Electronic address:

The quality of irrigation water used to cultivate produce that is consumed raw is an important issue with regard to food safety. In this study, the microbiological quality of potential irrigation water sources in Arizona was evaluated by testing for the presence of indicator and pathogenic bacteria. Reclaimed water samples were collected from two wastewater treatment plants and return flow samples were collected from two drainage canals and one return flow pond. Standard membrane filtration methods were used for detection of indicator bacteria. Water samples (n = 28) were filtered through cellulose ester membrane filters and bacterial populations were enumerated by placing the filters on selective agar. For detection of pathogens (Salmonella enterica, Listeria monocytogenes and Shiga toxin-producing E. coli (STEC)), water samples were filtered through Modified Moore swabs and enriched in Universal Pre-enrichment Broth, followed by selective enrichment broth for each pathogen. The enriched broth was streaked onto agar media selective for each pathogen. Presumptive colonies were confirmed by PCR/real-time PCR. Among the 14 reclaimed water samples from two sites, the ranges of recovered populations of E. coli, total coliforms, and enterococci were 0-1.3, 0.5-8.3 × 10, and 0-5.5 CFU/100 mL, respectively. No L. monocytogenes, Salmonella or STEC were found. In the 13 return flow water samples from 3 sites, the ranges of recovered populations of E. coli, total coliforms and enterococci were 1.9-5.3 × 10, 6.5 × 10-9.1 × 10, and 2.9-3.7× 10 CFU/100 mL, respectively. All samples were negative for L. monocytogenes. One (7.1%) of the return flow samples was positive for E. coli O145. Nine (64.3%) of the samples were positive for Salmonella. Both real-time PCR and culture-based methods were used for the detection of Salmonella and L. monocytogenes, and the results from the two methods were comparable. The findings of this study provide evidence that irrigation waters in Arizona, including reclaimed water and return flows, could be potential sources of bacterial contamination of produce. Additional work is needed to evaluate whether bacteria present in irrigation water sources transfer to the edible portion of irrigated plants and are capable of persisting through post-harvest activities.
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http://dx.doi.org/10.1016/j.envres.2018.11.048DOI Listing
March 2019

Survival of in Manure-Amended Soils Is Affected by Spatiotemporal, Agricultural, and Weather Factors in the Mid-Atlantic United States.

Appl Environ Microbiol 2019 03 20;85(5). Epub 2019 Feb 20.

University of Maryland Eastern Shore Department of Agriculture, Princess Anne, Maryland, USA.

Untreated biological soil amendments of animal origin (BSAAO), such as manure, are commonly used to fertilize soils for growing fruit and vegetable crops and can contain enteric bacterial foodborne pathogens. Little is known about the comparative longitudinal survival of pathogens in agricultural fields containing different types of BSAAO, and field data may be useful to determine intervals between manure application and harvest of produce intended for human consumption to minimize foodborne illness. This study generated 324 survival profiles from 12 different field trials at three different sites (UMES, PA, and BARC) in the Mid-Atlantic United States from 2011 to 2015 of inoculated nonpathogenic (gEc) and attenuated O157 (attO157) in soils which were unamended (UN) or amended with untreated poultry litter (PL), horse manure (HM), or dairy manure solids (DMS) or liquids (DML). Site, season, inoculum level (low/high), amendment type, management (organic/conventional), and depth (surface/tilled) all significantly ( < 0.0001) influenced survival duration (dpi100mort). Spatiotemporal factors (site, year, and season) in which the field trial was conducted influenced survival durations of gEc and attO157 to a greater extent than weather effects (average daily temperature and rainfall). Initial soil moisture content was the individual factor that accounted for the greatest percentage of variability in survival duration. PL supported greater survival durations of gEc and attO157, followed by HM, UN, and DMS in amended soils. The majority of survival profiles for gEc and attO157 which survived for more than 90 days came from a specific year (i.e., 2013). The effect of management and depth on dpi100mort were dependent on the amendment type evaluated. Current language in the Food Safety Modernization Act Produce Safety Rule states no objection to a 90- or 120-day interval between application of untreated BSAAO and harvest of crops to minimize transfer of pathogens to produce intended for human consumption with the intent to limit potential cases of foodborne illness. This regional multiple season, multiple location field trial determined survival durations of in soils amended with manure to determine whether this interval is appropriate. Spatiotemporal factors influence survival durations of more than amendment type, total amount of present, organic or conventional soil management, and depth of manure application. Overall, these data show poultry litter may support extended survival of compared to horse manure or dairy manure, but spatiotemporal factors like site and season may have more influence than manure type in supporting survival of beyond 90 days in amended soils in the Mid-Atlantic United States.
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http://dx.doi.org/10.1128/AEM.02392-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384115PMC
March 2019

Composting To Inactivate Foodborne Pathogens for Crop Soil Application: A Review.

J Food Prot 2018 11;81(11):1821-1837

4 U.S. Department of Agriculture, Agricultural Research Service, 10300 Baltimore Avenue, Building 201, Beltsville Area Research Center-East, Beltsville, Maryland 20705, USA (ORCID: http://orcid.org/0000-0002-8585-0308 [M.S.]).

Compost is organic material that has been degraded into a nutrient-stabilized humus-like substance through intense microbial activity, which can provide essential plant nutrients (nitrogen, phosphorus) to aid in the growth of fruits and vegetables. Compost can be generated from animal waste feedstocks; these can contain human pathogens, which can be inactivated through the heat and microbial competition promoted during the composting process. Outbreaks of infections caused by bacterial pathogens such as Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on fruit and vegetable commodities consumed raw emphasize the importance of minimizing the risk of pathogenic contamination on produce commodities. This review article investigates factors that affect the reduction and survival of bacterial foodborne pathogens during the composting process. Interactions with indigenous microorganisms, carbon:nitrogen ratios, and temperature changes influence pathogen survival, growth, and persistence in finished compost. Understanding the mechanisms of pathogen survival during the composting process and mechanisms that reduce pathogen populations can minimize the risk of pathogen contamination in the cultivation of fruits and vegetables.
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http://dx.doi.org/10.4315/0362-028X.JFP-18-217DOI Listing
November 2018

Conventional wastewater treatment and reuse site practices modify bacterial community structure but do not eliminate some opportunistic pathogens in reclaimed water.

Sci Total Environ 2018 Oct 26;639:1126-1137. Epub 2018 May 26.

Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health 4200 Valley Drive, College Park, MD 20742, United States. Electronic address:

Water recycling continues to expand across the United States, from areas that have access to advanced, potable-level treated reclaimed water, to those having access only to reclaimed water treated at conventional municipal wastewater treatment plants. This expansion makes it important to further characterize the microbial quality of these conventionally-treated water sources. Therefore, we used 16S rRNA gene sequencing to characterize total bacterial communities present in differentially-treated wastewater and reclaimed water (n = 67 samples) from four U.S. wastewater treatment plants and one associated spray irrigation site conducting on-site ultraviolet treatment and open-air storage. The number of observed operational taxonomic units was significantly lower (p < 0.01) in effluent, compared to influent, after conventional treatment. Effluent community structure was influenced more by treatment method than by influent community structure. The abundance of Legionella spp. increased as treatment progressed in one treatment plant that performed chlorination and in another that seasonally chlorinated. Overall, the alpha-diversity of bacterial communities in reclaimed water decreased (p < 0.01) during wastewater treatment and spray irrigation site ultraviolet treatment (p < 0.01), but increased (p < 0.01) after open-air storage at the spray irrigation site. The abundance of Legionella spp. was higher at the sprinkler system pumphouse at the spray irrigation site than in the influent from the treatment plant supplying the site. Legionella pneumophila was detected in conventionally treated effluent samples and in samples collected after ultraviolet treatment at the spray irrigation site, while Legionella feeleii persisted throughout on-site treatment at the spray irrigation site, and, along with Mycobacterium gordonae, was also detected at the sprinkler system pumphouse at the spray irrigation site. These data could inform the development of future treatment technologies and reuse guidelines that address a broader assemblage of the bacterial community of reclaimed water, resulting in reuse practices that may be more protective of public health.
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http://dx.doi.org/10.1016/j.scitotenv.2018.05.178DOI Listing
October 2018

An evaluation of the virulence and adherence properties of avian pathogenic .

One Health 2017 Dec 21;4:22-26. Epub 2017 Aug 21.

Department of Animal and Food Sciences, University of Delaware, 531 South College Avenue, 044 Townsend Hall, Newark, DE 19716, United States.

Avian pathogenic (APEC) cause disease primarly in poultry; however, the link between APEC and infections in humans is questionable. In this current study, a total of 100 APEC strains isolated from chickens in Delmarva were evaluated for the presence of virulence genes to investigate their zoonotic potential in humans. A total of 28 isolates possessed one Enterohaemorrhagic (EHEC) virulence factor each and 87 isolates possessed up to 5 extraintestinal pathogenic (ExPEC) virulence factors. Five APEC isolates exhibited stronger attachment to chicken breast than both human outbreak strains tested. Ten APEC isolates exhibited stronger attachment to human epithelial cells (HCT-8) than both outbreak strains. While the APEC isolates in this study were not found to possess all the virulence genes necessary to cause clinical illness in humans, their potential to acquire these genes in the environment as well as their ability to attach to food surfaces and human cells warrants further attention.
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http://dx.doi.org/10.1016/j.onehlt.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591385PMC
December 2017

Water for Agriculture: the Convergence of Sustainability and Safety.

Microbiol Spectr 2017 05;5(3)

Environmental Microbial Food Safety Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705.

Agricultural water is a precious and limited resource. Increasingly more water types and sources are being explored for use in irrigation within the United States and across the globe. As outlined in this chapter, the Produce Safety Rule (PSR) in the Food Safety and Modernization Act (FSMA) provide irrigation water standards for application of water to fruits and vegetables consumed raw. These rules for production and use of water will continue to develop and be required as the world experiences aspects of a changing climate including flooding as well as drought conditions. Research continues to assess the use of agricultural water types. The increased use of reclaimed water in the United States as well as for selected irrigation water needs for specific crops may provide increased water availability. The use of surface water can be used in irrigation as well, but several studies have shown the presence of some enteric bacterial pathogens (enterohemorrhagic , spp. and ) in these waters that may contaminate fruits and vegetables. There have been outbreaks of foodborne illness in the U.S., South America, Europe, and Australia related to the use of contaminated water in fruit and vegetable irrigation or washing. Unreliable water supplies, more stringent microbial water standards, mitigation technologies and expanded uses of reclaimed waters have all increased interest in agricultural water.
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http://dx.doi.org/10.1128/microbiolspec.PFS-0014-2016DOI Listing
May 2017

Survival of Salmonella Newport on Whole and Fresh-Cut Cucumbers Treated with Lytic Bacteriophages.

J Food Prot 2017 04;80(4):668-673

1 U.S. Department of Agriculture, Agricultural Research Service, Northeast Area, Environmental Microbial and Food Safety Laboratory, Beltsville, Maryland 20705.

Salmonella enterica associated with consumption of cucumbers ( Cucumis sativus ) has led to foodborne outbreaks in the United States. Whole and fresh-cut cucumbers are susceptible to S. enterica contamination during growing, harvesting, and postharvest handling. The application of lytic bacteriophages specific for S. enterica was evaluated to reduce Salmonella populations on cucumbers. Unwaxed cucumbers ('Lisboa' variety, or mini-cucumbers purchased at retail) were inoculated with Salmonella Newport (5 log CFU per cucumber) and were sprayed with 3.2 mL of phosphate-buffered saline (control) or 10 log PFU/ml of SalmoFresh, a Salmonella-specific bacteriophage preparation (phage), to deliver 4.76 × 10 PFU/cm. Cucumbers were stored at 10 or 22°C for 7 days. Inoculated mini-cucumbers were sliced with a sterile knife to investigate Salmonella transfer to mesocarp, and cut pieces were stored at 4°C for 2 days. Populations (log CFU per cucumber) of Salmonella Newport on phage-treated whole cucumbers were significantly (P < 0.05) smaller (2.44 ± 0.94) than on control-treated cucumbers (4.27 ± 0.37) on day 0. Populations on phage-treated cucumbers stored at 10°C were 1.72 ± 0.77 and 1.56 ± 0.46, which were significantly lower than those on control-treated cucumbers (3.20 ± 0.48 and 2.33 ± 0.25) on days 1 and 4, respectively. Between days 0 and 1, populations on control-treated cucumbers stored at 10 and 22°C declined by 1.07 and 2.47 log CFU per cucumber, respectively. At 22°C, Salmonella Newport populations declined by 2.37 log CFU per cucumber between days 0 and 1. Phage application to whole cucumbers before slicing did not reduce the transfer of Salmonella Newport to fresh-cut slices. Lytic phage application may be a potential intervention to reduce Salmonella populations on whole cucumbers.
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http://dx.doi.org/10.4315/0362-028X.JFP-16-449DOI Listing
April 2017

Importance of Soil Amendments: Survival of Bacterial Pathogens in Manure and Compost Used as Organic Fertilizers.

Microbiol Spectr 2016 08;4(4)

University of Maryland Eastern Shore, Department of Agriculture, Food, and Resource Science, Princess Anne, MD 21853.

Biological soil amendments (BSAs) such as manure and compost are frequently used as organic fertilizers to improve the physical and chemical properties of soils. However, BSAs have been known to be a reservoir for enteric bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC), Salmonella spp., and Listeria spp. There are numerous mechanisms by which manure may transfer pathogens to growing fruits and vegetables, and several outbreaks of infections have been linked to manure-related contamination of leafy greens. In the United States several commodity-specific guidelines and current and proposed federal rules exist to provide guidance on the application of BSAs as fertilizers to soils, some of which require an interval between the application of manure to soils and the harvest of fruits and vegetables. This review examines the survival, persistence, and regrowth/resuscitation of bacterial pathogens in manure, biosolids, and composts. Moisture, along with climate and the physicochemical properties of soil, manure, or compost, plays a significant role in the ability of pathogens to persist and resuscitate in amended soils. Adaptation of enteric bacterial pathogens to the nonhost environment of soils may also extend their persistence in manure- or compost-amended soils. The presence of antibiotic-resistance genes in soils may also be increased by manure application. Overall, BSAs applied as fertilizers to soils can support the survival and regrowth of pathogens. BSAs should be handled and applied in a manner that reduces the prevalence of pathogens in soils and the likelihood of transfer of food-borne pathogens to fruits and vegetables. This review will focus on two BSAs-raw manure and composted manure (and other feedstocks)-and predominantly on the survival of enteric bacterial pathogens in BSAs as applied to soils as organic fertilizers.
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http://dx.doi.org/10.1128/microbiolspec.PFS-0010-2015DOI Listing
August 2016

Survival and Growth of Listeria monocytogenes on Fresh-Cut "Athena" and "Rocky Ford" Cantaloupes During Storage at 4°C and 10°C.

Foodborne Pathog Dis 2016 11 22;13(11):587-591. Epub 2016 Aug 22.

2 Environmental Microbial and Food Safety Laboratory, United States Department of Agriculture, Agricultural Research Service , Beltsville, Maryland.

Cantaloupes, marketed as "Rocky Ford," were implicated in the U.S. multistate outbreak of listeriosis in 2011, which caused multiple fatalities. Listeria monocytogenes can survive on whole cantaloupes and can be transferred to the flesh of melons. The growth of L. monocytogenes on fresh-cut "Athena" and "Rocky Ford" cantaloupe cultivars during refrigerated storage was evaluated. Fresh-cut cubes (16.4 cm) from field-grown cantaloupes were each inoculated with 5 log CFU/mL of a multi-strain mixture of L. monocytogenes and stored at 4°C or 10°C. Inoculated fresh-cut cubes were also: (1) continuously stored at 4°C for 3 days; (2) temperature-abused (TA: 25°C for 4 h) on day 0; or (3) stored at 4°C for 24 h, exposed to TA on day 1, and subsequently stored at 4°C until day 3. L. monocytogenes populations on fresh-cut melons continuously stored at 4°C or 10°C were enumerated on selected days for up to 15 days and after each TA event. Brix values for each cantaloupe variety were determined. L. monocytogenes populations on fresh-cut cantaloupe cubes stored at 4°C increased by 1.0 and 3.0 log CFU/cube by day 7 and 15, respectively, whereas those stored at 10°C increased by 3.0 log CFU/cube by day 7. Populations of L. monocytogenes on fresh-cut cantaloupes stored at 10°C were significantly (p < 0.05) greater than those stored at 4°C during the study. L. monocytogenes showed similar growth on fresh-cut "Athena" and "Rocky Ford" cubes, even though "Athena" cubes had significantly higher Brix values than the "Rocky Ford" fruit. L. monocytogenes populations on fresh-cut cantaloupes exposed to TA on day 1 and then refrigerated were significantly greater (0.74 log CFU) than those stored continuously at 4°C for 3 days. Storage at 10°C or exposure to TA events promoted growth of L. monocytogenes on fresh-cut cantaloupe during refrigerated storage.
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http://dx.doi.org/10.1089/fpd.2016.2160DOI Listing
November 2016

Survival and growth of Listeria monocytogenes on whole cantaloupes is dependent on site of contamination and storage temperature.

Int J Food Microbiol 2016 Oct 25;234:65-70. Epub 2016 Jun 25.

United States Department of Agriculture, Agricultural Research Service, Environmental Microbial and Food Safety Laboratory, Beltsville, MD 20705, USA. Electronic address:

Whole cantaloupes (Cucumis melo L.), marketed as 'Rocky Ford', were implicated in a large multi-state outbreak of listeriosis in the United States in 2011; however, survival and growth of Listeria monocytogenes on whole cantaloupes remains relatively unexplored. The research presented here evaluated three different storage temperatures, two sites of contamination of cantaloupes, and two cantaloupe varieties to determine their effect on the survival of L. monocytogenes. 'Athena' and 'Rocky Ford' cantaloupe cultivars were grown in soil and harvested, and individual melons subsequently received a multi-strain inoculum of L. monocytogenes (6 log CFU/melon), which were then stored at 4°C, 10°C, and 25°C. Changes in L. monocytogenes populations on the rinds and stem scars of cantaloupes stored at each temperature were determined at selected times for up to 15days. An analysis of variance revealed that inoculation site and storage temperature significantly affected survival of L. monocytogenes on cantaloupes during storage (p<0.05), but cultivar did not influence L. monocytogenes (p>0.05). Populations of L. monocytogenes on stem scars of cantaloupes stored at 25°C increased by 1-2 log CFU/melon on day 1, and were significantly greater than those on cantaloupes stored at 4°C or 10°C (p<0.05), which remained constant or increased by approximately 0.3 log CFU/melon, respectively, over the same time period. A decrease of 2-5 log CFU/melon of L. monocytogenes occurred on the rinds of cantaloupes during storage by day 7, and were not significantly different at the three different storage temperatures (p>0.05). In trials performed in rind juice extracts, populations of L. monocytogenes decreased by 3 log CFU/mL when stored at 25°C by day 3, but grew by 3-4 log CFU/mL when stored at 4°C over 7days. Overall, site of contamination and storage temperature influenced the survival of L. monocytogenes on cantaloupes more than cantaloupe cultivar type.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2016.06.030DOI Listing
October 2016

Survival and Persistence of Nonpathogenic Escherichia coli and Attenuated Escherichia coli O157:H7 in Soils Amended with Animal Manure in a Greenhouse Environment.

J Food Prot 2016 06;79(6):913-21

Department of Agriculture, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA.

Animal manure provides benefits to agriculture but may contain pathogens that contaminate ready-to-eat produce. U.S. Department of Agriculture National Organic Program standards include 90- or 120-day intervals between application of manure and harvest of crop to minimize risks of pathogen contamination of fresh produce. Data on factors affecting survival of Escherichia coli in soils under greenhouse conditions are needed. Three separate studies were conducted to evaluate survival of nonpathogenic E. coli (gEc) and attenuated E. coli O157:H7 (attO157) inoculated at either low (4 log CFU/ml) or high (6 log CFU/ml) populations over 56 days. Studies involved two pot sizes (small, 398 cm(3); large, 89 liters), three soil types (sandy loam, SL; clay loam, CL; silt loam, SIL), and four amendments (poultry litter, PL; dairy manure liquids, DML; horse manure, HM; unamended). Amendments were applied to the surface of the soil in either small or large containers. Study 1, conducted in regularly irrigated small containers, showed that populations of gEc and attO157 (2.84 to 2.88 log CFU/g) in PL-amended soils were significantly (P < 0.05) greater than those in DML-amended (0.29 to 0.32 log CFU/g [dry weight] [gdw]) or unamended (0.25 to 0.28 log CFU/gdw) soils; soil type did not affect E. coli survival. Results from study 2, in large pots with CL and SIL, showed that PL-amended soils supported significantly higher attO157 and gEc populations compared with HM-amended or unamended soils. Study 3 compared results from small and large containers that received high inoculum simultaneously. Overall, in both small and large containers, PLamended soils supported higher gEc and attO157 populations compared with HM-amended and unamended soils. Populations of attO157 were significantly greater in small containers (1.83 log CFU/gdw) than in large containers (0.65 log CFU/gdw) at week 8, perhaps because small containers received more regular irrigation than large pots. Regular irrigation of small pots may have affected E. coli persistence in manure-amended soils. Overall, PL-amended soils in both small and large containers supported E. coli survival at higher populations compared with DML-, HM-, or unamended soils.
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http://dx.doi.org/10.4315/0362-028X.JFP-15-421DOI Listing
June 2016

Metrics Proposed To Prevent the Harvest of Leafy Green Crops Exposed to Floodwater Contaminated with Escherichia coli.

Appl Environ Microbiol 2016 07 13;82(13):3746-3753. Epub 2016 Jun 13.

Department of Nutrition and Food Science, University of Maryland, College Park, Maryland, USA

Unlabelled: The California Leafy Green Products Handler Marketing Agreement (LGMA) requires leafy green crops within 9 m of the edge of a flooded field not be harvested due to potential contamination (California Leafy Green Products Handler Marketing Board, Commodity Specific Flood Safety Guidelines for the Production and Harvest of Lettuce and Leafy Greens, 2012). Further, previously flooded soils should not be replanted for 60 days. In this study, the suitability of the LGMA metrics for farms in the Mid-Atlantic region of the United States was evaluated. The upper end of a spinach bed (in Beltsville, MD) established on a -5% grade was flooded with water containing 6 log CFU/ml Escherichia coli to model a worst-case scenario of bacterial movement through soil. Escherichia coli prevalence in soil and on foliar tissue was determined by most probable number (MPN) analysis at distances up to 9 m from the edge of the flood for 63 days. While E. coli was quickly detected at the 9-m distance within 1 day in the spring trial and within 3 days in the fall trial, no E. coli was detected on plants outside the flood zone after 14 days. On day 63 for the two trials, E. coli populations in the flood zone soil were higher in the fall than in the spring. Regression analysis predicted that the time required for a 3-log MPN/g (dry weight) decrease in E. coli populations inside the flood zone was within the 60-day LGMA guideline in the spring but would require 90 days in the fall. Overall, data suggest that the current guidelines should be revised to include considerations of field and weather conditions that may promote bacterial movement and survival.

Importance: This study tracked the movement of Escherichia coli from floodwater across a horizontal plane of soil and the potential for the contamination of distant leafy green produce. The purpose of this study was to address the validity of the California Leafy Green Products Handler Marketing Agreement recommendations for the harvest of leafy green crops after a flooding event. These recommendations were based on the turning radius of farming equipment but did not take into consideration the potential subsurface movement of pathogens in the water through soil. This research shows that further considerations of field slope, temperature, and additional rainfall events may be necessary to provide appropriate guidelines to prevent the harvest of leafy green crops contaminated by enteric pathogens in floodwaters. This study may be used to provide a framework for comprehensive recommendations to growers for good harvesting practices after a flooding event.
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http://dx.doi.org/10.1128/AEM.00052-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907190PMC
July 2016

Survival of Salmonella enterica in Dried Turkey Manure and Persistence on Spinach Leaves.

J Food Prot 2015 Oct;78(10):1791-9

Department of Nutrition and Food Science, Center for Food Safety and Security Systems, College of Agriculture and Natural Resources, University of Maryland, College Park, Maryland 20742, USA.

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 μm) 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 μm) 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.
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http://dx.doi.org/10.4315/0362-028X.JFP-15-047DOI Listing
October 2015

Comparison of U.S. Environmental Protection Agency and U.S. Composting Council microbial detection methods in finished compost and regrowth potential of Salmonella spp. and Escherichia coli O157:H7 in finished compost.

Foodborne Pathog Dis 2014 Jul 9;11(7):555-67. Epub 2014 May 9.

1 Environmental Microbial and Food Safety Laboratory, Henry A. Wallace Beltsville Area Research Center, Agricultural Research Service , United States Department of Agriculture, Beltsville, Maryland.

Bacterial pathogens may survive and regrow in finished compost due to incomplete thermal inactivation during or recontamination after composting. Twenty-nine finished composts were obtained from 19 U.S. states and were separated into three broad feedstock categories: biosolids (n=10), manure (n=4), and yard waste (n=15). Three replicates of each compost were inoculated with ≈ 1-2 log CFU/g of nonpathogenic Escherichia coli, Salmonella spp., and E. coli O157:H7. The U.S. Environmental Protection Agency's (EPA) protocols and U.S. Composting Council's (USCC) Test Methods for the Examination of Composting and Compost (TMECC) were compared to determine which method recovered higher percentages of inoculated E. coli (representing fecal coliforms) and Salmonella spp. from 400-g samples of finished composts. Populations of Salmonella spp. and E. coli O157:H7 were determined over 3 days while stored at 25°C and compared to physicochemical parameters to predict their respective regrowth potentials. EPA Method 1680 recovered significantly (p=0.0003) more inoculated E. coli (68.7%) than TMECC 07.01 (48.1%) due to the EPA method using more compost in the initial homogenate, larger transfer dilutions, and a larger most probable number scheme compared to TMECC 07.01. The recoveries of inoculated Salmonella spp. by Environmental Protection Agency Method 1682 (89.1%) and TMECC 07.02 (72.4%) were not statistically significant (p=0.44). The statistically similar recovery percentages may be explained by the use of a nonselective pre-enrichment step used in both methods. No physicochemical parameter (C:N, moisture content, total organic carbon) was able to serve as a sole predictor of regrowth of Salmonella spp. or E. coli O157:H7 in finished compost. However, statistical analysis revealed that the C:N ratio, total organic carbon, and moisture content all contributed to pathogen regrowth potential in finished composts. It is recommended that the USCC modify TMECC protocols to test larger amounts of compost in the initial homogenate to facilitate greater recovery of target organisms.
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http://dx.doi.org/10.1089/fpd.2013.1698DOI Listing
July 2014

Lytic bacteriophages: Potential interventions against enteric bacterial pathogens on produce.

Authors:
Manan Sharma

Bacteriophage 2013 Apr;3(2):e25518

United States Department of Agricultural; Agricultural Research Service; Environmental Microbial and Food Safety Laboratory; Henry A Wallace Beltsville Agricultural Research Center; Beltsville, MD USA.

Foodborne illnesses resulting from the consumption of produce commodities contaminated with enteric pathogens continue to be a significant public health issue. Lytic bacteriophages may provide an effective and natural intervention to reduce bacterial pathogens on fresh and fresh-cut produce commodities. The use of multi-phage cocktails specific for a single pathogen has been most frequently assessed on produce commodities to minimize the development of bacteriophage insensitive mutants (BIM) in target pathogen populations. Regulatory approval for the use of several lytic phage products specific for bacterial pathogens such as O157:H7, spp. and in foods and on food processing surfaces has been granted by various agencies in the US and other countries, possibly allowing for the more widespread use of bacteriophages in the decontamination of fresh and minimally processed produce. Research studies have shown lytic bacteriophages specific for O157:H7, spp. and have been effective in reducing pathogen populations on leafy greens, sprouts and tomatoes.
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http://dx.doi.org/10.4161/bact.25518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821672PMC
April 2013