Levels of Salmonella enterica and Listeria monocytogenes in Alternative Irrigation Water Vary Based on Water Source on the Eastern Shore of Maryland

ABSTRACT Irrigation water sources have been shown to harbor foodborne pathogens and could contribute to the outbreak of foodborne illness related to consumption of contaminated produce. Determining the probability of and the degree to which these irrigation water sources contain these pathogens is paramount. The purpose of this study was to determine the prevalence of Salmonella enterica and Listeria monocytogenes in alternative irrigation water sources. Water samples (n = 188) were collected over 2 years (2016 to 2018) from 2 reclaimed water plants, 3 nontidal freshwater rivers, and 1 tidal brackish river on Maryland’s Eastern Shore (ESM). Samples were collected by filtration using modified Moore swabs (MMS) and analyzed by culture methods. Pathogen levels were quantified using a modified most probable number (MPN) procedure with three different volumes (10 liters, 1 liter, and 0.1 liter). Overall, 65% (122/188) and 40% (76/188) of water samples were positive for S. enterica and L. monocytogenes, respectively. For both pathogens, MPN values ranged from 0.015 to 11 MPN/liter. Pathogen levels (MPN/liter) were significantly (P < 0.05) greater for the nontidal freshwater river sites and the tidal brackish river site than the reclaimed water sites. L. monocytogenes levels in water varied based on season. Detection of S. enterica was more likely with 10-liter filtration compared to 0.1-liter filtration. The physicochemical factors measured attributed only 6.4% of the constrained variance to the levels of both pathogens. This study shows clear variations in S. enterica and L. monocytogenes levels in irrigation water sources on ESM. IMPORTANCE In the last several decades, Maryland’s Eastern Shore has seen significant declines in groundwater levels. While this area is not currently experiencing drought conditions or water scarcity, this research represents a proactive approach. Efforts, to investigate the levels of pathogenic bacteria and the microbial quality of alternative irrigation water are important for sustainable irrigation practices into the future. This research will be used to determine the suitability of alternative irrigation water sources for use in fresh produce irrigation to conserve groundwater.

E nhanced awareness of the health benefits of fresh produce led to an average 4.5% per year increase in the consumption of fruits and vegetables worldwide in 1990 to 2004 (1, 2). While considered healthy, this increase in fresh produce consumption does not come without risks to public health due in part to the fact that these food products are usually consumed raw or with minimum processing (3). Consequently, as consumption of fresh produce has risen, outbreaks of foodborne illnesses associated with these foods have risen as well. According to the Centers for Disease Control and Prevention (CDC), 46% of all foodborne illnesses and 23% of foodborne illness related deaths in the United States annually can be attributed to ingestion of contaminated produce (4). While determining the source of produce-associated outbreaks can be difficult, produce contamination can occur at any point from farm to fork. One suspected route of contaminated produce is irrigation water (5)(6)(7). This research focuses on irrigation water sources on the Eastern Shore of Maryland (ESM), located in the mid-Atlantic region of the United States. ESM is an agriculturally intensive area, generating over 70% and 44% of Maryland's total agricultural and fresh produce sales, respectively, in 2017 (8).
Globally, the agriculture sector is the largest consumer of available freshwater, responsible for approximately 70% of total freshwater withdrawals (9). However, climate change and water scarcity are placing severe stress on available freshwater irrigation water sources, especially in drought-stricken areas (10). Declines in annual precipitation in the western United States are expected to reduce water supplies for irrigation during the summer and fall growing seasons (11). While the mid-Atlantic is currently not experiencing water scarcity, this is predicted to change in the coming years (12)(13)(14). In this region, groundwater is the most commonly used water source for agricultural irrigation, accounting for 53% of irrigation water withdrawals (14,15), and increased demand has led to a steady decline in groundwater levels in this region (14). In 2005, it was reported that the Coastal Plain region of Maryland, including ESM, had a 2-foot per year decline in groundwater levels between 1980 and 2005, potentially affecting the longterm sustainability of groundwater resources in this area (12). Moreover, ESM is estimated to be at moderate risk for decreases in irrigation water supplies due to drought in the future (13). With the demand for water in the agriculture sector expected to increase by 2050 and the continued depletion of groundwater-fed aquifers, safe use of alternative water sources for fresh produce irrigation has become an area of great interest (16,17).
There have been a number of studies showing variations in the microbial quality of irrigation water sources in the mid-Atlantic region. Micallef et al. (18) found 7.7% (n = 39) and 15% (n = 13) of pond water samples and irrigation ditch water samples to be positive for Salmonella on mid-Atlantic tomato farms in 2009 and 2010, respectively. Weller and associates (19) found that 12% (n = 10) and 6% (n = 4) of irrigation water samples were positive for L. monocytogenes and Salmonella, respectively, collected from 10 farms in New York state over a 6-week period. These authors also found that 63% and 6% of irrigation water samples in 2014 (19) and 2017 (20), respectively, from the same site in New York were positive for L. monocytogenes. Truitt and coworkers (21) found an overall prevalence of Salmonella of 19% (n = 400) in preharvest production waters on the Eastern Shore of Virginia. Of surface river waters sampled on ESM, 65% were positive for S. enterica in 2015 and 2016 (6). More recently, Sharma et al. (22) found that 50% and 31% of nontraditional irrigation water sources in the mid-Atlantic tested positive for S. enterica and L. monocytogenes, respectively.
The study presented here utilizes the same methodology as our previous research (22) but with different sampling sites in different geographic areas. In that study, the irrigation water sites sampled were mainly on the Western Shore of Maryland (WSM). While in the same region, these two geographic areas are very distinct from each other in many regards, including climate, agricultural land use, topography, soil composition, and population. It has been suggested that different environmental factors in different regions may affect Salmonella prevalence (5). As seen in the Weller studies previously mentioned (19,20), variation can be seen even within the same site at different points in time. Thus, the current study investigates whether there may be variations the prevalence of these two pathogens in an area with different geography, land use, and water types from our previous study.
In addition, this research uses a subset of sites used in our previous research (23). That previous study reported a low prevalence (2.35%) of Shiga-toxigenic Escherichia coli in these water sources. The focus of the current research is the two pathogenic bacteria that rank in the top five in terms of the economic burden they place on public health in the United States, Salmonella enterica and Listeria monocytogenes (24). From 2010 to 2017, 85 multistate outbreaks associated with fresh produce occurred in the United States (25). Of these 85, 56 were attributable to Salmonella, resulting in 3,778 illnesses, 778 hospitalizations, and 16 deaths; 4 were attributable to L. monocytogenes, leading to 173 illnesses, 169 hospitalizations, and 37 deaths (25). The overall goal of this study was to determine the microbiological quality, with respect to the prevalence of S. enterica and L. monocytogenes, in alternative irrigation water sources (26) on ESM.
Water type and seasonality affected recovery of pathogens. When compared by water type, S. enterica and L. monocytogenes levels were significantly (P , 0.05) lower in reclaimed water than in nontidal freshwater and tidal brackish water (Fig. 2). No significant (P . 0.05) differences were detected in pathogen levels between the nontidal freshwater and tidal brackish water sites. Seasonality did not affect S. enterica levels, with statistically similar (P . 0.05) levels found in all seasons (Fig. 3). In contrast, L. monocytogenes levels were significantly higher in spring and winter (P = 0.0069 and P = 0.0304, respectively) than in fall (Fig. 3).
Volume of water filtered affected the likelihood of Salmonella enterica recovery. Table 2 shows the odds ratios for recovery of S. enterica and L. monocytogenes by volume filtered. For S. enterica, the odds of recovery (2.15) were significantly (P = 0.0129) higher when 10 liters of water was filtered than when 0.1 liter was filtered. Filtering 1 liter of water had 1.82 times the odds of recovering S. enterica compared to filtering 0.1 liter of water, and filtering 10 liters of water had 2.15 times the odds of recovering S. enterica compared to filtering 0.1 liter of water. For L. monocytogenes, there were no statistically significant differences in the odds of recovery by volume of water filtered.
Ordinal relationships between the pathogens and environmental characteristics. The ordinal relationships between S. enterica and L. monocytogenes and the 10 environmental characteristics (water type, conductivity, salinity, oxygen-reducing potential,  dissolved oxygen, turbidity, nitrate, temperature, barometric pressure, and pH) tested are shown in the redundancy analysis (RDA) plot (Fig. 4). An analysis of variance using permutation was used to determine the significance of the constraining variables (environmental characteristics) and categorical values (water types) in the RDA model. Out of the environmental characteristics tested, pH and barometric pressure were determined to be significant (P = 0.008 and P = 0.049, respectively) factors in relation to S. enterica and L. monocytogenes levels. Both pathogens were negatively associated with increasing barometric pressure and pH (blue arrows are pointing away from the pathogen points [red squares]). Figure 4 shows that while most of the water samples are clustered around the center of the RDA ordination graph, several reclaimed water samples had a positive association with both pathogens, pointing to further investigation of both pathogens in reclaimed water compared to other water types. The total constrained variance accounted for by the water physicochemical factors in the RDA plot (6.4%) is represented by the eigenvectors RDA1 (x axis) and RDA2 (y axis).

DISCUSSION
ESM is currently experiencing decline in groundwater levels, and this is predicted to worsen in the future. In an effort to conserve groundwater, exploration of alternative water sources for irrigation has become a key funding priority of the U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (27). With groundwater being the primary water type used for irrigation on ESM, alternative water sources in this region include reclaimed water, untreated surface water, and brackish water (26).  The results of this study demonstrated levels of S. enterica and L. monocytogenes much lower in the reclaimed water samples than in the nontidal freshwater rivers and the tidal brackish river water samples. These data are consistent with our previous research (22).
Since the reclaimed water was tested after undergoing primary, secondary, and tertiary treatments to remove pathogens and other contaminants prior to lagoon storage, lower pathogen levels were expected. The use of reclaimed water for agricultural irrigation may offer several advantages: (i) it constitutes a reliable and renewable water source that is climate-resilient, (ii) it can be an affordable source of irrigation water, and (iii) its use would alleviate the burden on ground water sources (27,28).
The prevalence rates of S. enterica (65%) and L. monocytogenes (40%) in the current study were similar to those from Cooley et al. (5), who found S. enterica and L. monocytogenes prevalence to be 65% and 43%, respectively, in agricultural watersheds in central California. However, the prevalence levels in the current study were greater than those reported in our previous research for distinct sites located elsewhere (22), where the occurrence of S. enterica and L. monocytogenes was 50% and 31%, respectively, using the same sampling protocol and sampling dates. In that study, the prevalence of both S. enterica and L. monocytogenes was lower in reclaimed waters and in ponds than in rivers. For the current research, sampling sites were all located on ESM. This area is densely populated with poultry operations. All of the surface water sites sampled were within 4 km of several operational poultry facilities (Table 3 [29]). In addition, MA03 was within 1.6 km of a wastewater treatment discharge facility. Previous studies have shown that surface waters close to confined poultry feeding operations are susceptible to pathogen contamination (30,31). Vereen et al. (32) found that Salmonella detection frequencies were positively associated with the number of poultry houses in subwatersheds in Georgia and found that Salmonella prevalence increased near wastewater treatment plant discharge points (32). Moreover, runoff or dust from poultry farms or from field poultry litter application (6) may have contributed to the higher incidence of these pathogens found in these water sources.
S. enterica and L. monocytogenes incidence in rivers was greater in this study than in other studies evaluating the presence of these pathogens in pond surface waters in this region. Micallef   These arrows are pointing away from the pathogens (red squares), indicating a negative association between these parameters and the two pathogens. Shorter distances between dots (S. enterica, L. monocytogenes, water types) and water quality parameters (blue arrows) indicate higher levels of that characteristic relative to categorical (water types) or response (pathogen) variables. 20 agricultural ponds on the Eastern Shore of Virginia in 2015 and 2016 to be 19% (n = 400). This variation in prevalence between microbial quality of river and pond surface water was also reported for indicator bacteria (29) and for creek versus irrigation pond sites (33) and is of critical importance for food safety. Moreover, other factors may confound the data and make direct comparisons more challenging. This may include year to year variation and season of collection (33), as well as sample volumes analyzed. The current research evaluated three sample volumes (0.1 liters, 1 liter, and 10 liters). Filtering 1 liter and 10 liters was 1.82 and 2.15 times more likely to recover S. enterica than filtering 0.1 liters, respectively. Our previous research (22) showed that filtering 10 liters was 43.5 and 25.5 times more likely to recover S. enterica than filtering 1 liter and 0.1 liter, respectively. The relatively small increase in the likelihood of recovering S. enterica when filtering 10 liters versus 1 liter of water from these sites in the present study compared to the sites in our previous research indicates the greater prevalence of S. enterica in the specific river sources we evaluated. These results indicate that both the volume filtered and the volume analyzed can affect the recovery of S. enterica and L. monocytogenes.
The lack of a seasonal effect on S. enterica prevalence is in agreement with other studies (3,34). However, significantly higher levels of Salmonella spp. are frequently reported during the summer compared to the other seasons (33). L. monocytogenes populations in water were significantly greater in winter and spring than fall in the current study. These results are consistent with Cooley et al. (5), whose results also showed greater L. monocytogenes prevalence in winter and spring than fall. Similarly, several investigators have found that L. monocytogenes was more frequently detected in waters with colder temperatures (3,22).
The results of the RDA analysis show that little variance in S. enterica and L. monocytogenes levels was attributable to the water physicochemical factors measured, with only 6.4% of the variance constrained in the RDA. This finding is consistent with other studies (22,35). McEgan et al. (35) found weak linear relationships between Salmonella levels and water physicochemical indicators in surface waters in central Florida; however, they did find site-specific correlations in some cases. Sharma et al. (22) reported poor correlations between water physiochemical factors and S. enterica and L. monocytogenes, with a total constrained variance of 13.8%. Weller et al. (20) found that interactions between environmental factors affected the likelihood of pathogen detection in agricultural waters in Arizona and New York. These authors concluded that the food safety risks associated with use of a water source for irrigation are dependent on environmental conditions at the time of water use (20). The variability in these results emphasizes the regionality of microbial water quality characteristics. Therefore, it is not redundant to investigate how local environmental conditions affect the survival and persistence of pathogens in irrigation water and what indicators of microbial quality to use for various irrigation water types. This study shows that S. enterica and L. monocytogenes are present in at high levels in alternative irrigation water sources on ESM. Several produce-related foodborne illness outbreaks have been traced back to this agricultural area (36,37). From a microbial perspective, due to lower pathogen loads, reclaimed water may offer an affordable and sustainable agricultural irrigation water source in this region. However, due to the small sample sized used in this study, further research is needed. The difference in incidence of positive samples compared to previous studies in other geographic areas stresses the need for regional studies that evaluate the unique conditions and environmental prevalence of foodborne pathogens in irrigation water sources. This research can be used to determine the most suitable alternative water sources for use in fresh produce irrigation in this region, combining food safety and conservation goals. Moreover, the results of this research will help identify water sources that require on-farm mitigation strategies to reduce bacterial populations and improve the quality of those water sources for irrigation.

MATERIALS AND METHODS
Experimental sites. Water samples were collected over 2 years (October 2016 to October 2018) from six sites (four surface water and two reclaimed water sites) on Maryland's Eastern Shore. The four surface water sites which included: three nontidal freshwater rivers (MA03, n = 36; MA07, n = 36; MA09, n = 37) and one tidal/brackish river (MA08, n = 36), were sampled twice monthly during the growing season (October 2016, June-October 2017, May-October 2018) and once a month during the off season (November 2016-May 2017, November 2017-April 2018). The reclaimed irrigation water sources (MA01, n = 13; MA02, n = 10) were only in use during the growing season; therefore, sampling at these sites was conducted from April 2017 to October 2018 (Table 3; 29).
Modified Moore swab design. On-site water collection was conducted using MMS adapted from Sbodio et al. (38) and modified in previous research (22,23). MMS were created using grade no. 90 cheesecloth (Lion Services, Inc., Charlotte, NC, USA) folded in thirds lengthwise and rolled into a cylindrical shape 16 cm long and 4 cm in diameter. Autoclaved MMS was aseptically inserted in a 29.2-cm long and 3.81-cm diameter polyvinyl chloride (PVC) cartridge. The PVC cartridge was designed with a mesh screen on one side for water inlet and debris filtration and a 3.81-cm male barbed PVC fitting on the opposite end for connection to a water pump.
Water sample collection. This study utilized a modified MPN approach using three volumes (0.1 liters, 1 liter, and 10 liters) collected in triplicate (22). For the 0.1-and 1-liter samples, water was collected in a sterile 1-liter bottle, transferred to a sterile graduated cylinder, and measured to the target volume. The target volume of water was then poured through the mesh screen of a sterile cartridge containing an MMS and allowed to flow through by gravity filtration. For the 10-liter samples, the cartridge was inserted into a PVC flotation device allowing for collection of samples from a depth of 15 cm to minimize sediment disturbance. The cartridge was connected to a water pump (Honda model no. WX10TA; Honda Power Equipment, Alpharetta, GA, USA), and 10 liters of water was actively pumped through the MMS. At the reclaimed water sites, 20-liter water samples were collected from spigots near field discharge points into a sterile 20-liter carboy and treated with 20 ml of 10% sodium thiosulfate (Fisher Scientific, Washington, DC, USA) to neutralize any free chlorine in the water. Water was then filtered as described above. After filtration, MMS were placed in labeled Whirl-Pak bags (Nasco, Madison, WI, USA) and transported on ice to the laboratory.
At each sample collection site, the water physicochemical parameters water temperature (°C), dissolved oxygen (mg/liter), conductivity (mS/cm), pH, oxygen reducing potential (mV), salinity (PNU), and turbidity (FNU) were also measured using an EXO2 multiparameter water quality sonde (YSI, Yellow Springs, OH, USA). Precipitation amounts (1, 7, and 14 days prior to sampling) and ambient temperature were retrieved from Weather Underground. Weather data were obtained from the weather stations closest to each sampling site, with an average distance of 21 km (range, 3 to 50 km). For reclaimed water