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Published online 4 January 2008
Published in J Environ Qual 37:266-271 (2008)
DOI: 10.2134/jeq2007.0019
© 2008 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Waste Management

EPA Worst Case Water Microcosms for Testing Phage Biocontrol of Salmonella

Michael R. McLaughlin* and John P. Brooks

USDA, Agricultural Research Service, Waste Management and Forage Research Unit, POB 5367, Mississippi State, MS 39762. Journal article number J-11061 of the Mississippi Agricultural and Forestry Experiment Station. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. This work was prepared by employees of the U.S. Government as part of their official duties and is in the public domain and may be used without further permission

* Corresponding author (mike.mclaughlin{at}ars.usda.gov).

Received for publication January 8, 2007.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
A microplate method was developed as a tool to test phages for their ability to control Salmonella in aqueous environments. The method used EPA (U.S. Environmental Protection Agency) worst case water (WCW) in 96-well plates. The WCW provided a consistent and relatively simple defined turbid aqueous matrix, high in total organic carbon (TOC) and total dissolved salts (TDS), to simulate swine lagoon effluent, without the inconvenience of malodor and confounding effects from other biological factors. The WCW was originally defined to simulate high turbidity and organic matter in water for testing point-of-use filtration devices. Use of WCW to simulate lagoon effluent for phage testing is a new and innovative application of this matrix. Control of physical and chemical parameters (TOC, TDS, turbidity, temperature, and pH) allowed precise evaluation of microbiological parameters (Salmonella and phages). In a typical application, wells containing WCW were loaded with Salmonella enterica susp. enterica serovar Typhimurium (ATCC14028) and treated with phages alone and in cocktail combinations. Mean Salmonella inactivation rates (k, where the lower the value, the greater the inactivation) of phage treatments ranged from –0.32 to –1.60 versus –0.004 for Salmonella controls. Mean log10 reductions (the lower the value, the greater the reduction) of Salmonella phage treatments were –1.60 for phage PR04-1, –2.14 for phage PR37-96, and –2.14 for both phages in a sequential cocktail, versus –0.08 for Salmonella controls. The WCW microcosm system was an effective tool for evaluating the biocontrol potential of Salmonella phages.

Abbreviations: ATCC, American Type Culture Collection • BA, blood agar • EPA, (US) Environmental Protection Agency • ISO, International Organization for Standardization • MOI, multiplicity of infection • MPN, most probable number • NTU, nephelometric turbidity units • PBS, phosphate buffered saline • phage, bacteriophage • SM, suspension medium • TDS, total dissolved salts • TOC, total organic carbon • TSA, trypticase soy agar • TSB, trypticase soy broth • WCW, worst case water


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
BACTERIOPHAGES (phages) are viral pathogens of bacteria. They are cultured in their respective host bacteria using conventional microbiological procedures and typically have very specific host ranges restricted to one or a few bacterial species. Phages have been used in a variety of applications to exploit their exquisite host specificity, including use as indicators of the presence of their bacterial hosts (Kuhn et al., 2002a, 2002b) and as indicators of bacterial (manure) contamination (Miller et al., 1998). Typing phages have been widely used in identifying and classifying bacterial strains of human bacterial pathogens, including Salmonella (Welkos et al., 1974; Farmer et al., 1975; Anderson et al., 1977; Sinton et al., 1998; Brenner et al., 1999; Leclerc et al., 2000). Phages offer potential for targeted biological control of bacterial pathogens in human, animal, and plant diseases (Lederberg 1996; Alisky et al., 1998; Lorch 1999; Kudva et al., 1999; Borah et al., 2000; Leverentz et al., 2001; Schuch et al., 2002), and have been used experimentally to treat Escherichia coli infections in broilers (Huff et al., 2002a, 2002b, 2005), and against poultry strains of Clostridium perfringens (Siragusa et al., 2004).

Scientific and commercial interest in using phages for biocontrol of bacterial diseases is growing. Many recent reviews document interest in phages as biocontrol agents in food (Greer, 2005; Hudson et al., 2005), for phage therapy (Thiel, 2004; Brussow, 2005; Skurnik and Strauch, 2006) and for wastewater treatment (Withey et al., 2005). Phages have been applied experimentally to reduce Salmonella contamination in a variety of food matrices including chickens (Toro et al., 2005), on chicken skin (Goode et al., 2003), sprout seeds (Pao et al., 2004), on fresh-cut fruit (Leverentz et al., 2001), and in the manufacture and storage of cheddar cheese (Modi et al., 2001). In August 2006, the U.S. Food and Drug Administration announced approval of a phage treatment for prevention and control of Listeria on ready-to-eat meat and poultry products and the company producing the product announced plans to release similar products for E. coli O157:H7 and Salmonella within the next year (http://www.intralytix.com/Intral_News_PR081906.htm; verified 4 Oct. 2007).

Because bacterial contamination of meat, poultry, and dairy products is frequently linked to fecal contamination, the control of human bacterial pathogens during animal production naturally focuses on manure management. In the work reported here, a model system was developed, tested and described for simulating the environment of a swine manure lagoon without the problems of odor and other microbes incumbent in lagoon effluent. The model used EPA worst case water, a defined medium originally developed and approved for testing point-of-use microbiological water purifiers (USEPA, 1987). Like effluent, the worst case water contained high levels of dissolved salts, organic carbon, and turbidity (Gerba and Naranjo, 2000). Control of Salmonella by phages was demonstrated in the worst case water model system and is believed to be a new application of worst case water tests. The model provides a useful tool for studying phages and defining the requirements for their successful use in controlling Salmonella in other more complex aqueous matrices. Test results including Salmonella inactivation rates and log10 reductions following phage treatment are presented.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results and Discussion
 Conclusions
 REFERENCES
 
Salmonella and Phage Culture
Salmonella and phage isolates are listed in Table 1 . Freeze dried primary cultures of Salmonella were reconstituted in trypticase soy broth (TSB) overnight at 35°C, and stored in 1- to 2-mL aliquots at –70°C. Fresh secondary cultures were prepared weekly from ice crystal scrapings of a primary culture streaked on blood agar (BA: trypticase soy agar, TSA, +5% sheep red blood cells; HealthLink, Jacksonville, FL) in 100 x 15 mm Petri dishes. Plates were inverted and incubated overnight at 35°C, visually checked for Salmonella purity and uniformity, and held at room temperature. Working cultures were prepared daily by transfer of single isolated colonies from BA plates to TSB culture tubes. Cultures in TSB were incubated 6 h without aeration in loosely capped glass vials at 35°C. Under these conditions cultures reached approximately 1011 cfu L–1. Working cultures produced by this protocol were used as sources of Salmonella for 16 worst case water experiments and as host cells in phage plaque assays.


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  		Table 1. Salmonella and phage isolates used in the present study.

 
Cultures of Salmonella test cells for worst case water experiments were further prepared in one of two ways. In the first series of eight experiments, stationary or "starved" cells were prepared by growing the bacteria to exponential phase (approximately 6 h) in TSB in a 100-mL flask at 35°C. This culture was aseptically decanted into four sterile 50 mL centrifuge tubes and centrifuged at 5000 x g for 30 min to pellet the cells. The supernatant was decanted and cells were resuspended in an equal volume of sterile phosphate buffered saline (PBS) and centrifuged as before. This procedure was repeated three times to remove residual TSB, and then the washed cells were resuspended in PBS and stored at 4°C. Washed and stored cell suspensions were used for 3 to 4 wk then replaced with a fresh preparation.

In the second series of eight experiments Salmonella test cells were grown to exponential phase immediately before each experiment. To produce these fresh exponential phase cells, Salmonella were first grown in 1.0% peptone in worst case water overnight at 35°C, then 0.1 mL was removed and inoculated into 10 mL of 1.0% peptone in worst case water and incubated at 35°C for 4 h. Approximately 1.5 mL of the fresh exponential phase cell suspension was removed and centrifuged in a microcentrifuge at 5000 x g for 5 min. Supernatants were removed after centrifugation and replaced by an equal volume of sterile PBS. This washing procedure was repeated three times and cells were resuspended in sterile PBS for use in experiments.

Phage Lysate Production
Phages PR04-1 and PR37-96, from our laboratory collection of swine lagoon phages (Table 1), were increased by inoculation of exponential phase broth cultures of the respective Salmonella host strain as described (McLaughlin and Balaa, 2006). These phages are members of the Podoviridae (linear double-stranded DNA genomes with icosahedral capsids and short non-contractile tails). Both phages produced lytic infections in Salmonella host cells (McLaughlin et al., 2006). Lysates were clarified by centrifugation (5000 x g 10 min at 5°C), decanted, and filtered through 0.45 µm cellulose acetate filters into sterile glass or polypropylene vials, treated with chloroform (to 2.5% v/v) and stored at 5°C (Adams, 1959). Phage titers were determined by plaque assay of an appropriate dilution series for each lysate. Phage preparations were diluted in suspension medium (SM: 0.01 mol L–1 MgSO4; 0.1 mol L–1 NaCl; 0.05 mol L–1 Tris, pH 7.5) to appropriate levels for each experiment and multiplicity of infection (MOI) ratios from 0.01 to 86, with most experiments (10 of 16) at a mean of 4.9 ± 1.9.

Phage Counts
Phages were detected and enumerated in plaque assays (Adams, 1959). Briefly, assays were done by mixing 100 µL of each phage dilution in aqueous media with 100 µL of fresh exponential phase Salmonella host culture in TSB into 5.0 mL of soft (0.75% agar) TSA, which had been previously melted and held at 45°C in a water bath. Test suspensions were mixed by vortex mixer and dispensed uniformly over the surface of 20 mL of hard (1.5% agar) TSA in 100-mm-diameter plates. Soft agar overlays were allowed to solidify at room temperature then plates were inverted and incubated overnight at 35°C. Plaque forming units (pfu) were counted 4 to 15 h later.

EPA Worst Case Water
The worst case water matrix with defined total dissolved solids (TDS = 1500 mg L–1), total organic carbon (TOC = 10 mg L–1), and turbidity (30 NTU) was prepared from sea salts, humic acid, and fine test dust, respectively, according to the standardized guide protocol (USEPA, 1987; Gerba and Naranjo, 2000). Sea salts (S9883; Sigma, St. Louis, MO) were prepared in distilled water, to which humic acid (H16752; Sigma-Aldrich, St. Louis, MO) and fine test dust (ISO 12103-1; Powder Technology Inc., Burnsville, MN) were added to standard TDS, TOC, and turbidity levels, as listed above. The final pH of the worst case water was 5.3.

Experimental Microcosms
All experiments were conducted using Salmonella and phage preparations added to worst case water in sterile 96-well flat bottom microplates (cat. no. 167008; Nunc, Roskilde, Denmark). In preliminary experiments, phage titers declined due to adsorption of phages to microplate well surfaces, but Salmonella titers were not affected. Subsequent preliminary experiments showed that phage adsorption was blocked by pretreatment of test wells with peptone. Additional tests showed no effect from adsorbed peptone on Salmonella growth. Therefore, the standard procedure for test plate preparation in all experiments reported here included a pretreatment peptone blocking step. Blocking comprised incubation of 350 µL per well of sterile 1.0% peptone in distilled water for 2 h at 35°C, followed by rinsing with an equal volume of sterile distilled water three times for 3 min each. In other preliminary experiments worst case water was shown to have no effect on Salmonella or phage titer during 24 h incubation at 35°C.

Test components were typically assigned to wells in adjacent paired rows or columns to allow collection of two replicate samples, one from each of two different paired wells for each successive time interval. Treatments were separated by one to two rows or columns of empty wells to minimize effects of aerosol contamination across treatments during pipetting. In a typical experiment, a multichannel pipetter was used to load test wells sequentially with 200 µL of worst case water, then 50 µL of Salmonella cells in PBS (or PBS alone in phage control wells without Salmonella) and 50 µL of phage in SM (or SM alone in Salmonella control wells without phage). Concentrations of Salmonella and phage were varied to suit the respective experimental protocol, but averaged (16 experiments) 3.0 x 1010 cfu L–1 and 1.7 x 1010 pfu L–1, respectively. Test components were mixed by gentle pipetting 10 times, plate lids were replaced and plates were incubated inside a moist chamber (sealed plastic bag with a moist paper towel) at 35°C. Samples (100 µL each) were withdrawn from different replicate pairs of wells at successive time intervals, typically 0, 1, 2, 4, 6, and 24 h post loading, for Salmonella and phage analyses.

Salmonella Counts
Salmonella population counts were determined using a standard spread plate technique. Briefly, a 0.1-mL aliquot was removed from each well and placed into 0.9 mL of sterile saline (0.85% NaCl) and mixed on a vortex mixer. Following mixing, the samples were diluted to extinction and plated accordingly onto TSA in duplicate plates, using 0.1 mL per plate as the inoculum. Plates were incubated at 35°C for approximately 18 to 24 h and colonies counted thereafter.

Salmonella Inactivation and Statistical Analysis
Salmonella inactivation rates were calculated using first-order kinetics based on Chick's Law of disinfection as:

Formula
where Nt = Salmonella titer (cfu L–1) at sample time t (h); N0 = Salmonella titer (cfu L–1) at the onset of the experiment (sample time t = 0 h); k, the inactivation rate, was derived as the slope of the respective regression line; and b was derived as the y-axis intercept of the respective regression line (Hurst, 1991). Negative k values characterize inactivation, while positive k values indicate growth. Linear regression analysis was performed using log10 reduction values, defined as Log10 (Nt/N0), as the dependent variable, and elapsed time, t in hours, as the independent variable. Log10 reduction values used to develop regression equations for estimating inactivation rates were based on sample counts of sampling times from t = 0 to the time (h) of maximum decline in Salmonella levels (i.e., the initial time interval of greatest inactivation) before Salmonella regrowth began in each respective experiment. Inactivation rate and log10 reduction means were subjected to analysis of variance (ANOVA, {alpha} = 0.05) and means from ANOVA tests with significant F tests were compared by least significant difference (LSD). All statistical tests were completed using the Microsoft Excel Statistical Package (Microsoft Corp., Redmond, WA) and all differences were at the p < 0.05 level.


   Results and Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
Conclusions
 REFERENCES
 
Adding phage to the microcosm significantly reduced initial Salmonella titer (Fig. 1A ). This reduction in Salmonella titer was due to the inclusion of phage and not due to potential die-off factors associated with Salmonella in a microcosm state. Salmonella titers in control wells without phage showed no reductions during the first 6 h, when changes in phage-treated wells were most pronounced, and declined only about 0.5 log10 cfu L–1 over the 24-h experimental period. Phage titers increased 1 to 2 log10 cfu L–1 during the first 2 h in wells of phage-treated Salmonella but remained unchanged in control wells (Fig. 1B), demonstrating the viability and stability of the phage in the worst case water matrix. Preliminary tests of MOI rate either with additional peptone in the worst case water matrix (data not shown) or without peptone (Fig. 1A) showed faster and greater reduction of Salmonella with MOI {approx} 5, than with MOI < 1 or > 10. In the experiments reported here the mean MOI = 4.9 (± 1.9) produced the steepest log10 reduction in Salmonella. Log10 reduction values (Table 2 ) of Salmonella inoculated with phage were greater than those for Salmonella controls without phage. Furthermore, Salmonella inactivation rates (Table 3 ) were higher (i.e., lower k value) with phage than without phage. Interestingly, use of phage PR37-96 produced a lower inactivation rate (higher or less negative k value) when compared to PR04-1 under similar microcosm conditions (Table 3) despite producing a greater log10 reduction (Table 2). This may have been due to a slower viral replication cycle, or lack of ideal conditions met for this particular phage and bacterial host combination. Ideal conditions are influenced by many factors, including growth state of the host bacterium, pH, and specific host-phage binding properties. Probably due to the overriding effect of the higher inactivation rate of phage PR04-1, sequential inoculations of the microcosm with the two phages in both possible orders, produced no differences in Salmonella log10 reduction or inactivation rates between the two sequences (data not shown).


Figure 1
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  		Fig. 1. Changes in Salmonella and phage PR04-1 titers in worst case water microcosms without peptone. (A) Effect of multiplicity of infection rate, MOI = (pfu L–1)/(cfu L–1), on exponential phase Salmonella cell populations. Open circle ({circ}) no phage (± SE, n = 8); open diamond (Figure 1) MOI = 0.3 (± SE, n = 2); open square ({square}) MOI = 4.5 (± SE, n = 4); open triangle ({Delta}) MOI = 21.4 (± SE, n = 2). (B) Changes in phage titers: open diamond (Figure 1) with Salmonella (MOI = 4.5); and open square ({square}) without Salmonella; (± SE, n = 4).

 

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  		Table 2. Log10 reduction of Salmonella post inoculation with phage PR04-1, PR37-96, phage cocktail, or no phage (control).

 

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  		Table 3. Inactivation rates (k) of Salmonella post inoculation with phage PR04–1, PR37–96, phage cocktail, or no phage (control).

 
Although experimental samples were collected and analyzed at intervals as long as 24 to 96 h post loading for all 16 experiments, Salmonella inactivation was most pronounced during the first 1 to 4 h post phage loading. Log10 reduction data presented here (Fig. 2 ) are from samples collected up to 24 h post loading in six experiments (n = 4) in which Salmonella cells were grown to exponential phase before inoculation and added without soluble peptone as a growth promoter within the worst case water matrix. Exponential phase cells were added to the microcosms without additional nutrients to simulate a lagoon environment. We suggest that in the natural environment, Salmonella shed in feces would be at or near exponential phase growth, but once bacterial cells are flushed into the effluent of a lagoon, competition from other bacterial populations for relatively fewer nutrients makes it impossible to sustain exponential growth and contributes to keeping Salmonella populations low. Indeed, this hypothesis is supported by data from Hill and Sobsey (2003) which showed Salmonella concentrations in untreated flushed swine waste were higher by 1 to 2 log10 MPN 100 mL–1 (most probable number) than those in the respective single-stage primary treatment lagoons. Chinivasagam et al. (2004) reported Salmonella often present in lagoon effluent at <1000 cfu L–1. The presence of Salmonella-specific lytic phages is also believed to be a contributing factor in reducing Salmonella populations in swine effluent lagoons (McLaughlin et al., 2006). In the lagoon environment, rapid phage lysis of newly introduced exponentially growing cells and increased competition from other bacteria would be a formidable combination restricting Salmonella population growth.


Figure 2
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  		Fig. 2. Log10 reduction of exponential phase Salmonella cells in worst case water without peptone. Closed square ({blacksquare}) with phage treatment and open diamond (Figure 2) without phage treatment ( ± SE, n = 4). Phage treatments were: (A) PR04-1; (B) PR37-96; and (C) a cocktail of PR04-1 and PR37-96.

 
It is known that phage infection of stationary-phase cells is not productive, i.e., does not result in lysis and release of progeny phage until host cell growth resumes (Chibani-Chennoufi et al., 2004). It is likely, therefore, that phages infect stationary cells in effluent and that any subsequent influx of nutrients sufficient to renew growth of bacterial host cells would also result in phage population growth, through lysis of the already infected cells and release of multiple new phage progeny. In the experiments reported here, following the initial inactivation phase, Salmonella exhibited a regrowth phase (Fig. 2, 3 ) in which Salmonella titers that had been reduced by phage lysis often recovered to levels approximately equal to those before phage treatment. Salmonella in these experimental wells often grew to levels greater than in control wells (without phage). This was believed to be due to continued growth of Salmonella cells which escaped phage infection and which were capable of scavenging nutrients liberated by phage-lysed cells. In a brief investigation of these surviving cells, selected Salmonella colonies from treatment wells where regrowth had occurred were tested and found to be susceptible to phage infection and to form plaques when inoculated with phage. In the lagoon environment, however, such regrowth would not be expected because of nutrient scavenging competition from other indigenous autochthonous and zymogenous bacteria. Although this hypothesis requires further investigation, results of experiments with phages in two-species bacterial communities have shown that phages can provide long-term suppression of target bacteria (Harcombe and Bull, 2005), supporting the hypothesis that competition from other bacterial species can enhance phage treatment.


Figure 3
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  		Fig. 3. Effects of peptone (0.1%) and phage (PR04-1) on growth of exponential phase Salmonella cells in worst case water microcosms (± SE, n = 4). (A) Peptone effect without phage. (B) Peptone effect with phage. (C) Phage effect with peptone. (D) Phage effect without peptone. Symbols: closed diamond ({diamondsuit}), +phage + peptone; open diamond (Figure 3), +phage–peptone; closed square ({blacksquare}),–phage +peptone; open square ({square}),–phage–peptone.

 
Though determination of the ideal conditions for phage infection and subsequent bacterial inactivation was not the objective of the present study, under conditions of bacterial cell starvation, little or no phage-induced bacterial inactivation was observed. When peptone was added to the microcosms, bacterial inactivation rates increased. This demonstrated that for this type of phage host system, the host must be viable and capable of growth when nutrients become available, in order for phage lysis to occur. Bacterial cell populations excreted from a host animal via feces, may remain in growth phase for only a short time (Woods, 1976). That observation and results of the present study suggest that phages capable of inactivating starved or stationary host cells should also be investigated.


   Conclusions
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
REFERENCES
 
The present study demonstrated that EPA worst case water was a useful matrix for simulating swine lagoon effluent in tests of phage biocontrol of Salmonella. The 96-well microplate Salmonella-phage test described here is a new application of the worst case water matrix. The relative simplicity of the test and worst case water matrix proved advantageous in this initial study of parameters necessary for successful application of phage biocontrol of Salmonella. The microplate format was easy to use in the laboratory and the worst case water matrix was uniform across experiments, chemically and microbiologically defined, and free of malodors associated with lagoon effluents. A potential limitation of this experimental matrix, i.e., lack of competition from other microbes present in lagoon effluents, may be overcome in the future by adding multiple host and nonhost bacterial species to the experimental matrix.


   ACKNOWLEDGMENTS
 
The technical assistance of Renotta K. Smith is gratefully acknowledged.


   NOTES
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ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.


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M. R. McLaughlin and J. P. Brooks
Recovery of Salmonella from Bermudagrass Exposed to Simulated Wastewater
J. Environ. Qual., January 13, 2009; 38(1): 337 - 342.
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