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Evaluation of the microbial safety and quality of Louisiana strawberries after flooding
Food Control 110 (2020) 106970
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Evaluation of the microbial safety and quality of Louisiana strawberries after flooding
T
Shifa Shiraza, Dorra Djebbi-Simmonsa, Mohammed Alhejailia, Kaleb Danosb, Marlene Janesa, Kathryn Fontenotb, Wenqing Xua,∗ a b
School of Nutrition and Food Sciences, Louisiana State University Agriculture Center, Baton Rouge, LA, 70803, USA School of Plant, Environmental and Soil Sciences, Louisiana State University Agriculture Center, Baton Rouge, LA, 70803, USA
ARTICLE INFO
ABSTRACT
Keywords: Flooding Strawberries Soil Generic E. coli Coliform
Adverse weather such as flooding affects Louisiana strawberry production. Floodwater is an ideal medium for the growth of foodborne pathogens and may cause contamination of soil, water, and fresh produce. To evaluate the microbial safety of strawberries after flooding, four flooding scenarios were investigated. Five raised beds were exposed to a simulated flood event. Treatments included High Flood High Contamination (HFHC), High Flood Low Contamination (HFLC), Low Flood High Contamination (LFHC), Low Flood Low Contamination (LFLC) and a Control (C). High flood groups (HF) were exposed to 890 L of floodwater where strawberries were fully submerged, while low flood groups (LF) were exposed to 600 L of floodwater where strawberries did not come into direct contact with the floodwater. Three strains of generic Escherichia coli were spiked into the floodwater to establish a baseline population of approximately 106 colony forming units per liter (CFU/L) (high contamination) and 102 CFU/L (low contamination). Strawberry plants in all treatments were flooded for 4 h until floodwater receded. Strawberries were harvested when mature and sampled during shelf-life at 4 °C at 0, 48, 96, and 144 h. Soil samples were collected in the field every 48 h for one week. Even though the generic E. coli was not detected (< 10 CFU/g) in strawberry samples, it was detected in soil samples within 96 h after flooding. In soil samples, generic E. coli was higher in HFHC samples (1.6 log CFU/g) compared to HFLC samples (1.1 log CFU/g) at harvest. Additionally, 1.0 to 2.8 log CFU/g of coliform were present in the strawberries and soil at 0, 48, 96, and 144 h in all treatment beds.
1. Introduction Strawberries are produced throughout the United States. California, Florida, and Oregon are the three leading states in terms of total acres grown. Even though Louisiana is not a major production state (USDA NASS, 2018), strawberries are an important fruit to the local economy. The majority of Louisiana's strawberry production occurs in Tangipahoa and Livingston parishes, where approximately 400–500 acres of strawberries are grown annually (Schloemann, 2005, pp. 1–73). However, in recent years, strawberry acreage has decreased. In 2014, eighty one growers produced 367 acres of strawberries. Despite low acreage, the strawberry industry generated a gross farm value (GFV) of $23.7 million (Louisiana State University Agricultural Center, 2014). Strawberries are one of the most celebrated fruit crops in Louisiana. Each year in April, the town of Ponchatoula puts on the state's largest strawberry festival to mark the significance of this crop. Strawberries are the official state fruit of Louisiana (LA Secretary of Louisiana
∗
Secretary of State, 2016). Perishable commodities, including strawberries, are susceptible to excessive water exposure. Excessive water poses problems for Louisiana growers because the state receives an average of 60 inches of rainfall a year (US Climate Data, 2017). In March 2016, more than 16 inches of rain occurred in a two-day period leading to severe flooding in southeast Louisiana (Yan & Flores, 2016). This flooding event resulted in staggering loss of strawberry production, interrupted peak harvest period and raised health concerns. Furthermore, hurricane-associated storm intensity and rainfall rates are projected to increase as the climate continues to warm (NASA, 2019). Floodwater serves as an ideal medium for bacteria, viruses, protozoa, and helminthes (WHO, 2004). If the agricultural field is adjacent to a livestock farm, industrial areas or untreated sewage and wastewater, floodwaters can intermingle with possible sources of microbial contamination (Miraglia et al., 2009). Contaminated floodwater may lead to the contamination of produce, soil and plants with foodborne
Corresponding author. School of Nutrition and Food Sciences, 269 Knapp Hall, Louisiana State University, Baton Rouge, LA, 70803, USA. E-mail address: [email protected] (W. Xu).
https://doi.org/10.1016/j.foodcont.2019.106970 Received 3 February 2019; Received in revised form 21 October 2019; Accepted 28 October 2019 Available online 31 October 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Field experimental design. Five raised beds were exposed to a simulated flooding event. Treatments included High Flood High Contamination (HFHC), High Flood Low Contamination (HFLC), Low Flood High Contamination (LFHC), Low Flood Low Contamination (LFLC) and a Control (C).
Strawberries were irrigated by hand twice a day for the first 3 weeks to allow roots to establish. After root establishment, strawberries were irrigated automatically using drip emitters (Rain Bird, Azusa, CA) for 15 min per day (one emitter per three strawberry plants). Between planting and the first harvest, strawberry plants were sprayed twice with Quadris Top (Syngenta Crop Protection, St. Gabriel, LA) and Pristine WG (BASF Corporation, Geismar, LA) fungicides at rates listed in the Southern Region Small Fruit Consortium Production Manuals (SRSFC, 2017). The presence of Botrytis cinerea-a gray mold was observed before the harvest. Along with fungicide application, strawberries exhibiting gray mold symptoms were removed from the plants and discarded.
pathogens such as E. coli O157:H7 and Listeria monocytogenes (Taylor et al., 2011). Exposure to higher levels of pathogens in floodwater raises public health concerns (EPA, 2001). Fresh produce grown in flooded fields may serve as a potential vehicle for foodborne outbreaks. The Food and Drug Administration (FDA, 2009) states that if the edible portion of a crop has been exposed to floodwaters, it is considered adulterated and should not enter human food channels. There is no practical method to recondition the edible portion of a crop to provide reasonable assurance of human food safety. For crops where floodwater did not contact the edible portions of the crops, growers are directed to evaluate crop safety for human consumption on a case-bycase basis (FDA, 2009). Unfortunately, it demands time, resources, funding and food safety training from growers to make informed decisions. Three questions were posed in this study. The first question was: if the floodwater come into contact with strawberry plants but not the fruit, is the final product still safe? To answer this question, we conducted microbial analysis of strawberries in and near the floodwater. We also evaluated the microbial contamination in soil samples to assess the potential cross-contamination between the soil and strawberries that would mature after floodwater receded. The second question was: if flooded strawberries were harvested, will the contamination level change during shelf-life? To address this concern, we investigated how the microbial population on strawberries might change during shelf-life when they were harvested right after flooding. The third question was: if fruit was still green or not mature when the flood event occurred, will they be free of contamination after they mature? To answer the last question, we differentiated mature and immature strawberries after flooding for the microbial analysis.
2.2. Inoculum preparation Three strains of generic E. coli (ATCC® 23716™, 25922™ and 55974™) were used as indicators for fecal contamination and stored at −80 °C until use. All three strains were bio-safety level one (BSL-1) organisms. ATCC® 23716™ is a K-2 wild type, ATCC® 25922™ and has been widely used as control for antimicrobial susceptibility testing. ATCC® 55974™ was designated from ATCC® 11775™. The latter has been used in water testing but has been adjusted to a BSL-2 level strain since October 2016. After thawing, ten μL of each strain culture was transferred to fresh Tryptic Soy Broth (TSB) (Hardy Diagnostics, Santa Maria, CA) and incubated at 37 °C for 18 h. Then ten μL of each culture was transferred again to fresh TSB and incubated at 37 °C for 24 h yielding 109 CFU/mL. To prepare the cocktail inoculum, equal volume of each generic E. coli strain was diluted and mixed to establish the baseline of approximately 108 CFU/mL for future inoculation.
2. Materials and methods
2.3. Field setup and flooding
2.1. Strawberry production
Five raised beds (4 ft. L × 8 ft. W × 24 in. H) were constructed with treated lumber and lined with black Visqueen to retain floodwater. The field setup is shown in Fig. 1. The Control treatment was flooded with Baton Rouge municipal water. Twelve inches of water (890 L) was introduced into the control bed to fully submerge the strawberries. Immediately before flooding the treatment beds, generic E. coli cocktails was diluted using the municipal water in two, food-grade, and high density polyethylene buckets to generate 106 or 102 CFU/mL of inoculum. Fresh collected cow manure (~0.4 g/L water) was stirred into the water to increase the organic load. High Flood High Contamination (HFHC) strawberries were flooded with 12 inches of water (890 L) spiked with 890 mL of 106 CFU/mL generic E. coli. High Flood Low Contamination (HFLC) strawberries were flooded with 12 inches of
Strawberries plants were planted and maintained at the Louisiana State University Agricultural Center (LSU AgCenter) Botanic Gardens using standard growing procedures in Louisiana (Fontenot, Johnson, Morgan, & Ivey, 2014). Strawberries were planted in October and were managed through March. Some strawberry producers place a plastic barrier between the plants and soil. In this study, however, in order to access the ability of soil to retain the contamination, plastic mulch was not used. Three bareroot ‘St. Festival’ strawberry plants were planted in Rose medium purchased in bulk at Cleggs Nursery in Baton Rouge, Louisiana. Strawberries were fertilized with Peters 20-20-20 at a rate of 200 parts per million (ppm) of nitrogen (N) four times prior to harvest. 2
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2.6. Enumeration of fecal indicators
Table 1 Influence of flooding on coliform populations on mature strawberries during a shelf-life study at 4 °C. Treatments
0h
48 h
96 h
144 h
C LFLC LFHC HFLC HFHC
ND ND 1.5 ± 0.3a 1.1 ± 0.5a 1.0 ± 0.7a
ND ND ND ND ND
ND ND ND ND ND
ND ND ND ND ND
To estimate levels of fecal indicators in floodwater, mature/immature strawberries, and soil samples, a 1:10 dilution of 25 mL or g of samples in 225 mL of 0.1% peptone water was prepared and homogenized using a stomacher for 1 min to generate a uniform distribution. One mL of each of the mixtures (mature/immature strawberries and soil) was plated on 3 M™ Petrifilm™ E. coli/Coliform Count Plates to determine coliforms and E. coli population. Petrifilms were incubated at 35 ± 2 °C for 48 h to determine colony counts.
Values are the average log CFU/g ± standard deviation. ND= Not detected. Below Detection Limit (10 CFU/g). y (C) = Control, (LFLC) = Low Flood Low Contamination, (LFHC) = Low Flood High Contamination, (HFLC) = High Flood Low Contamination, (HFHC) = High Flood High Contamination. z Sampling time at which indicator bacteria population were measured after flooding. Means in columns with different letters are significant at (P ≤ 0.05).
2.7. Statistical analysis All experiments were conducted twice as two independent trials with triplicates in each trial. The data of the indicator bacteria in the mature/immature strawberries and soil was converted into logarithmic units (log CFU/g) and analyzed using SAS®. Duncan's Multiple Range test was used to determine differences between high flood (HF) and low flood (LF) treatments, as well as high contamination (HC) and low contamination (LC) treatments against the Control at different time points. Significant differences were reported when p≤ 0.05.
water (890 L) spiked with 890 mL of 102 CFU/mL generic E. coli. Low Flood High Contamination (LFHC) strawberries were flooded with 8 inches of water (600 L) spiked with 600 mL of 106 CFU/L generic E. coli. Low Flood Low Contamination (LFLC) strawberries were flooded with 8 inches of water (600 L) spiked with 600 mL of 102 CFU/L generic E. coli. In the two raised beds with high flood treatments, the floodwater completely submerged the strawberries. While in the two raised beds with low flood treatments, the floodwater came in contact with the plants but not the fruit. Baseline population levels of generic E. coli and total coliform in the floodwater were evaluated. Floodwater remained in contact with the plants for 4 h, then was removed from the raised beds using an electric pump (Xtreme Pump, Thibodaux, LA) and a handheld pump (Xtreme Pump, Thibodaux, LA). Samples were collected as described below.
3. Results and discussion 3.1. Indicator bacteria baseline levels in floodwater Total coliform and generic E. coli were used as indicators of sanitary quality of water and recent fecal contamination. In high contamination beds (HFHC and LFHC), the baseline levels of generic E. coli and total coliform in floodwater were 5.7 ± 1.9 and 5.9 ± 0.8 log CFU/L, respectively. In low contamination beds (HFLC and LFLC), the baseline levels of generic E. coli and total coliform in floodwaters were 2.2 ± 0.7 and 3.6 ± 0.9 log CFU/L, respectively. Population levels of generic E. coli in the floodwater contained bacteria from cow manure and spiked generic E. coli.
2.4. Strawberry sample collection All mature fruit from each raised bed was harvested and placed into labeled Rubbermaid® rigid containers (one bed per container). Samples were then transported to the laboratory on ice within 1 h, where the strawberries were equally divided and placed into designated Genpak® clamshell boxes and stored in the refrigerator at 4 °C until microbial analysis. Immature green strawberries were left in the field after flooding and allowed to mature for one week. To differentiate two growth stages, later harvested strawberries will be referred to as immature strawberries throughout the paper. After maturing, “immature strawberries” were also sampled, transported, and stored in the same manner as mature strawberries. Mature strawberry and immature strawberry samples were analyzed at 0, 48, 96 and 144 h after harvest. At each time point, 25 g of strawberries were randomly picked from the each clamshell box for microbial analysis.
3.2. Indicator bacteria in mature strawberries during shelf life after flooding Generic E. coli was not detected on the mature strawberries after harvest or during shelf life with a detection limit of 10 CFU/g. The results in Table 1 showed the coliform populations on mature strawberries at the time of harvest and at 48 h increments until 144 h. Immediately after flooding, the coliform population on strawberries reached 1.5 log CFU/g, 1.1 log CFU/g, and 1.0 log CFU/g in LFHC, HFLC and HFLC, respectively. Coliform was not detected in the control group as well as LFLC flooded strawberries. This indicated that heavy flooding or highly contaminated floodwater may have introduced coliform to strawberries. Presence of total coliform was not intended to detect fecal contamination, but rather to reflect general hygiene. However, the presence of total coliform may also be independent from flooding, since some species in total coliform are naturally found in plant materials and soil. The coliform population fell under detection limit and remained so during storage at refrigerator temperature (4 °C) for 144 h. The decrease in coliform on the surface of strawberries may be attributed to multiple reasons. The surface of a whole strawberry is waxy and dry, which may reduce bacteria survival (Flessa, Lusk, & Harris, 2005). Researchers (Yu, Newman, Archbold, & Hamilton-Kemp, 2001) reported that a significant reduction of E. coli O157:H7 (1.3–2.3 log CFU/g) on intact strawberry surfaces was found after 3 days under refrigeration storage. They suggested that the reduced surface survival during shelf life may be a result of desiccation, nutrient deprivation or growth competition.
2.5. Soil sample collection Soil samples were collected in the field at 0, 48, 96 and 144 h after flooding. Two hundred g of soil were collected from ten random spots within each raised bed and divided into two separate Nasco™ WhirlPak™ Easy-To-Close sterile collection bags (100 g per bag) for analysis. Soil samples were transported to the laboratory on ice and were stored in refrigerator at 4 °C until further analysis. Microbial analysis was conducted within 12 h of the sampling. At each time point, 25 g of soil was taken from each bag for microbial analysis.
3
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ranged from 1.0 to 4.3 log CFU/g. Other studies found total coliforms ranged from 2.7 log CFU/g to 8.2 log CFU/g in mixed salad vegetables and up to 5 log CFU/g on fresh lettuce (Najafi & Bahreini, 2012).
Table 2 Influence of flooding on coliform population on immature strawberries during a shelf-life study at 4 °C. Treatments
0h
48 h
C LFLC LFHC HFLC HFHC
ND ND ND ND ND
2.2 1.3 1.4 2.0 1.8
± ± ± ± ±
0.4Aa 1.2a 1.0Aa 0.5a 0.8Aa
96 h
144 h
1.0 ± 0.9Ba ND 1.1 ± 1.0Aa ND ND
ND ND ND ND 1.1 ± 1.0A
3.4. Indicator bacteria in soil after flooding Soil samples were taken every 48 h from the strawberry field after floodwaters receded. As shown in Table 3, generic E. coli (1.1–1.6 log CFU/g) was detected in soil subjected to treatments LFLC, LFHC, HFLC and HFHC but not the control. After 48 h, E. coli was detected only in the soil that was flooded with high microbial load (HFHC and LFHC). The generic E. coli population in the HFHC (1.6 log CFU/g) was greater than that of soil in HFLC (1.1 log CFU/g) immediately after flooding. Generic E. coli levels did not change significantly between 0 h (1.6 log CFU/g) and 48 h (1.5 log CFU/g) when subjected to HFHC water treatments but was not detected at 96 h and 144 h. Similarly, the generic E. coli levels of soil subjected to the HFLC treatment was 1.1 log CFU/g at 0 h but fell below the detection limit after 48 h and remained low throughout 144 h. The level of fecal contamination had a significant effect on the E. coli population in the high flood treatments only immediately after harvest. In this study, results indicated that even when the edible portion of strawberries did not come in direct contact with floodwater, there was potential risk for them to pick up contaminants from the soil for up to two days after floodwater recede if left in the field. Results in Table 3 also showed coliform population in soil after flooding. High levels of coliform were present in all water treatments ranging from 1.0 log CFU/g to 2.8 log CFU/g. However, coliform population decreased throughout the 144 h in the control and low flood treatments (LFLC and LFHC). Yet, high flood treatments (HFLC and HFHC) displayed presence of coliform population even at the 144 h (> 2 log CFU/g) regardless of the initial contamination level. This indicated that initial contamination levels may not have contributed to coliform populations in the soil. Flooding may cause contamination of fresh produce if the soil has higher levels of generic E. coli and coliform population and if there was splashing of soil particles onto the berries during a rainfall (Delbeke et al., 2015). Our study indicated that soil contamination does not necessarily indicate contamination of the strawberries. Since most strawberries are grown on protected plastic covered rows to assist
Values are the average log CFU/g ± standard deviation. ND= Not detected. Below Detection Limit (10 CFU/g). y (C) = Control, (LFLC) = Low Flood Low Contamination, (LFHC) = Low Flood High Contamination, (HFLC) = High Flood Low Contamination, (HFHC) = High Flood High Contamination. z Sampling time at which indicator bacteria population were measured after flooding. Means in columns with different lowercase letters are different at (P ≤ 0.05). Means in rows with different uppercase letters are different at (P ≤ 0.05). Immature fruit were immature at the time of the flood event but left on the plants to mature for one week after the flood event.
3.3. Indicator bacteria in immature strawberries during shelf life after flooding Generic E. coli was not detected in any of the immature strawberries. The coliform population was present in all floodwater treatments at 48 h as illustrated in Table 2. Significant reduction was observed in the control samples at 96 h of refrigerated storage (4 °C). Although coliform population in the LFHC treatment did not decrease between 48 h and 96 h it was reduced to non-detected by 144 h. Immature strawberries subjected to water treatment HFHC were positive for coliforms at the 48 h (1.8 log CFU/g) and 144 h (1.1 log CFU/g) storage periods. Compared to mature strawberries harvested immediately after the flood event, the overall coliform population of the immature strawberries was higher after storage for 48 h in all water treatments and at 96 h in the control and LFHC. Interestingly, none of the immature strawberry samples showed detectable coliform at 0 h. The reason for the later increase of the coliform population was not clear. Several fresh produce studies have indicated coliform population varies in wide ranges. Johnston et al. (2005) found total coliforms on green leaves and herbs
Table 3 Influence of flooding on indicator bacteria population in soil during specific sampling times. E. coli Population at Specific Sampling Timesz Treatmentsy
0h
C LFLC LFHC HFLC HFHC
ND 1.2 1.2 1.1 1.6
Treatments C LFLC LFHC HFLC HFHC
48 h
96 h
144 h
ND ND 1.5 ± 0.4Aa ND 1.5 ± 0.5Aa
ND ND ND ND ND
ND ND ND ND ND
Coliform Population at Specific Sampling Times 0h 48 h
96 h
144 h
2.4 2.6 2.7 2.4 2.8
2.0 2.4 2.3 2.2 2.2
± ± ± ±
± ± ± ± ±
0.2 ab 0.8Aab 0.5b 0.3Aa
0.2Ab 0.2Aab 0.2Aa 0.4Ab 0.2Aa
1.7 2.2 2.4 2.2 2.1
± ± ± ± ±
1.2ABb 1.0ABa 0.7Aa 0.4Aa 0.5Cab
± ± ± ± ±
0.3Ac 0.2Aa 0.2Aab 0.3Abc 0.3BCab
1.0 1.5 1.9 2.2 2.4
± ± ± ± ±
1.1Bc 1.0Bbc 0.2Bab 0.1Aab 0.2Ba
Values are the average log CFU/g ± standard deviation. ND= Not detected. Below Detection Limit (10 CFU/g). Means in columns with different lowercase letters are different at (P ≤ 0.05). Means in rows with different uppercase letters are different at (P ≤ 0.05). y (C) = Control, (LFLC) = Low Flood Low Contamination, (LFHC) = Low Flood High Contamination, (HFLC) = High Flood Low Contamination, (HFHC) = High Flood High Contamination. z Sampling time at which indicator bacteria population were measured after flooding. 4
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picking, soil is not the only contributing factor to cause contamination in strawberries (Delbeke et al., 2015). Moreover, it is very unlikely for pathogens to internalize through irrigation water or the soil into the strawberries through the roots with naturally contaminated water and with low numbers of pathogens (Holvoet, Sampers, Seynnaeve, & Uyttendaele, 2014).
hygiene indicators and enteric pathogens in irrigation water, soil and lettuce and the impact of climatic conditions on contamination in the lettuce primary production. International Journal of Food Microbiology, 171, 21–31. Johnston, L. M., Jaykus, L. A., Moll, D., Martinez, M. C., Anciso, J., Mora, B., et al. (2005). A field study of the microbiological quality of fresh produce. Journal of Food Protection, 68(9), 1840–1847. Louisiana Secretary of State (2016). https://www.sos.la.gov/HistoricalResources/ PublishedDocuments/LouisianaFactsBooklet.pdf, Accessed date: 31 January 2019. Louisiana State University Agricultural Center (2014). Louisiana AgSummary. http://apps. lsuagcenter.com/agsummary/PowerUsers/2014statetotals.pdf, Accessed date: 31 January 2019. Miraglia, M., Marvin, H. J., Kleter, G. A., Battilani, P., Brera, C., Coni, E., et al. (2009). Climate change and food safety: An emerging issue with special focus on Europe. Food and Chemical Toxicology, 47(5), 1009–1021. Najafi, M. B. H., & Bahreini, M. (2012). Microbiological quality of mixed fresh-cut vegetable salads and mixed ready- to-eat fresh herbs in Mashhad, Iran. International Conference on Nutrition and Food Sciences IPCBEE, 39, 62–66 (2012). National Aeronautics and Space Administration (2019). https://climate.nasa.gov/effects/ , Accessed date: 28 April 2019. Schloemann, S. (2005). Crop profile for strawberries in Louisiana. https://ipmdata. ipmcenters.org/documents/cropprofiles/LAstrawberries.pdf, Accessed date: 31 January 2019. Southern Region Small Fruit Consortium (2017). http://www.smallfruits.org/assets/ documents/ipm-guides/Fungicide-Selection.pdf, Accessed date: 31 January 2019. Taylor, J., Lai, K. M., Davies, M., Clifton, D., Ridley, I., & Biddulph, P. (2011). Flood management: Prediction of microbial contamination in large-scale floods in urban environments. Environment International, 37(5), 1019–1029. United States Department of Agriculture National Agricultural Statistics Service (2018). Noncitrus fruits and nuts 2017 summary. https://www.nass.usda.gov/Publications/ Todays_Reports/reports/ncit0618.pdf, Accessed date: 31 January 2019. U.S. (2017). Climate data. http://www.usclimatedata.com/climate/louisiana/unitedstates/3188, Accessed date: 31 January 2019. U.S. Environmental Protection Agency. (2001). Source water protection practices bulletin managing septic systems to prevent contamination of drinking water. https://www. oregon.gov/deq/FilterDocs/EPASWPPracticesBulletin_SanitarySewer.pdf, Accessed date: 31 January 2019. U.S. Food and Drug Administration. (2009). Safety of food affected by hurricanes, flooding, and power outages. https://www.fda.gov/Food/RecallsOutbreaksEmergencies/ Emergencies/ucm112723.htm, Accessed date: 31 January 2019. World Health Organization (2004). Guidelines for drinking-water quality. Accessed https:// apps.who.int/iris/bitstream/handle/10665/44584/9789241548151_eng. pdf;jsessionid=ADE282309307728457DDA2DBCD5B2A1A?sequence=1, Accessed date: 31 January 2019. Yan, H., & Flores, R. (2016). Louisiana flood: Worst US disaster since hurricane sandy, red cross says. CNNhttp://www.cnn.com/2016/08/18/us/louisiana-flooding/index. html, Accessed date: 31 January 2019. Yu, K., Newman, M. C., Archbold, D. D., & Hamilton-Kemp, T. R. (2001). Survival of Escherichia coli O157:H7 on strawberry fruit and reduction of the pathogen population by chemical agents. Journal of Food Protection, 64(9), 1334–1340.
4. Conclusion The level of flooding and initial contamination did not affect the microbial counts on flooded strawberries since generic E. coli was not detected in the fruit. However, generic E. coli was detected in soil samples up to 48 h. This implied that even when the edible portion of strawberries did not come in direct contact with floodwater, there was potential risk for them to pick up contaminants from the soil for up to two days after floodwater receded. Declaration of competing interest No Conlict of Interest. Acknowledgments This project was supported by the U.S. Department of Agriculture's (USDA) Agriculture Marketing Service through grant agreement16SCBGP-LA-0053. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA. References Delbeke, S., Ceuppens, S., Hessel, C. T., Castro, I., Jacxsens, L., De Zutter, L., et al. (2015). Microbial safety and sanitary quality of strawberry primary production in Belgium: Risk factors for Salmonella and shiga toxin-producing Escherichia coli contamination. Applied and Environmental Microbiology, 81(7), 2562–2570. Flessa, S., Lusk, D. M., & Harris, L. J. (2005). Survival of Listeria monocytogenes on fresh and frozen strawberries. International Journal of Food Microbiology, 101(3), 255–262. Fontenot, K., Johnson, C., Morgan, A., & Ivey, M. L. (2014). Strawberries. http://www. lsuagcenter.com/NR/rdonlyres/F364192A-744D-4E43-B95E-E9EBD75CDF4E/ 100278/Pub3364Strawberries4Cweb.pdf, Accessed date: 31 January 2019. Holvoet, K., Sampers, I., Seynnaeve, M., & Uyttendaele, M. (2014). Relationships among
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Evaluation of the microbial safety and quality of Louisiana strawberries after flooding
T
Shifa Shiraza, Dorra Djebbi-Simmonsa, Mohammed Alhejailia, Kaleb Danosb, Marlene Janesa, Kathryn Fontenotb, Wenqing Xua,∗ a b
School of Nutrition and Food Sciences, Louisiana State University Agriculture Center, Baton Rouge, LA, 70803, USA School of Plant, Environmental and Soil Sciences, Louisiana State University Agriculture Center, Baton Rouge, LA, 70803, USA
ARTICLE INFO
ABSTRACT
Keywords: Flooding Strawberries Soil Generic E. coli Coliform
Adverse weather such as flooding affects Louisiana strawberry production. Floodwater is an ideal medium for the growth of foodborne pathogens and may cause contamination of soil, water, and fresh produce. To evaluate the microbial safety of strawberries after flooding, four flooding scenarios were investigated. Five raised beds were exposed to a simulated flood event. Treatments included High Flood High Contamination (HFHC), High Flood Low Contamination (HFLC), Low Flood High Contamination (LFHC), Low Flood Low Contamination (LFLC) and a Control (C). High flood groups (HF) were exposed to 890 L of floodwater where strawberries were fully submerged, while low flood groups (LF) were exposed to 600 L of floodwater where strawberries did not come into direct contact with the floodwater. Three strains of generic Escherichia coli were spiked into the floodwater to establish a baseline population of approximately 106 colony forming units per liter (CFU/L) (high contamination) and 102 CFU/L (low contamination). Strawberry plants in all treatments were flooded for 4 h until floodwater receded. Strawberries were harvested when mature and sampled during shelf-life at 4 °C at 0, 48, 96, and 144 h. Soil samples were collected in the field every 48 h for one week. Even though the generic E. coli was not detected (< 10 CFU/g) in strawberry samples, it was detected in soil samples within 96 h after flooding. In soil samples, generic E. coli was higher in HFHC samples (1.6 log CFU/g) compared to HFLC samples (1.1 log CFU/g) at harvest. Additionally, 1.0 to 2.8 log CFU/g of coliform were present in the strawberries and soil at 0, 48, 96, and 144 h in all treatment beds.
1. Introduction Strawberries are produced throughout the United States. California, Florida, and Oregon are the three leading states in terms of total acres grown. Even though Louisiana is not a major production state (USDA NASS, 2018), strawberries are an important fruit to the local economy. The majority of Louisiana's strawberry production occurs in Tangipahoa and Livingston parishes, where approximately 400–500 acres of strawberries are grown annually (Schloemann, 2005, pp. 1–73). However, in recent years, strawberry acreage has decreased. In 2014, eighty one growers produced 367 acres of strawberries. Despite low acreage, the strawberry industry generated a gross farm value (GFV) of $23.7 million (Louisiana State University Agricultural Center, 2014). Strawberries are one of the most celebrated fruit crops in Louisiana. Each year in April, the town of Ponchatoula puts on the state's largest strawberry festival to mark the significance of this crop. Strawberries are the official state fruit of Louisiana (LA Secretary of Louisiana
∗
Secretary of State, 2016). Perishable commodities, including strawberries, are susceptible to excessive water exposure. Excessive water poses problems for Louisiana growers because the state receives an average of 60 inches of rainfall a year (US Climate Data, 2017). In March 2016, more than 16 inches of rain occurred in a two-day period leading to severe flooding in southeast Louisiana (Yan & Flores, 2016). This flooding event resulted in staggering loss of strawberry production, interrupted peak harvest period and raised health concerns. Furthermore, hurricane-associated storm intensity and rainfall rates are projected to increase as the climate continues to warm (NASA, 2019). Floodwater serves as an ideal medium for bacteria, viruses, protozoa, and helminthes (WHO, 2004). If the agricultural field is adjacent to a livestock farm, industrial areas or untreated sewage and wastewater, floodwaters can intermingle with possible sources of microbial contamination (Miraglia et al., 2009). Contaminated floodwater may lead to the contamination of produce, soil and plants with foodborne
Corresponding author. School of Nutrition and Food Sciences, 269 Knapp Hall, Louisiana State University, Baton Rouge, LA, 70803, USA. E-mail address: [email protected] (W. Xu).
https://doi.org/10.1016/j.foodcont.2019.106970 Received 3 February 2019; Received in revised form 21 October 2019; Accepted 28 October 2019 Available online 31 October 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Field experimental design. Five raised beds were exposed to a simulated flooding event. Treatments included High Flood High Contamination (HFHC), High Flood Low Contamination (HFLC), Low Flood High Contamination (LFHC), Low Flood Low Contamination (LFLC) and a Control (C).
Strawberries were irrigated by hand twice a day for the first 3 weeks to allow roots to establish. After root establishment, strawberries were irrigated automatically using drip emitters (Rain Bird, Azusa, CA) for 15 min per day (one emitter per three strawberry plants). Between planting and the first harvest, strawberry plants were sprayed twice with Quadris Top (Syngenta Crop Protection, St. Gabriel, LA) and Pristine WG (BASF Corporation, Geismar, LA) fungicides at rates listed in the Southern Region Small Fruit Consortium Production Manuals (SRSFC, 2017). The presence of Botrytis cinerea-a gray mold was observed before the harvest. Along with fungicide application, strawberries exhibiting gray mold symptoms were removed from the plants and discarded.
pathogens such as E. coli O157:H7 and Listeria monocytogenes (Taylor et al., 2011). Exposure to higher levels of pathogens in floodwater raises public health concerns (EPA, 2001). Fresh produce grown in flooded fields may serve as a potential vehicle for foodborne outbreaks. The Food and Drug Administration (FDA, 2009) states that if the edible portion of a crop has been exposed to floodwaters, it is considered adulterated and should not enter human food channels. There is no practical method to recondition the edible portion of a crop to provide reasonable assurance of human food safety. For crops where floodwater did not contact the edible portions of the crops, growers are directed to evaluate crop safety for human consumption on a case-bycase basis (FDA, 2009). Unfortunately, it demands time, resources, funding and food safety training from growers to make informed decisions. Three questions were posed in this study. The first question was: if the floodwater come into contact with strawberry plants but not the fruit, is the final product still safe? To answer this question, we conducted microbial analysis of strawberries in and near the floodwater. We also evaluated the microbial contamination in soil samples to assess the potential cross-contamination between the soil and strawberries that would mature after floodwater receded. The second question was: if flooded strawberries were harvested, will the contamination level change during shelf-life? To address this concern, we investigated how the microbial population on strawberries might change during shelf-life when they were harvested right after flooding. The third question was: if fruit was still green or not mature when the flood event occurred, will they be free of contamination after they mature? To answer the last question, we differentiated mature and immature strawberries after flooding for the microbial analysis.
2.2. Inoculum preparation Three strains of generic E. coli (ATCC® 23716™, 25922™ and 55974™) were used as indicators for fecal contamination and stored at −80 °C until use. All three strains were bio-safety level one (BSL-1) organisms. ATCC® 23716™ is a K-2 wild type, ATCC® 25922™ and has been widely used as control for antimicrobial susceptibility testing. ATCC® 55974™ was designated from ATCC® 11775™. The latter has been used in water testing but has been adjusted to a BSL-2 level strain since October 2016. After thawing, ten μL of each strain culture was transferred to fresh Tryptic Soy Broth (TSB) (Hardy Diagnostics, Santa Maria, CA) and incubated at 37 °C for 18 h. Then ten μL of each culture was transferred again to fresh TSB and incubated at 37 °C for 24 h yielding 109 CFU/mL. To prepare the cocktail inoculum, equal volume of each generic E. coli strain was diluted and mixed to establish the baseline of approximately 108 CFU/mL for future inoculation.
2. Materials and methods
2.3. Field setup and flooding
2.1. Strawberry production
Five raised beds (4 ft. L × 8 ft. W × 24 in. H) were constructed with treated lumber and lined with black Visqueen to retain floodwater. The field setup is shown in Fig. 1. The Control treatment was flooded with Baton Rouge municipal water. Twelve inches of water (890 L) was introduced into the control bed to fully submerge the strawberries. Immediately before flooding the treatment beds, generic E. coli cocktails was diluted using the municipal water in two, food-grade, and high density polyethylene buckets to generate 106 or 102 CFU/mL of inoculum. Fresh collected cow manure (~0.4 g/L water) was stirred into the water to increase the organic load. High Flood High Contamination (HFHC) strawberries were flooded with 12 inches of water (890 L) spiked with 890 mL of 106 CFU/mL generic E. coli. High Flood Low Contamination (HFLC) strawberries were flooded with 12 inches of
Strawberries plants were planted and maintained at the Louisiana State University Agricultural Center (LSU AgCenter) Botanic Gardens using standard growing procedures in Louisiana (Fontenot, Johnson, Morgan, & Ivey, 2014). Strawberries were planted in October and were managed through March. Some strawberry producers place a plastic barrier between the plants and soil. In this study, however, in order to access the ability of soil to retain the contamination, plastic mulch was not used. Three bareroot ‘St. Festival’ strawberry plants were planted in Rose medium purchased in bulk at Cleggs Nursery in Baton Rouge, Louisiana. Strawberries were fertilized with Peters 20-20-20 at a rate of 200 parts per million (ppm) of nitrogen (N) four times prior to harvest. 2
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2.6. Enumeration of fecal indicators
Table 1 Influence of flooding on coliform populations on mature strawberries during a shelf-life study at 4 °C. Treatments
0h
48 h
96 h
144 h
C LFLC LFHC HFLC HFHC
ND ND 1.5 ± 0.3a 1.1 ± 0.5a 1.0 ± 0.7a
ND ND ND ND ND
ND ND ND ND ND
ND ND ND ND ND
To estimate levels of fecal indicators in floodwater, mature/immature strawberries, and soil samples, a 1:10 dilution of 25 mL or g of samples in 225 mL of 0.1% peptone water was prepared and homogenized using a stomacher for 1 min to generate a uniform distribution. One mL of each of the mixtures (mature/immature strawberries and soil) was plated on 3 M™ Petrifilm™ E. coli/Coliform Count Plates to determine coliforms and E. coli population. Petrifilms were incubated at 35 ± 2 °C for 48 h to determine colony counts.
Values are the average log CFU/g ± standard deviation. ND= Not detected. Below Detection Limit (10 CFU/g). y (C) = Control, (LFLC) = Low Flood Low Contamination, (LFHC) = Low Flood High Contamination, (HFLC) = High Flood Low Contamination, (HFHC) = High Flood High Contamination. z Sampling time at which indicator bacteria population were measured after flooding. Means in columns with different letters are significant at (P ≤ 0.05).
2.7. Statistical analysis All experiments were conducted twice as two independent trials with triplicates in each trial. The data of the indicator bacteria in the mature/immature strawberries and soil was converted into logarithmic units (log CFU/g) and analyzed using SAS®. Duncan's Multiple Range test was used to determine differences between high flood (HF) and low flood (LF) treatments, as well as high contamination (HC) and low contamination (LC) treatments against the Control at different time points. Significant differences were reported when p≤ 0.05.
water (890 L) spiked with 890 mL of 102 CFU/mL generic E. coli. Low Flood High Contamination (LFHC) strawberries were flooded with 8 inches of water (600 L) spiked with 600 mL of 106 CFU/L generic E. coli. Low Flood Low Contamination (LFLC) strawberries were flooded with 8 inches of water (600 L) spiked with 600 mL of 102 CFU/L generic E. coli. In the two raised beds with high flood treatments, the floodwater completely submerged the strawberries. While in the two raised beds with low flood treatments, the floodwater came in contact with the plants but not the fruit. Baseline population levels of generic E. coli and total coliform in the floodwater were evaluated. Floodwater remained in contact with the plants for 4 h, then was removed from the raised beds using an electric pump (Xtreme Pump, Thibodaux, LA) and a handheld pump (Xtreme Pump, Thibodaux, LA). Samples were collected as described below.
3. Results and discussion 3.1. Indicator bacteria baseline levels in floodwater Total coliform and generic E. coli were used as indicators of sanitary quality of water and recent fecal contamination. In high contamination beds (HFHC and LFHC), the baseline levels of generic E. coli and total coliform in floodwater were 5.7 ± 1.9 and 5.9 ± 0.8 log CFU/L, respectively. In low contamination beds (HFLC and LFLC), the baseline levels of generic E. coli and total coliform in floodwaters were 2.2 ± 0.7 and 3.6 ± 0.9 log CFU/L, respectively. Population levels of generic E. coli in the floodwater contained bacteria from cow manure and spiked generic E. coli.
2.4. Strawberry sample collection All mature fruit from each raised bed was harvested and placed into labeled Rubbermaid® rigid containers (one bed per container). Samples were then transported to the laboratory on ice within 1 h, where the strawberries were equally divided and placed into designated Genpak® clamshell boxes and stored in the refrigerator at 4 °C until microbial analysis. Immature green strawberries were left in the field after flooding and allowed to mature for one week. To differentiate two growth stages, later harvested strawberries will be referred to as immature strawberries throughout the paper. After maturing, “immature strawberries” were also sampled, transported, and stored in the same manner as mature strawberries. Mature strawberry and immature strawberry samples were analyzed at 0, 48, 96 and 144 h after harvest. At each time point, 25 g of strawberries were randomly picked from the each clamshell box for microbial analysis.
3.2. Indicator bacteria in mature strawberries during shelf life after flooding Generic E. coli was not detected on the mature strawberries after harvest or during shelf life with a detection limit of 10 CFU/g. The results in Table 1 showed the coliform populations on mature strawberries at the time of harvest and at 48 h increments until 144 h. Immediately after flooding, the coliform population on strawberries reached 1.5 log CFU/g, 1.1 log CFU/g, and 1.0 log CFU/g in LFHC, HFLC and HFLC, respectively. Coliform was not detected in the control group as well as LFLC flooded strawberries. This indicated that heavy flooding or highly contaminated floodwater may have introduced coliform to strawberries. Presence of total coliform was not intended to detect fecal contamination, but rather to reflect general hygiene. However, the presence of total coliform may also be independent from flooding, since some species in total coliform are naturally found in plant materials and soil. The coliform population fell under detection limit and remained so during storage at refrigerator temperature (4 °C) for 144 h. The decrease in coliform on the surface of strawberries may be attributed to multiple reasons. The surface of a whole strawberry is waxy and dry, which may reduce bacteria survival (Flessa, Lusk, & Harris, 2005). Researchers (Yu, Newman, Archbold, & Hamilton-Kemp, 2001) reported that a significant reduction of E. coli O157:H7 (1.3–2.3 log CFU/g) on intact strawberry surfaces was found after 3 days under refrigeration storage. They suggested that the reduced surface survival during shelf life may be a result of desiccation, nutrient deprivation or growth competition.
2.5. Soil sample collection Soil samples were collected in the field at 0, 48, 96 and 144 h after flooding. Two hundred g of soil were collected from ten random spots within each raised bed and divided into two separate Nasco™ WhirlPak™ Easy-To-Close sterile collection bags (100 g per bag) for analysis. Soil samples were transported to the laboratory on ice and were stored in refrigerator at 4 °C until further analysis. Microbial analysis was conducted within 12 h of the sampling. At each time point, 25 g of soil was taken from each bag for microbial analysis.
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ranged from 1.0 to 4.3 log CFU/g. Other studies found total coliforms ranged from 2.7 log CFU/g to 8.2 log CFU/g in mixed salad vegetables and up to 5 log CFU/g on fresh lettuce (Najafi & Bahreini, 2012).
Table 2 Influence of flooding on coliform population on immature strawberries during a shelf-life study at 4 °C. Treatments
0h
48 h
C LFLC LFHC HFLC HFHC
ND ND ND ND ND
2.2 1.3 1.4 2.0 1.8
± ± ± ± ±
0.4Aa 1.2a 1.0Aa 0.5a 0.8Aa
96 h
144 h
1.0 ± 0.9Ba ND 1.1 ± 1.0Aa ND ND
ND ND ND ND 1.1 ± 1.0A
3.4. Indicator bacteria in soil after flooding Soil samples were taken every 48 h from the strawberry field after floodwaters receded. As shown in Table 3, generic E. coli (1.1–1.6 log CFU/g) was detected in soil subjected to treatments LFLC, LFHC, HFLC and HFHC but not the control. After 48 h, E. coli was detected only in the soil that was flooded with high microbial load (HFHC and LFHC). The generic E. coli population in the HFHC (1.6 log CFU/g) was greater than that of soil in HFLC (1.1 log CFU/g) immediately after flooding. Generic E. coli levels did not change significantly between 0 h (1.6 log CFU/g) and 48 h (1.5 log CFU/g) when subjected to HFHC water treatments but was not detected at 96 h and 144 h. Similarly, the generic E. coli levels of soil subjected to the HFLC treatment was 1.1 log CFU/g at 0 h but fell below the detection limit after 48 h and remained low throughout 144 h. The level of fecal contamination had a significant effect on the E. coli population in the high flood treatments only immediately after harvest. In this study, results indicated that even when the edible portion of strawberries did not come in direct contact with floodwater, there was potential risk for them to pick up contaminants from the soil for up to two days after floodwater recede if left in the field. Results in Table 3 also showed coliform population in soil after flooding. High levels of coliform were present in all water treatments ranging from 1.0 log CFU/g to 2.8 log CFU/g. However, coliform population decreased throughout the 144 h in the control and low flood treatments (LFLC and LFHC). Yet, high flood treatments (HFLC and HFHC) displayed presence of coliform population even at the 144 h (> 2 log CFU/g) regardless of the initial contamination level. This indicated that initial contamination levels may not have contributed to coliform populations in the soil. Flooding may cause contamination of fresh produce if the soil has higher levels of generic E. coli and coliform population and if there was splashing of soil particles onto the berries during a rainfall (Delbeke et al., 2015). Our study indicated that soil contamination does not necessarily indicate contamination of the strawberries. Since most strawberries are grown on protected plastic covered rows to assist
Values are the average log CFU/g ± standard deviation. ND= Not detected. Below Detection Limit (10 CFU/g). y (C) = Control, (LFLC) = Low Flood Low Contamination, (LFHC) = Low Flood High Contamination, (HFLC) = High Flood Low Contamination, (HFHC) = High Flood High Contamination. z Sampling time at which indicator bacteria population were measured after flooding. Means in columns with different lowercase letters are different at (P ≤ 0.05). Means in rows with different uppercase letters are different at (P ≤ 0.05). Immature fruit were immature at the time of the flood event but left on the plants to mature for one week after the flood event.
3.3. Indicator bacteria in immature strawberries during shelf life after flooding Generic E. coli was not detected in any of the immature strawberries. The coliform population was present in all floodwater treatments at 48 h as illustrated in Table 2. Significant reduction was observed in the control samples at 96 h of refrigerated storage (4 °C). Although coliform population in the LFHC treatment did not decrease between 48 h and 96 h it was reduced to non-detected by 144 h. Immature strawberries subjected to water treatment HFHC were positive for coliforms at the 48 h (1.8 log CFU/g) and 144 h (1.1 log CFU/g) storage periods. Compared to mature strawberries harvested immediately after the flood event, the overall coliform population of the immature strawberries was higher after storage for 48 h in all water treatments and at 96 h in the control and LFHC. Interestingly, none of the immature strawberry samples showed detectable coliform at 0 h. The reason for the later increase of the coliform population was not clear. Several fresh produce studies have indicated coliform population varies in wide ranges. Johnston et al. (2005) found total coliforms on green leaves and herbs
Table 3 Influence of flooding on indicator bacteria population in soil during specific sampling times. E. coli Population at Specific Sampling Timesz Treatmentsy
0h
C LFLC LFHC HFLC HFHC
ND 1.2 1.2 1.1 1.6
Treatments C LFLC LFHC HFLC HFHC
48 h
96 h
144 h
ND ND 1.5 ± 0.4Aa ND 1.5 ± 0.5Aa
ND ND ND ND ND
ND ND ND ND ND
Coliform Population at Specific Sampling Times 0h 48 h
96 h
144 h
2.4 2.6 2.7 2.4 2.8
2.0 2.4 2.3 2.2 2.2
± ± ± ±
± ± ± ± ±
0.2 ab 0.8Aab 0.5b 0.3Aa
0.2Ab 0.2Aab 0.2Aa 0.4Ab 0.2Aa
1.7 2.2 2.4 2.2 2.1
± ± ± ± ±
1.2ABb 1.0ABa 0.7Aa 0.4Aa 0.5Cab
± ± ± ± ±
0.3Ac 0.2Aa 0.2Aab 0.3Abc 0.3BCab
1.0 1.5 1.9 2.2 2.4
± ± ± ± ±
1.1Bc 1.0Bbc 0.2Bab 0.1Aab 0.2Ba
Values are the average log CFU/g ± standard deviation. ND= Not detected. Below Detection Limit (10 CFU/g). Means in columns with different lowercase letters are different at (P ≤ 0.05). Means in rows with different uppercase letters are different at (P ≤ 0.05). y (C) = Control, (LFLC) = Low Flood Low Contamination, (LFHC) = Low Flood High Contamination, (HFLC) = High Flood Low Contamination, (HFHC) = High Flood High Contamination. z Sampling time at which indicator bacteria population were measured after flooding. 4
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picking, soil is not the only contributing factor to cause contamination in strawberries (Delbeke et al., 2015). Moreover, it is very unlikely for pathogens to internalize through irrigation water or the soil into the strawberries through the roots with naturally contaminated water and with low numbers of pathogens (Holvoet, Sampers, Seynnaeve, & Uyttendaele, 2014).
hygiene indicators and enteric pathogens in irrigation water, soil and lettuce and the impact of climatic conditions on contamination in the lettuce primary production. International Journal of Food Microbiology, 171, 21–31. Johnston, L. M., Jaykus, L. A., Moll, D., Martinez, M. C., Anciso, J., Mora, B., et al. (2005). A field study of the microbiological quality of fresh produce. Journal of Food Protection, 68(9), 1840–1847. Louisiana Secretary of State (2016). https://www.sos.la.gov/HistoricalResources/ PublishedDocuments/LouisianaFactsBooklet.pdf, Accessed date: 31 January 2019. Louisiana State University Agricultural Center (2014). Louisiana AgSummary. http://apps. lsuagcenter.com/agsummary/PowerUsers/2014statetotals.pdf, Accessed date: 31 January 2019. Miraglia, M., Marvin, H. J., Kleter, G. A., Battilani, P., Brera, C., Coni, E., et al. (2009). Climate change and food safety: An emerging issue with special focus on Europe. Food and Chemical Toxicology, 47(5), 1009–1021. Najafi, M. B. H., & Bahreini, M. (2012). Microbiological quality of mixed fresh-cut vegetable salads and mixed ready- to-eat fresh herbs in Mashhad, Iran. International Conference on Nutrition and Food Sciences IPCBEE, 39, 62–66 (2012). National Aeronautics and Space Administration (2019). https://climate.nasa.gov/effects/ , Accessed date: 28 April 2019. Schloemann, S. (2005). Crop profile for strawberries in Louisiana. https://ipmdata. ipmcenters.org/documents/cropprofiles/LAstrawberries.pdf, Accessed date: 31 January 2019. Southern Region Small Fruit Consortium (2017). http://www.smallfruits.org/assets/ documents/ipm-guides/Fungicide-Selection.pdf, Accessed date: 31 January 2019. Taylor, J., Lai, K. M., Davies, M., Clifton, D., Ridley, I., & Biddulph, P. (2011). Flood management: Prediction of microbial contamination in large-scale floods in urban environments. Environment International, 37(5), 1019–1029. United States Department of Agriculture National Agricultural Statistics Service (2018). Noncitrus fruits and nuts 2017 summary. https://www.nass.usda.gov/Publications/ Todays_Reports/reports/ncit0618.pdf, Accessed date: 31 January 2019. U.S. (2017). Climate data. http://www.usclimatedata.com/climate/louisiana/unitedstates/3188, Accessed date: 31 January 2019. U.S. Environmental Protection Agency. (2001). Source water protection practices bulletin managing septic systems to prevent contamination of drinking water. https://www. oregon.gov/deq/FilterDocs/EPASWPPracticesBulletin_SanitarySewer.pdf, Accessed date: 31 January 2019. U.S. Food and Drug Administration. (2009). Safety of food affected by hurricanes, flooding, and power outages. https://www.fda.gov/Food/RecallsOutbreaksEmergencies/ Emergencies/ucm112723.htm, Accessed date: 31 January 2019. World Health Organization (2004). Guidelines for drinking-water quality. Accessed https:// apps.who.int/iris/bitstream/handle/10665/44584/9789241548151_eng. pdf;jsessionid=ADE282309307728457DDA2DBCD5B2A1A?sequence=1, Accessed date: 31 January 2019. Yan, H., & Flores, R. (2016). Louisiana flood: Worst US disaster since hurricane sandy, red cross says. CNNhttp://www.cnn.com/2016/08/18/us/louisiana-flooding/index. html, Accessed date: 31 January 2019. Yu, K., Newman, M. C., Archbold, D. D., & Hamilton-Kemp, T. R. (2001). Survival of Escherichia coli O157:H7 on strawberry fruit and reduction of the pathogen population by chemical agents. Journal of Food Protection, 64(9), 1334–1340.
4. Conclusion The level of flooding and initial contamination did not affect the microbial counts on flooded strawberries since generic E. coli was not detected in the fruit. However, generic E. coli was detected in soil samples up to 48 h. This implied that even when the edible portion of strawberries did not come in direct contact with floodwater, there was potential risk for them to pick up contaminants from the soil for up to two days after floodwater receded. Declaration of competing interest No Conlict of Interest. Acknowledgments This project was supported by the U.S. Department of Agriculture's (USDA) Agriculture Marketing Service through grant agreement16SCBGP-LA-0053. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA. References Delbeke, S., Ceuppens, S., Hessel, C. T., Castro, I., Jacxsens, L., De Zutter, L., et al. (2015). Microbial safety and sanitary quality of strawberry primary production in Belgium: Risk factors for Salmonella and shiga toxin-producing Escherichia coli contamination. Applied and Environmental Microbiology, 81(7), 2562–2570. Flessa, S., Lusk, D. M., & Harris, L. J. (2005). Survival of Listeria monocytogenes on fresh and frozen strawberries. International Journal of Food Microbiology, 101(3), 255–262. Fontenot, K., Johnson, C., Morgan, A., & Ivey, M. L. (2014). Strawberries. http://www. lsuagcenter.com/NR/rdonlyres/F364192A-744D-4E43-B95E-E9EBD75CDF4E/ 100278/Pub3364Strawberries4Cweb.pdf, Accessed date: 31 January 2019. Holvoet, K., Sampers, I., Seynnaeve, M., & Uyttendaele, M. (2014). Relationships among
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