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Journal of Environmental Quality 31:1702-1709 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Surface Water Quality

Salmonella typhimurium Survival and Viability is Unaltered by Suspended Particles in Freshwater

Ryan P. Maki*,a,b and Randall E. Hicksa

a Department of Biology, 10 University Drive, University of Minnesota-Duluth, Duluth, MN 55812
b South Florida Water Management District, P.O. Box 24680, West Palm Beach, FL 33416-4680

* Corresponding author (rmaki{at}sfwmd.gov)

Received for publication May 21, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rolling microcosm experiments were conducted to determine whether suspended particles affect the survival and viability of a model pathogen, Salmonella choleraesuis, serotype typhimurium (American Type Culture Collection no. 23567), in a freshwater microbial community. Water from the Duluth, MN harbor of Lake Superior (including native microorganisms) was inoculated with clay, silt, or flocculent organic particles in a range of concentrations and a streptomycin-resistant strain of S. typhimurium. Microcosms (incubated at 20°C) were rolled horizontally (3 rpm) and sampled periodically for total bacteria and total, viable, and culturable S. typhimurium. Total S. typhimurium abundance decreased rapidly in all experiments (8.5–73.1% d-1). Total bacteria did not decrease as rapidly as the S. typhimurium population in any experiment, suggesting that a microcosm effect was not responsible for the decline in S. typhimurium populations. Loss rates of attached and free cells were similar, indicating that attachment to particles did not enhance the persistence of Salmonella cells beyond our minimum detectable differences. After eight days, only 0.1 to 11.9% of the initial S. typhimurium inocula were detected by direct counts. Suspended particles had a minimal effect on the survival and viability of S. typhimurium; the losses of total, viable, or culturable Salmonella were generally the same across particle treatments and concentrations. Silt and flocculent particles affected loss rates of total and viable S. typhimurium similarly to inorganic particles (clay). It appears unlikely that suspended particles would provide a means for S. typhimurium to persist at hazardous levels in freshwater.

Abbreviations: ANOVA, analysis of variance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SUSPENDED PARTICLES may play a fundamental role in the contamination of receiving waters with pathogenic bacteria. Bacteria may be affected in a number of ways by the presence of suspended particles. Bacteria that attach to particles in aquatic systems are often larger and more productive than free bacteria (Crump et al., 1998; Kirchman and Mitchell, 1982; Torreton, 1999) due to bacterial utilization of nutrients attached to the particles (Crump et al., 1998; Kirchman and Mitchell, 1982; Hoppe et al., 1988). Since starving or injured cells are less likely to remain viable than healthy cells (McFeters, 1997; Smith et al., 1994), the utilization of nutrients from suspended particles could also lead to a higher proportion of cells remaining viable. More bacteria, including S. typhimurium, remain viable in high nutrient treatments than populations of the same species in low nutrient environments (Smith et al., 1994). Although many enteric bacterial populations are known to decline in receiving waters (Gonzalez et al., 1992; Gurijala and Alexander, 1990; McFeters and Stuart, 1972) and coliform concentrations have been correlated with suspended particles (Gary and Adams, 1985; Sherer et al., 1988), no one has examined whether the presence of suspended particles alters the survival or viability of bacterial pathogens in freshwater microbial communities.

Understanding the influence of suspended particles on the survival and viability of pathogenic bacteria is of critical importance to human health because of current problems with overpopulation, contaminated runoff, and changing wastewater treatment policies. Many wastewater treatment facilities have eliminated chlorine addition from their treatment process to reduce formation and discharge of trihalomethanes, which can be toxic or genotoxic to humans and animals (Bitton, 1994; Western Lake Superior Sanitary District, 1993). This practice may increase the load of living and uninjured pathogens discharged with post-treatment wastewater.

Salmonella typhimurium, the model pathogen used in this study, is widespread and common in both agricultural runoff and human wastewater (up to 80 cells mL-1). Salmonella typhimurium is the most common Salmonella serotype found in humans suffering from infectious gastroenteritis (McCormick et al., 1993). It has been estimated that 1% of the human population excretes Salmonella at any one time (Sterritt and Lester, 1988). Salmonella spp. are often present in wastewater and have a minimum infective dose of 104 to 107 cells (Bitton, 1994). With an estimated three million infections every year (Spector, 1998), Salmonella spp. are clearly a threat to human health in bodies of water receiving wastewater effluent.

The primary objective of this study was to determine whether the concentration of different suspended particles changes the survival or viability of S. typhimurium in a freshwater microbial community. Attached and free Salmonella cells were monitored separately to determine if attachment to suspended particles influenced the survival of S. typhimurium populations. An additional objective was to determine whether suspended particles containing an organic component (i.e., silt and flocculent organic particles) affect the survival and viability of S. typhimurium differently than inorganic suspended particles (i.e., clay).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Four rolling microcosm experiments were conducted to test the effects of three suspended particle types: clay, silt, and flocculent organic particles. The experiment with clay particles was conducted in 1997; the experiments with silt and flocculent particles were performed in 1998. Sediments were collected from areas that contribute suspended particles to the Duluth, MN, harbor of Lake Superior. Clay sediment was collected from the bank of the Nemadji River at its mouth into the Duluth harbor. Samples were kept cool (4°C) and transported back to the laboratory in sterile Whirlpak bags (Nasco, Fort Atkinson, WI). The clay was mixed with Milli-Q water (Millipore Corp., Bedford, MA) to form a suspension. Large particles were allowed to settle, and the overlying suspension was centrifuged (15 min, 2500 x g). The top (approximately 1 mm) of the pellet was used in the microcosms. At the outset of the experiment, 67% of the particles were 2 µm in diameter or smaller (Table 1) as determined by laser particle counter (Spectrex Corp., Redwood City, CA). Silt was collected by sampling surficial sediment with an Ekman grab sampler (Wildlife Supply Co., Buffalo, NY) in Kimball's Bay of the Duluth harbor. The sediment was centrifuged (10 min, 1000 x g), and the top (approximately 1 mm) of the pellet was used in the microcosms. This experimental sediment was at least 75% silt sized particles at the outset of the experiment as determined by settling column analysis (Table 1). Experimental flocs were generated in the laboratory by rotating harbor water in 1-L bottles at 3 rpm for 48 h in a 20°C incubator (modified from Shanks and Edmondson, 1989). Each bottle generated an average of 3.05 mg (dry weight) of flocculent organic material. Flocs were gathered by gently pouring the bottle contents through a plankton net (80-µm pore size) and then rinsed off the netting into the experimental microcosms with Milli-Q water.


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  		Table 1. Particle size distributions at the outset of the microcosm experiments with clay and silt particles.

 
The water used to fill the microcosms and provide the native microbial community was collected from the surface (0–2 m) of the Duluth harbor. Water and clay were collected in early November for the experiment with clay particles. In the flocculent particle experiment, water was collected and particles generated in mid-August. Water and silt were collected in mid-June and again in mid-November for the two separate experiments with silt particles. Most water was stored (4°C) in 18.9-L (5-gallon) carboys for less than 24 h before being used in the microcosms. Water for the flocculent organic particle experiment was held up to 6 d. The longer storage was necessary due to the slow generation of flocculent organic particles for the treatment microcosms. All water, except for the water used to generate flocculent organic particles, was filtered through 5-µm-pore-size polycarbonate (Millipore) filters before being added to microcosms. This filtration eliminated large particles that could have interfered with the experimental particle additions but did not remove all native microbes. Water used in the flocculent organic particle treatments was left unfiltered to enhance the generation of flocculent particles.

Model Pathogen
A streptomycin-resistant strain of S. typhimurium was chosen to selectively plate this strain from the experimental microcosms. Cultures were grown in nutrient broth (Difco, 1984) for 24 h on a shaker table (100 rpm). Cultures were sampled to estimate cell density by epifluorescent microscopy and rinsed twice before being added to the microcosms to avoid enriching the experimental environment with additional nutrients. Rinsing consisted of centrifuging cultures in 1.5-mL centrifuge tubes (3 min, 1000 x g), removing the overlying nutrient broth, and resuspending the cells in Milli-Q water (cells were resuspended in autoclaved harbor water in the clay particle experiment).

Experimental Design
Sterile, 1-L Pyrex bottles were rotated horizontally (3 rpm) on two tissue culture rotators (Lab-Line Instruments, Melrose Park, IL) in a 20°C incubator in the dark to keep particles suspended for the duration of the experiments. Eight harbor water-filled microcosms were divided into controls (no particles added) and three to five particle treatments for each experiment. Only the control (n = 3) and highest treatment (n = 2) microcosms were replicated in the experiment with clay particles. In the experiments with silt particles, only the control (n = 2) and highest particle treatments (n = 2) microcosms were replicated. Each treatment in the experiment with flocculent organic particles had two replicate microcosms. Experimental particle concentrations were set within the range of suspended particle concentrations (0.5–180 mg L-1) delivered to the Duluth harbor of Lake Superior by the St. Louis River (Minnesota Pollution Control Agency, 1984). Salmonella typhimurium was added to a final density of 1 to 5 x 106 cells mL-1 to achieve approximately a 1:1 ratio with the native bacteria.

Sampling Methods
Samples were taken periodically to enumerate total bacteria and total, viable, and culturable S. typhimurium. Initially, the microcosms were sampled every 48 h beginning with the S. typhimurium inoculation (time 0 h). After the initial 4 to 6 d, samples were taken at 4- to 7-d increments. Experiments were terminated once S. typhimurium population sizes remained constant in at least three consecutive samples or diminished to below the detection limits for direct and viable counts. The experiments lasted 24 to 41 d. Samples were collected by removing 10 mL of water and particles with a pipette after the microcosms were gently inverted for 10 s to evenly distribute particles. Sterile 5-mL tips used in the sampling procedure were obliquely cut to accommodate the larger particles that formed over the course of each experiment. These 10-mL samples were subsampled to determine total bacteria by epifluorescent microscopy and DAPI staining (Porter and Feig, 1980) and total, viable, and culturable Salmonella. Total Salmonella cells were detected by epifluorescent microscopy after in situ hybridization with a fluorescently labeled (tetramethylrhodamine or Cy3) 23S rRNA oligonucleotide probe specific for the Salmonella genus (Hicks et al., 1992; Amann et al., 1995). The probe was developed by Nietupski et al. (1992). Free and attached cell abundances were both measured. Viable Salmonella cells were detected by the direct viable count procedure in combination with in situ hybridization (Kogure et al., 1979). Culturable S. typhimurium were enumerated by selective pour plating on streptomycin-amended (100 µL L-1), bismuth sulfite agar (Difco, 1984).

Particle aggregation in each experiment produced an array of particle sizes varying from 1 µm to approximately 1 mm in diameter. Some particles aggregated to a size that could not be sampled adequately during each experiment. These large particles were analyzed for Salmonella and weighed at the termination of the flocculent organic and silt particle experiments. Particle aggregation was monitored throughout the experiments by visual observation and weekly determinations of particle size distributions (1–100 µm) with a Spectrex laser particle counter (Hall, 1992).

Statistical Approach
All statistical analyses were performed with SAS (SAS Institute, 1996). Linear regressions were performed on natural log transformations of the number of cells or colony forming units (CFU) versus time to determine Salmonella loss rates (these regressions were also performed on data from the literature to allow accurate comparisons). Natural log transformations of the data placed more even emphasis on data from the entire experiment than nonlinear decay models, which emphasize the early time points (the sharpest part of the decay in these experiments). These loss rates were compared by weighted linear regression, weighted one-way analysis of variance (ANOVA), and weighted mixed-model ANOVA to determine the effects of suspended particles on Salmonella survival and viability and to compare loss of Salmonella cells existing in several physiological states (Wardlaw, 1985).

Loss rates of free and attached S. typhimurium subpopulations were calculated with data from 48 h after inoculation to the termination of the experiments. Salmonella typhimurium attachment rates were assumed to be greater than detachment rates at the outset of these experiments because only free S. typhimurium cells were added to the microcosms, which already contained particles uncolonized by the model pathogen (i.e., detachment rate = 0). Thus, data from the initial samples (i.e., time 0 h) were intentionally excluded in these analyses to decrease the chance of overestimating the loss of free cells due to higher attachment than detachment rates in the early hours of these experiments. Hence, the loss rates of free and attached cells are not directly comparable with the loss rate calculated for total S. typhimurium abundance. Loss rates for attached and free cells were compared by weighted, mixed-model ANOVAs. These loss rates were paired by microcosm to protect against skewing results by allowing individual bottle effects to influence the analyses.

Weighted, one-way ANOVA was used to determine whether Salmonella loss rates differed between control (no particles added) and high particle treatments. Weighted linear regression analyses were used to compare Salmonella loss rates across suspended particle gradients. Minimum detectable differences were calculated for the weighted ANOVA and regression analyses, at the {alpha} = 0.05, ß = 0.10 level, according to Eq. [17.18] in Zar (1999). A weighted, mixed-model ANOVA was used to examine differences in loss rates between total bacteria and total Salmonella and between total Salmonella and the viable and culturable portions of the population within each experiment. This was done to help distinguish the major factors affecting the decline of the Salmonella. Weighting was used to account for the inconsistent variability in the data without excluding data. Loss rates were accepted for use in the weighted regressions if they were based on at least two data points (samples in which Salmonella were observed). Weights were assigned as variance-1 except for some of the regressions based on only two points. For the six regressions based upon only two points that had standard errors less than 90% of the standard errors of regressions based on three or more data points, the weight of the regression was considered artificially high. In those cases, a surrogate weight was computed with a standard error equal to that of the regressions based on at least three data points that had standard errors greater than 10% and less than 90% of all such regressions. The 90% cutoff was arbitrarily set but effectively eliminated excessively high weights and allowed a more accurate comparison.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A great deal of particle aggregation was evident in each experiment. At the outset of the experiments (except the flocculent organic particle experiment), large suspended particles (approximately 1 mm in diameter) were not visible, but developed within 96 h. Aggregation was detected in a smaller particle range (1–100 µm) by laser particle counter in only some experiments. A preliminary experiment indicated that the aggregation of these suspended particles was not due to the S. typhimurium inoculum.

Total bacteria and total, viable, and culturable Salmonella declined significantly in all experiments (p < 0.05; Fig. 1) . The abundance of total, viable, and culturable Salmonella declined more rapidly (p < 0.05) than total bacteria in each experiment (Fig. 1). After 7 d, total Salmonella decreased to 11.9% of the initial population in the clay experiment and 0.2% in the flocculent particle experiment. Results were similar in the experiments with silt particles; Salmonella decreased to 0.1% of the initial inoculum within eight days in each experiment.



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  		Fig. 1. Decreases in the number of cells or colony forming units (CFU) in the control and highest particle treatments during the clay, flocculent, and silt particle experiments. Total (-{diamondsuit}-), viable (-{blacksquare}-), and culturable (-{blacktriangleup}-) Salmonella cells decreased similarly in all control and treatment microcosms. Total bacterial populations (x) persisted at a relatively constant level in all experiments.

 
Suspended particles had little measurable effect on the survival and viability of S. typhimurium. There was no significant difference in 9 of 12 comparisons of loss rates between the control and highest particle treatments for total, viable, or culturable Salmonella (p < 0.05; Tables 2–4). The highest clay treatment lead to a 13% higher loss rate of culturable Salmonella than that observed in the controls. However, the highest clay treatment had no effect on the loss of total or viable Salmonella. The highest treatment of flocculent organic particles lead to higher loss rates for total and viable Salmonella (301 and 14%, respectively) compared with control treatments but had no effect on the loss of culturable Salmonella. There were no differences detected in Salmonella loss rates between controls and the highest silt treatments.


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  		Table 2. Loss rates for total bacteria and total, viable, and culturable Salmonella in the clay experiment (duration 41 d).

 

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  		Table 4. Loss rates for total bacteria and total, viable, and culturable Salmonella in two silt particle experiments covering different particle concentrations (0–7.4 and 0–30.0 mg L-1).

 

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  		Table 3. Loss rates for total bacteria and total, viable, and culturable Salmonella in the flocculent particle experiment (duration 34 d).

 
The minimum detectable differences (MDDs) for the analyses above varied greatly between experiments and parameters. The MDDs are expressed as absolute differences in loss rates between control and highest particle treatments (see Tables 2–4) at the {alpha} = 0.05 and ß = 0.10 levels for the more sensitive of the ANOVA or weighted linear regression analyses in each case (see regression analyses below). These MDDs are reported only for those parameters that yielded no significant results for either the ANOVA or regression comparisons. For the experiment with clay particles, the MDD was 25.0% d-1 for viable Salmonella. For the experiment with flocculent organic particles, the MDD was 2.2% d-1 for colony forming units (CFU). For the first experiment with silt particles, the MDD was 94.1% d-1 for total Salmonella and 17.9% d-1 for CFU. For the second experiment with silt particles, the MDD was 92.0% d-1 for total Salmonella, 98.8% for viable Salmonella, and 14.7% d-1 for CFU.

The concentration of suspended particles had a minimal effect on the survival, viability, and culturability of S. typhimurium. In 8 of 12 comparisons, the slope of the weighted linear regression comparing loss rates across suspended particle concentrations was not significantly different from zero, indicating that the Salmonella loss rate was not increased or diminished with increasing particle concentration (Table 5). The loss rates were slightly affected in only 4 cases out of 12. Loss of culturable Salmonella increased 0.1% mg-1 d-1 with increasing clay concentration, but the loss of total Salmonella dropped 0.1% mg-1 d-1. Loss of viable Salmonella increased 3.3% mg-1 d-1 with increasing flocculent particle concentration and 6.4% mg-1 d-1 with increasing silt concentration (Silt Experiment I).


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  		Table 5. Relationship between Salmonella loss rates and suspended particle concentrations (experimental durations of 24 to 41 d).

 
In addition to determining differences in loss between treatments, loss rates were compared among different portions of the Salmonella populations within each experiment (Tables 2–4; comparing across the rows). Total and viable Salmonella demonstrated higher loss rates than culturable Salmonella (p < 0.05) cells in the flocculent and silt particle experiments (Tables 3 and 4). There was no difference between the loss rates of total and viable Salmonella. In the clay particle experiment, culturable Salmonella cells disappeared more rapidly than either viable or total Salmonella cells, and viable Salmonella cell were lost more rapidly than total Salmonella cells (p < 0.05; Table 2).

Cell attachment to suspended particles was observed in every treatment. Considering all experiments, 29.7% (SE 12.7) of the S. typhimurium cells attached to particles but individual attachment frequencies ranged from 0 to 100%. Like total bacteria and total Salmonella, the abundances of free and attached S. typhimurium subpopulations also decreased in a nonlinear fashion. Attached Salmonella cells disappeared at a rate similar to free cells (p > 0.05, Table 6). The minimum detectable differences for the attached versus free Salmonella loss comparisons for each experiment were 6.9% d-1 for the experiment with clay particles, 81.4% d-1 for the experiment with flocculent organic particles, and 49.5 and 58.2% d-1 for the first and second experiments with silt particles, respectively. The analyses of large suspended aggregates at the termination of each experiment revealed very low numbers of S. typhimurium. Less than 0.02% of the initial Salmonella were detected on the large aggregates for the experiments with the flocculent organic and silt particles.


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  		Table 6. Loss rates of free and attached Salmonella cells measured during the four suspended particle experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Salmonella typhimurium abundance decreased in a nonlinear fashion in all particle treatments in all experiments. Culturable S. typhimurium cells decreased at a rate similar to a laboratory strain of S. typhimurium (43% d-1 vs. our 24–53% d-1) in lake water containing native microbes (Liang et al., 1982). In contrast, culturable S. typhimurium cells diminished more slowly than an isolated Salmonella spp. declined in autoclaved sediments and sea water (Matson, 1993). Culturable Salmonella were not present 12 d after inoculation in that study (Matson, 1993), whereas they persisted at least 16 d and as many as 41 d in separate experiments in this study. Culturable S. typhimurium cells also declined more slowly than in a previous study (Gurijala and Alexander, 1990) of Escherichia coli in lake water (71% d-1), but similar to this species in two other studies (46% d-1 in Amy and Hiatt [1989] and 22% d-1 in Awong et al. [1990]). Total Salmonella cells were lost at a rate similar to E. coli in lake water during Brettar and Hofle's 1992 study (9–73% d-1 vs. 45% d-1, respectively; Brettar and Hofle, 1992).

A microcosm effect (i.e., a chemically contaminated or otherwise inadvertently lethal physical condition within the microcosms) was probably not the entire cause of the rapid decline in S. typhimurium populations because total bacterial populations did not decline nearly as rapidly as S. typhimurium populations. Such an effect would have been expected to cause a similarly rapid decrease in the total bacterial populations.

The ANOVA analyses comparing Salmonella loss rates between microcosms with no added particles and the highest particle treatments, as well as the regressions of Salmonella loss rates versus suspended particle concentrations, indicated that the presence of suspended particles had a minimal effect on the survival and viability of S. typhimurium. Both types of comparisons usually produced slopes not different from zero, indicating no detectable effect. This result was unexpected because coliform concentrations have been correlated with suspended particle loads in streams (Gary and Adams, 1985; Sherer et al., 1988), and Sherer et al. (1992) found that coliform survival was greater in microcosms containing water and settled sediment than in microcosms containing water but no sediment. Two possible explanations may shed light on these conflicting results. First, it may have been the case in each of the three studies above that the increased coliform abundance (and in the case of Sherer et al. [1992], survival) associated with high particle loads was due to increased coliform survival only in the settled sediments. In each of these studies the settled sediments were disturbed prior to sampling, suspending particles and coliforms. Our study differed in that the particles were in suspension for the duration of the experiment. In contrast, these results may be explained by inability of our strain of S. typhimurium to utilize nutrients on particles as effectively as the coliform bacteria in the above three studies.

Although total and viable Salmonella cells appeared to be lost faster in the highest flocculent particle treatment compared with the control microcosms, it is possible that the unfiltered lake water used to construct the high particle treatment allowed more predators than in the control microcosms and caused these increased loss rates. When weighted linear regressions were performed with these data (excluding the control microcosms), no differences (p > 0.05) in total or viable cells losses were observed with increasing particle concentrations. In only 4 of 12 comparisons of loss rates across particle concentrations did regression slopes differ from zero. These slopes were either nearly zero or somewhat positive (Table 5, -0.1 to 6.4% of population mg-1 d-1). These results indicated that the concentration of suspended particles did not slow the loss of total or viable S. typhimurium cells compared with cell losses in control microcosms and that particles were not the major cause of the decline of the S. typhimurium populations.

The fact that no particle type greatly affected the survival or viability of S. typhimurium indicated that the organic particles (silt and flocculent) did not have a greater effect than the inorganic clay particles. It is doubtful that the failure of S. typhimurium to persist longer in the presence of particles was due to the inability of the S. typhimurium strain to attach to particles since a great deal of particle attachment was observed in each experiment. This observation also indicated that S. typhimurium cells attached to particles may have been unable to effectively utilize the nutrients in these particles.

Our analyses indicated that a viable but nonculturable fraction may have developed within Salmonella populations only in the experiment with clay particles. The presence of a viable but nonculturable cell fraction in that experiment is somewhat questionable because some nonspecific binding of the probe labeled with tetramethylrhodamine isothiocyanate (TRITC) was suspected. If nonspecific binding had occurred, then viable rod-shaped bacteria of other genera might have been counted as viable Salmonella and artificially inflated the total and viable Salmonella population estimates. Alternatively, lower estimates of viable Salmonella compared with culturable Salmonella in the silt and flocculent particle experiments may indicate that some injured Salmonella cells that were unable to elongate during the direct viable count method were being resuscitated during the incubation period in bismuth sulfite agar (Wai et al., 1996). However, our work cannot confirm either of these alternatives.

Comparing the loss rates of particle-attached Salmonella with those of free Salmonella cells is a more complex comparison than the comparisons of total and viable Salmonella discussed previously. There are more factors that can influence the measured losses of free and attached cells in one microcosm than there are affecting the measured loss of total cells in one microcosm versus another. Two assumptions were made here. The reproductive rates of free and attached cells were assumed to be equal and the attachment and detachment rates were also assumed to be equal. If these assumptions were true, then S. typhimurium persistence in these experiments was not affected to a detectable extent by cell attachment to suspended particles.

A large portion of the Salmonella population attached to particles in each experiment, which indicates that the results were not skewed by a tendency of S. typhimurium to remain free in the water. Substantially different detachment than attachment rates would have to occur to overestimate the true loss of attached cells. Bacterial attachment and detachment rates in aquatic systems have been found to be similar (Leff et al., 1998). Thus, it is doubtful that the analysis of these experiments failed to reveal any real differences between the loss of free and attached Salmonella cells.

Starvation probably caused a portion of the decline in S. typhimurium observed in all of these experiments. It is often a major factor in the death or loss of viability of allochthonous bacteria discharged into oligotrophic waters (Barcina et al., 1997; Sherer et al., 1992). Increases in Salmonella abundance were not observed, making reductive cell division as a response to starvation doubtful in these experiments (Roszak and Colwell, 1987). Predation and competition with the native microbes cannot be eliminated either as causes of the rapid decline of S. typhimurium. Both of these factors are often found to be the most significant controls on allochthonous bacterial abundance in aquatic systems (Barcina et al., 1997; Gonzalez et al., 1992; Janakiraman and Leff, 1999; Muela et al., 1998).

Salmonella typhimurium did not proliferate or even maintain its population size in the presence of suspended particles. The persistence of total and viable Salmonella was not prolonged by the presence of any type or concentration of suspended particles. Similarly, at our level of detection, Salmonella cells attached to these particles did not persist longer than free Salmonella cells. Salmonella typhimurium population responses to organic particles were similar to the response to inorganic (clay) particles. Considering these experimental results, it appears that S. typhimurium would be unlikely to reach its infective level by accumulating and persisting on suspended particles in freshwater. It is also apparent that suspended particle load is not a good indicator of bacterial pathogen survival since the presence of particles neither greatly increased nor decreased the persistence of S. typhimurium in this study.


   ACKNOWLEDGMENTS
 
We thank Peter Aas, Jonathan Pundsack, Angela Malley, and Brendan Keough for their technical assistance. Vysis Corporation provided the 23S rRNA oligonucleotide probe sequence specific to the Salmonella genus. We are grateful to Dr. Ron Regal for guidance on the statistical analyses and to Dr. Howard Mooers for advice on particle measurements and manipulations. This manuscript was improved by the critical reviews of Lyle Shannon, Dr. Rich Axler, and three anonymous reviewers. This work is the result of research sponsored by the Minnesota Sea Grant College Program (Project R/NP-13) supported by the NOAA Office of Sea Grant, United States Dep. of Commerce, under grant number US DOC/NA46-RG0101. The U.S. Government is authorized to reproduce and distribute reprints for government purposes, not withstanding any copyright notation that may appear hereon. This paper is journal reprint number JR466 of the Minnesota Sea Grant College Program.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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