FEMS Microbiology Ecology, 91, 2015, fiv005 doi: 10.1093/femsec/fiv005 Advance Access Publication Date: 12 January 2015 Research Article
RESEARCH ARTICLE
Effects of transparent exopolymer particles and suspended particles on the survival of Salmonella enterica serovar Typhimurium in seawater Marion C. F. Davidson1 , Terra Berardi2 , Beatriz Aguilar2 , Barbara A. Byrne2 and Karen Shapiro2,∗ 1
´ erinaire ´ ´ eral ´ Ecole nationale vet d’Alfort, 7 Avenue du Gen de Gaulle, 94704 Maisons-Alfort Cedex, France and Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California Davis, One Shields Ave. Davis, CA 95616, USA 2
∗ Corresponding author: School of Veterinary Medicine, University of California Davis, Department of Pathology, Microbiology, and Immunology, One Shields Ave. Davis CA 95616, USA. Tel: +001-530-219-5476; Fax:+001-530752-3349; E-mail:
[email protected] One sentence summary: Results document prolonged survival of Salmonella, an important pathogen of people, in seawater, with sticky polysaccharides (TEP) favoring and suspended particles inhibiting environmental persistence of this bacterium. Editor: Patricia Sobecky
ABSTRACT The bacterium Salmonella enterica can infect marine mammals and has been increasingly implicated in seafood-borne disease outbreaks in humans. Despite the risk this zoonotic agent poses to animals and people, little is known regarding the environmental factors that affect its persistence in the sea. The goal of this study was to evaluate the impact of two constituents on the survival of Salmonella in the marine environment: transparent exopolymer particles (TEP) and suspended particles. A decay experiment was conducted by spiking Salmonella into bottles containing seawater, seawater with alginic acid as a source of TEP, filtered seawater or filtered seawater with alginic acid. Survival of Salmonella was monitored using culture followed by enrichment assays to evaluate if the bacteria entered a viable but non-cultivable (VBNC) state. Salmonella cell counts dropped significantly faster (P ≤ 0.05) in the unfiltered seawater samples with and without TEP. The slowest decay occurred in filtered seawater containing alginic acid, with VBNC Salmonella persisting for 17 months. These findings suggest that TEP may favor Salmonella survival while suspended particles facilitate its decay. Insight on the survival of allochthonous, zoonotic pathogens in seawater can guide monitoring, management and policy decisions relevant to wildlife and human public health. Key words: Salmonella enterica; fecal indicator bacteria; Escherichia coli; persistence; detection; ocean
INTRODUCTION Salmonella is one of the major pathogens causing food-borne (Mead et al., 1999) illnesses in both humans and animals. In the United States, this zoonotic bacterium has been reported as the leading cause of hospitalizations and deaths linked to contaminated food eaten between 2000 and 2008 (Scallan et al., 2011).
While food-borne salmonellosis in people has been traditionally attributed to consumption of contaminated meat or eggs, several recent outbreaks have been linked with ingestion of seafood (Iwamoto et al., 2010; Amagliani, Brandi and Schiavano 2012). For example, in July 2012, a multistate outbreak of Salmonella enterica serovars Bareilly and Nchanga was linked to the consumption of raw scraped ground tuna, with a total of 425 reported cases (CDC
Received: 8 July 2014; Accepted: 5 January 2015 C FEMS 2015. All rights reserved. For Permissions, please e-mail:
[email protected].
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2012). Less than a month later, a second Salmonella outbreak due to smoked salmon was reported in both the Netherlands and the United States, with about 866 people sickened (Friesema et al., 2012). Between 1997 and 2008, seafood consumption was responsible for 5% of all reported cases of Salmonella infections in the United States (Doyle et al., 2009). Seafood-associated outbreaks of salmonellosis have been caused by fish, shrimp, oysters and clams, all of which can acquire Salmonella from polluted waters (Iwamoto et al., 2010). Contamination of seafood may occur during the post-harvest processing or via contamination of marine animals in their natural habitats. While Salmonella is a fecal bacterium that is not indigenous to the marine environment, it has been identified in numerous seawater samples including in Spain (MartinezUrtaza et al., 2004), Morocco (Setti et al., 2009), Portugal (Dionisio et al., 2000) and the United States (Walters et al., 2013). In the latter study, 74 samples collected from 13 central California coastal sites between 2008 and 2009 tested positive for Salmonella (overall prevalence 30.7%). Salmonella enterica serovar Typhimurium was the most commonly detected serovar, followed by Heidelberg and Enteritidis. As these are the same serovars isolated from patients suffering from salmonellosis in California, the potential risk of Salmonella strains circulating in the marine environment to public health should not be discounted. Furthermore, the PFGE patterns of 75% of the queried isolates were indistinguishable from clinical entries in PulseNet, which highly suggests that they have the potential to be clinically relevant (Walters et al., 2013). Although the concentrations of Salmonella in live and raw fish are thought to be low, for some Salmonella serotypes the infective dose required to cause illness is very low at 1–10 cells (Huss, Reilly and Karim Ben Embarek 2000). Salmonella can also be concentrated by filter-feeding molluscs that are often eaten raw (Amagliani, Brandi and Schiavano 2012). A study of oysters harvested from 36 US bays showed that 7.4% of all tested oysters were contaminated with Salmonella (Brands et al., 2005). As oysters are often consumed raw, even a relatively low number of bacteria can pose a significant risk for infection. The risks linked to contamination of certain seafood species in the marine environment should therefore not be underestimated. In addition, because of the significant increase of seafood consumption in recent decades has been associated with consumers’ preferences for sushi and other minimally processed products, seafood-borne diseases have become a major health issue. The presence of Salmonella in the marine environment may originate from two sources: upstream contamination in watersheds that receive fecal pollution from human sewage, urban and agricultural runoff or in situ defecation by infected marine animals (Martinez-Urtaza, et al., 2004; Pommepuy et al., 2005). Indeed, In addition to causing disease in humans, Salmonella has been isolated from many marine animals such as marine birds, cetaceans, pinnipeds, reptiles and fish (Minette 1986; Berardi et al., 2014). A wide range of Salmonella serovars including Typhimurium, Enteritidis, Montevideo and Newport, which are known human pathogens, have been isolated from pinnipeds. The reported prevalence of Salmonella in pinnipeds can be as high as 25% in some specific areas such as the Channel Islands, California (Stoddard et al., 2008). Most marine mammals and birds are thought to carry the bacterium asymptomatically, harboring Salmonella and shedding it in their feces without displaying clinical symptoms (Minette 1986; Higgins 2000). By releasing bacteria into the marine environment, these animals may provide a reservoir for this zoonotic agent (Fenlon 1983; Huss, Reilly and Karim Ben Embarek 2000). However, some marine
species can also suffer from clinical salmonellosis, presenting with gastrointestinal symptoms and septicemia that can lead to their death (Dierauf and Gulland 2001). Therefore, a better understanding of Salmonella in the marine environment, including fecal sources, transport and survival in seawater, should assist with the monitoring and management of safe seafood harvest. Our work is focused on providing novel insight on the survival of Salmonella in the marine environment. Specifically, the goal of this study was to evaluate the impact of two seawater constituents on Salmonella persistence in coastal ecosystems: suspended particles and transparent exopolymer particles (TEP). Suspended particles are ubiquitous in the marine environment and prior studies have demonstrated that the association of Escherichia coli [a fecal indicator bacteria (FIB)] with particles such as aggregates favors its survival (Lyons et al., 2010). For Salmonella, one report indicated that suspended particles in freshwater systems do not impact the survival of the bacterium (Maki and Hicks 2002); however, to date such studies have not been performed in marine water. In a study evaluating Salmonella in marine laboratory mesocosms, the number of Salmonella cells associated with sinking aggregates was two to three orders of magnitude greater than the number in surrounding water; however, survival of the bacterium was not evaluated in this previous investigation (Shapiro et al., 2013). The second group of seawater constituents that may impact bacterial survival are TEP, which are diverse classes of sticky polysaccharide molecules produced by marine organisms including cyanobacteria, phytoplankton, invertebrates and algae (Passow and Alldredge 1994; Ramaiah, Yoshikawa and Furuya 2001; Heinonen, Ward and Holohan 2007). These gel-like substances have been shown to provide surfaces for colonization by bacteria (Passow 2002). TEP are sticky agents that are critically important for geochemical processes in the ocean, and can serve as a gluing agent for enhancing aggregate formation that drive vertical transport of matter and energy. As a carbon source, TEP can also provide nutrients to marine bacteria and a source of refuge from potential predators (Verdugo et al., 2004). However, investigations that address whether a zoonotic bacterium could also utilize these particles to enhance its persistence have been lacking to date. Therefore, this study aimed to test the hypothesis that TEP and suspended particles can enhance the survival of Salmonella in seawater.
MATERIALS AND METHODS To evaluate the impact of both TEP and suspended particles (including inorganic material, organic aggregates and plankton) on the survival of Salmonella, the decay of the bacterium was monitored over time in four different seawater treatments: unfiltered seawater (containing suspended particles and background levels of TEP), seawater with added alginic acid (containing suspended particles and a high concentration of TEP), filtered seawater (lacking both particles and TEP) and filtered seawater with added alginic acid (lacking particles but containing a high concentration of TEP). Alginic acid is a single type of TEP produced by giant kelp (Macrocystis pyrefera), a macroalga that is prevalent along the coast of California. The addition of alginic acid to seawater provided a controlled approach for assessing the impact of a single type of TEP on survival of Salmonella.
Sample collection Seawater (10 L) was obtained approximately 3 kilometers offshore of Cambria, CA (Latitude 35.559738◦ Longitude
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−121.145110◦ ) in June 2012. Collection of the sample several kilometers from the shore was performed to provide a sample that should yield relatively low concentrations of naturally present TEP, which can be high close to the shoreline due to discharge of freshwater, phytoplankton bloom events or presence of kelp forests (Passow and Alldredge 1994; Ramaiah, Yoshikawa and Furuya 2001). The sample was collected approximately 30 cm below the surface using a sterile polypropylene carboy. The sample was kept chilled on ice and transported overnight to the laboratory. Upon arrival, an aliquot was removed for physicochemical testing and the decay experiment was initiated within 24 hr of sample collection. TEP quantification in the original seawater sample and the different water treatments used in the experiment was performed using the alcian blue staining semi-quantitative colorimetric technique described by Passow and Alldredge (1995).
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with naturally occurring suspended particles while minimizing decay of the bacterium before the monitoring phase of the experiment has begun. Following this initiation phase and for the duration of the experiment, bottles were maintained upright in a stationary water bath within a walk-in refrigerator (hence, under mostly dark conditions) at 12◦ C, which represents an average surface seawater temperature in central CA coastal waters.
Salmonella detection
Salmonella enterica serotype Typhimurium labeled with a green fluorescent protein (GFP) and carbenicillin resistance (kindly provided by Dr Andreas Baumler, UC Davis—henceforth referred to as Salmonella) was inoculated at 107 colony-forming units (cfu) per mL into 150 mL of the four seawater treatment types: unfiltered seawater, filtered seawater (0.2 μm), unfiltered seawater with added alginic acid (Thermo Fisher Scientific Inc.) at a target concentration of 100 mg per L and filtered seawater with added alginic acid at the same concentration. The concentration of added alginic acid represents a high concentration of TEP measured in natural seawater as documented in studies worldwide (Passow 2002). For each of the four seawater treatment types, three replicates were tested (12 bottles total). An additional bottle served as a negative control and consisted of unfiltered seawater; this sample was maintained alongside the treatment bottles for the entire duration of the experiment and tested for presence of Salmonella at the same time points (Fig. 1).
To evaluate the decay of Salmonella in each bottle, serial dilutions were prepared to achieve a solution that would produce countable colonies on selective agar. Water bottles were gently inverted several times and aliquots (100–1000 μL) removed for inoculation onto Luria-Bertani agar containing carbenicillin (0.1 mg mL−1 ). Spread plates were prepared by diluting aliquots as needed to achieve countable colonies, and GFP-producing colonies were enumerated following 24 hr incubation at 37◦ C. Plating was performed daily for the first week, twice a week for the subsequent three months and once a week until no growth was observed for three consecutive weeks. Following three consecutive negative growth results from a specific bottle, further enrichment assays were performed to evaluate if Salmonella entered a viable but non-cultivable (VBNC) state. To revive VBNC ˜ Salmonella cells, a protocol was adapted from Morinigo et al. (1990). In brief, a 10 mL aliquot was concentrated onto a mixed cellulose membrane (0.45 μm pore size Microfil funnel, Millipore, Billerica, MA) and placed in 100 mL buffered peptone water (BPW 1%) on a rotating shaking incubator for 24 hr at 37◦ C. After the incubation, 100 μL of the enriched BPW solution were transferred to 10 mL Rappaport-Vassiliadis (RV) medium and incubated at 43◦ C for 72 hr. Enriched RV medium was then streaked onto xylose lysine deoxycholate agar and incubated at 37◦ C for 24 hr. Presence of black (sulfur-reducing) colonies that fluoresced green under UV illumination were determined to yield a positive growth of spiked GFP Salmonella.
Experimental conditions
Statistical analysis
Immediately following the addition of the Salmonella cells, experimental bottles were placed on a horizontal roller apparatus and rolled for 48 hr at 4◦ C. This procedure was performed to produce water movement and internal currents that would promote association of particles, which is reported to produce the most realistic marine aggregates under laboratory conditions (Shanks and Edmondson 1989). The lower temperature for the initiation phase was designed to allow for Salmonella to become associated
Negative binomial regression was used to determine whether (1) presence of suspended particles and/or (2) alginic acid as a source of TEP were significant predictors for the numbers of Salmonella cells recovered from seawater over time. Each bottle was set as a cluster effect to adjust for repeated sampling within replicates, and the sampling time (day) was incorporated as an exposure variable to compare cell counts for each time point among bottles. The Kruskal–Wallis (KW) non-parametric test
Sample preparation
Figure 1. Experimental design of the Salmonella survival experiment. A single seawater sample was used to prepare three replicates (150 mL) of each of the four seawater treatment types: filtered seawater with and without alginic acid as a source of TEP and unfiltered seawater with and without alginic acid. The filtration of seawater provided an identical water matrix that lacked suspended particles larger than 0.2 μm or TEP producers such as phytoplankton. All samples except for the negative control were inoculated at 107 cfu mL−1 with a GFP strain of Salmonella. Samples were maintained at 12◦ C in the dark for the duration of the experiment (18 months).
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was applied to test for differences in the decay rate of Salmonella among the different treatments by evaluating the number of days taken to achieve each log reduction (1 to 7) of Salmonella cells. Significant KW analyses were then followed by a nonparametric post hoc pair-wise comparison (Siegel and Castellan 1988). Statistical analyses were performed using STATA (College Station). Significance was determined at P ≤ 0.05 for all statistical analyses.
RESULTS The physicochemical water characteristics of the seawater used in the decay experiment were: 33.1 ppt salinity, 7.80 pH, 1.0 nephlometric turbidity units turbidity, 4.2 mg.L−1 total suspended solids (TSS), with the carbon and nitrogen fraction contributing to TSS < 0.1 mg.L−1 each. TEP concentrations in both the filtered and unfiltered seawater samples were not detectable. In filtered and unfiltered seawater with added alginic acid, TEP concentrations ranged between 2758 and 2945 μL gum zanthan equivalents per liter (within values for maximal TEP recorded in natural waters (as reviewed by Passow 2002). Overall, the decay of Salmonella was slower in filtered seawater samples that lacked suspended particles, as displayed in Fig. 2. Negative binomial regression results indicated that lack of suspended particles (due to filtration) and presence of TEP in the form of added alginic acid were significantly associated with higher numbers of Salmonella cells recovered over the entire duration of the study (P ≤ 0.05). Decay of Salmonella was first detected in unfiltered seawater without added alginic on day 6, and
with added alginic acid on day 9. By day 12, a 3-log reduction of Salmonella concentration was observed in both unfiltered seawater treatments, with and without alginic acid. Over the entire duration of the experiment, survival of Salmonella was significantly longer in filtered seawater containing alginic acid (Fig. 2 and KW P ≤ 0.05). Survival of Salmonella also appeared to be higher in the filtered seawater not containing added alginic acid: on the 10th week (day 72) a 6-log reduction was measured in both unfiltered seawater treatments, while both filtered seawater types had decayed by less than one order of magnitude. For the full duration of the experiment, there were no statistically significant differences in Salmonella decay in unfiltered samples with or without alginic acid (KW P > 0.05). Salmonella cells have the ability to enter a VBNC state in which cell metabolism diminishes and the cells fail to grow under traditional cultivation techniques (Roszak, Grimes and Colwell 1984). This survival mechanism is triggered by stress (i.e. starvation) under adverse environmental conditions. In the current study, VBNC Salmonella cells were present in unfiltered seawater until the 18th (122 days) and 32nd (219 days) week in samples with and without alginic acid, respectively (Table 1). Cultivable Salmonella was present in filtered seawater without alginic acid for 43 weeks (296 days), but VBNC cells were not detected in this water type. The longest survival of Salmonella occurred in filtered seawater containing alginic acid, with cultivable cells present for 44 (313 days) and 48 weeks (342 days) in two of the replicates and 62 weeks (439 days) in the third replicate, which also contained VBNC bacteria for 17 months (74 weeks, 522 days). Overall, the presence of VBNC Salmonella
Figure 2. Survival of Salmonella Typhimurium in seawater. Average concentration of Salmonella cells detected in different seawater treatments (n = 3) over time. Error bars denote 95% confidence intervals. Salmonella was initially inoculated at 107 cfu per mL into treatment bottles, and was not detected in a non-inoculated negative control seawater sample for the entire duration of the experiment.
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Table 1. Successful revival (yes/no) for detection of viable but not cultivable (VBNC) Salmonella cells in each replicate from the decay study. Attempts for revival began after three consecutive attempts to directly culture the bacterium failed, and continued until two revival attempts in a row did not yield growth. Dashes denote dates when revival was not attempted because Salmonella was still directly cultivable from the sample. Sample type Seawater + alginic acid
Seawater Replicate Experimental week 16 18 23 26 32 36 43 48 50 51 53 55 66 71 74 76 78
Filtered seawater
Filtered seawater + alginic acid
1
2
3
1
2
3
1
2
3
1
2
3
– Yes Yes Yes Yes No No
Yes Yes Yes Yes Yes No No
– – – – No No
– – No No
– – – No No
Yes Yes No No
– – – – No No
– – – – – – – No No
– – – – – – No No
– – – – – – – – – – Yes Yes No Yes Yes No No
– – – – – – – No No
– – – – – – – – – No No
cells was detected in one of the three replicates containing unfiltered seawater and alginic acid; one of the three replicates containing filtered seawater and alginic acid; and two of the three replicates containing seawater without alginic acid (Table 1). During the entire duration of the experiment, Salmonella was not detected in the negative control sample, confirming that contamination between samples during the experiment was unlikely.
DISCUSSION Results from this study suggest that suspended particles have a negative impact on the survival of Salmonella in seawater, while TEP may facilitate the persistence of this zoonotic pathogen. We initially hypothesized that maintaining Salmonella in non-filtered seawater would have a positive impact, due to a prior report documenting the enhanced survival of aggregateassociated bacteria such as E. coli via utilization of organic nutrients and provision of refuge from predators (Lyons et al., 2010). However, the finding that suspended particles promoted faster decay of Salmonella did not support this hypothesis. One plausible explanation is that survival of Salmonella was compromised by either nutrient depletion and/or predation by other aquatic organisms that are commonly present in non-filtered seawater. For instance, various studies indicate that the main predators of bacteria in the marine environment are protozoa (Barcina et al., 1991; Gonzalez et al., 1992; Rozen and Belkin 2001). Lytic bacteria such as Bdellovibrio and like organisms can also contribute to the removal of allochthonous bacteria from aquatic systems (Taylor et al., 1974; Barcina, Lebaron and Vives-Rego 1997; Jurkevitch 2006). Protozoa and lytic bacteria are mostly larger than 0.2 μm and were therefore removed from the water
by the filtration process. This reduction in predators may explain why Salmonella survived longer in the filtered seawater samples. The increased survival of Salmonella in filtered seawater samples also suggests that in the specific seawater tested here, nutrient competition and/or predation due to other microorganisms may have had a larger negative impact on the survival of Salmonella than any potential positive impact provided by organic particles. The results from the current study therefore suggest that there may be important differences between the effects of suspended particles or organic aggregates on the survival of Salmonella and E. coli. This finding is particularly important because many bacterial decay studies and water quality monitoring practices are based on the quantification of E. coli as a marker of pathogenic fecal bacterial contamination and survival (US EPA 2000; Field and Samadpour 2007; US EPA 2012). Additional studies should be performed to simultaneously evaluate the survival of both bacteria under identical seawater conditions to further test whether E. coli is a suitable indicator for the survival of Salmonella in the marine environment. Although alginic acid appeared to favor Salmonella survival in filtered seawater, this effect was not statistically significant (Fig. 2). However, the absence of a statistically significant difference may be due to the small replicate numbers used in this study (three replicates per seawater type), which resulted in diminished statistical power for identifying differences in survival between water groups. A greater number of replicates would be needed to more definitively evaluate whether TEP favors Salmonella survival in filtered seawater. Overall, the study findings suggest that areas with high concentrations of TEP and low abundance of suspended particles may represent marine habitats that could promote the persistence of Salmonella. Because TEP production in the ocean is typically associated with release from phytoplankton, cyanobacteria or macroalgae,
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the probability for high TEP concentrations in low suspended particle conditions may be rare. Yet, observations of high TEP concentrations during low phytoplankton cell counts are documented (Passow and Alldredge 1995; Passow et al., 2001). Under such conditions, the prolonged persistence of Salmonella following pollution events could favor contamination of fish and mollusks with Salmonella, which in turn may lead to an increase in risk of this zoonotic pathogen entering the food chain of both humans and higher trophic level marine animals. Viable Salmonella cells were detected in unfiltered seawater for 32 weeks (219 days) and in filtered seawater for 17 months (74 weeks or 522 days), with both filtration and presence of TEP serving as significant predictors for the survival of this zoonotic bacterium (negative binomial regression P < 0.05). The survival durations of culturable cells reported in this study are longer than previously reported in the literature for Salmonella Typhimurium (12–54 days (Nabbut and Kurayiyyah 1972; Lebaron and Joux 1994; Smith, Howington and McFeters 1994)), as well as for FIB such as E. coli [30–54 days reported by Smith, Howington and McFeters (1994) or 20 days reported by Solecki et al. (2011)]. The variable experimental conditions and detection methods utilized across these different studies may explain the prolonged Salmonella survival duration reported here. The survival of allochthonous bacteria in aquatic systems depends on various biotic and abiotic environmental factors (Barcina, Lebaron and Vives-Rego 1997). The main biotic factors are predation, grazing and competition, and protection conferred by selected plasmids (Barcina, Lebaron and Vives-Rego 1997, 1992). Abiotic factors including osmotic stress (salinity), nutrient deprivation, temperature and especially exposure to visible light and UV radiation (Davies and Evison 1991) have been shown to induce the most negative impact on the survival of enteric bacteria in seawater. Light is a significant determinant for duration of survival at shallow depths where light penetrates the water column (Kapuscinski and Mitchell 1981; Davies and Evison 1991; Gourmelon 1995; Gourmelon et al., 1997; Rozen and Belkin 2001). In this study, the seawater samples were maintained in a water bath within a walk-in refrigerator to provide a stable temperature at 12◦ C, and thus storage occurred under mostly dark conditions. The slow decay of Salmonella observed in this study may therefore be due to diminished visible light and UV-induced degradation. Salinity may also negatively impact the survival of allochthonous bacteria, as documented by studies showing decreased survival of E. coli in seawater with increasing salinities (Anderson, Rhodes and Kator 1979; Troussellier et al., 1998). Since the salinity concentration present in this study represents real osmotic conditions (33 ppt), this parameter is not likely to contribute to longer than expected survival of Salmonella under natural conditions in our regional coastal environment. Temperature is a third abiotic factor that can impact the survival of Salmonella, though its impact on the survival of allochthonous bacteria in aquatic systems has been at times contradictory. However, most of the studies performed in the presence of natural microflora demonstrate that bacterial survival is lower at higher temperatures, which is hypothesized to occur due to increased predation activity from protozoans (Flint 1987; Barcina, Lebaron and Vives-Rego 1997; Blaustein, Pachepsky and Hill 2013). In this study, temperature-mediated impacts on protozoan predation may have impacted Salmonella survival in the unfiltered seawater samples. The temperature used in this study (12◦ C) represents an average temperature in central California coastline waters, and was lower than temperatures used in most other studies (Barcina, Lebaron and Vives-Rego 1997; Rozen and
Belkin 2001). For instance, one investigation that reported survival of Salmonella for only 19 days in seawater was performed at 20◦ C (Joux, Lebaron and Troussellier 1997). If indeed a lower temperature decreases the predatory actions of the protozoa, it may also partly explain why the bacterium survived for 32 weeks (219 days) in our unfiltered seawater samples. However, it is unlikely that temperature’s effects on predation contributed to the long persistence of the bacteria in the filtered seawater samples. Finally, an additional factor that could influence reported survival durations of Salmonella across different studies is the selected method used to detect and/or quantify the bacterium, such as cfu counts or flow cytometry. The consequences of the disparities in the methods applied in decay experiments have not been documented, but differences in assay sensitivity could impact results even if water conditions were similar across different investigations. In addition, in certain studies that demonstrated Salmonella survival for up to 54 (Smith, Howington and McFeters 1994) or 35 (Lebaron and Joux 1994) days, the experiments were terminated at that specific time point even though Salmonella cells were still present in a VBNC state. Thus, longer survival durations may have been reported had the experiments been continued. Moreover, the specific strain of Salmonella and the concentration of the initial inoculum may also impact survival reported across different studies. Here we utilized a GFPcontaining S. enterica serovar Typhimurium strain that allowed differentiation from other sulfur-reducing bacteria that may be present in seawater (e.g. Proteus spp.). Confirmation of green fluorescing colonies also ensured that the enumerated colonies represent only the strain introduced into the experimental samples.
CONCLUSION In the seawater samples evaluated in this study, suspended particles had a negative impact on the survival of Salmonella Typhimurium, while TEP favored the bacterium’s survival in filtered samples. As prior investigations report enhanced survival of E. coli due to presence of suspended particles, the present results suggest that commonly used FIB organisms may not serve as suitable indicators for the persistence of Salmonella in seawater. Moreover, the long duration of Salmonella survival in seawater described here (up to 17 months or 522 days) suggests that the associated health risks for susceptible marine animals and humans from fecally polluted marine waters may have been underestimated (Easton et al., 2005). In future investigations, additional environmental factors such as the presence of light should also be taken into consideration to characterize zoonotic pathogen survival and the associated risks for wildlife and human health. While visible and UV light are important variables for bacterial survival, many seafood species live in deeper waters under mostly dark conditions, where Salmonella may survive longer than previous studies report. Ultimately, reducing and mitigating contamination of the marine environment by fecal bacteria and implementing efficient treatment of sewage and storm water drainage should be targeted to reduce illness due to Salmonella in animals and people.
ACKNOWLEDGEMENTS The authors thank Ryen Morey and Anna Naranjo for their assistance with Salmonella culture assays, Fernanda Mazzillo for per-
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forming TEP quantification and Tad Doane for performing water physicochemical analyses.
FUNDING M. Davidson was supported by the Merial Veterinary Scholar Program 2012. Project funding was provided by NSF Ecology of Infectious Disease grant OCE-1065990. Conflict of interest statement. None declared.
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