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Applied and Environmental Microbiology, September 2005, p. 5254-5259, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5254-5259.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Impact of Phages on Two-Species Bacterial Communities

W. R. Harcombe* and J. J. Bull

Section of Integrative Biology, Institute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712

Received 18 December 2004/ Accepted 5 April 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A long history of experimental work has shown that addition of bacteriophages to a monoculture of bacteria leads to only a temporary depression of bacterial levels. Resistant bacteria usually become abundant, despite reduced growth rates relative to those of phage-sensitive bacteria. This restoration of high bacterial density occurs even if the phages evolve to overcome bacterial resistance. We believe that the generality of this result may be limited to monocultures, in which the resistant bacteria do not face competition from bacterial species unaffected by the phage. As a simple case, we investigated the impact of phages attacking one species in a two-species culture of bacteria. In the absence of phages, Escherichia coli B and Salmonella enterica serovar Typhimurium were stably maintained during daily serial passage in glucose minimal medium (M9). When either of two E. coli-specific phages (T7 or T5) was added to the mixed culture, E. coli became extinct or was maintained at densities that were orders of magnitude lower than those before phage introduction, even though the E. coli densities with phage reached high levels when Salmonella was absent. In contrast, the addition of a phage that attacked only Salmonella (SP6) led to transient decreases in the bacterial number whether E. coli was absent or present. These results suggest that phages can sometimes, although not always, provide long-term suppression of target bacteria.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacteriophages are predators of bacteria. Soon after the discovery of phages in the 1910s scientists attempted to use them as agents to cure bacterial infection (phage therapy) (5, 6). By the 1940s, phage therapy was considered a failure in the West and was abandoned in favor of antibiotics, although some Eastern European countries nurtured the technology and kept it to the present (18, 26).

Western interest in phage therapy is undergoing a revival (15, 18, 26). The greatest interest lies in using phage to treat infections, a technique that may face considerable economic hurdles because of the enormous cost of clinical trials coupled with the typically narrow host ranges of most phages. It has also been proposed that phage might be applied environmentally to depress the abundance of bacteria before they cause infection (11, 15, 21). For example, phage could be used to kill infectious bacteria in livestock feedlots (11) or on surfaces in hospitals. The advantage of this approach is that there should be fewer regulatory concerns, which should translate into a greatly reduced cost of implementation (8).

A critical obstacle for environmental phage treatment is the evolution of bacterial resistance to the phage. When large continuous cultures of bacteria have been treated with phages, resistance invariably evolves, and the concentrations of bacteria return to nearly their former levels (4, 12). This outcome has been obtained (i) despite the demonstrable fitness cost of phage resistance in many bacteria (12, 13) and (ii) even when multiple rounds of phage evolution allow the phage to enter an evolutionary arms race with bacteria (2, 20).

These in vitro laboratory experiments throw doubt on the idea that the release of phages can suppress the levels of target bacteria. Yet the experiments share one major limitation: they have all been conducted with single species of bacteria (usually Escherichia coli) growing essentially in monoculture (2, 12, 13, 20). Outside the laboratory, environmental populations of bacteria rarely exist in monocultures; rather, they typically exist in complex microbial communities. The ascent of phage-resistant bacteria could be profoundly affected by competition from other microbial species in ways that cannot be anticipated from monocultures. There is some support for this idea from studies of cyanobacteria in natural environments. Several studies found that cyanobacterial densities can be significantly reduced by phages (10, 22, 28). However, this result has been challenged by findings showing that cyanobacteria are largely resistant and therefore unaffected by phages (29).

Here we address the effect of interspecies competition on bacteriophage control of bacterial populations. A pair of bacterial species was maintained during serial transfers in minimal media. Phages specific to one host were introduced to observe the impact on the target species given interspecific bacterial competition.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains.
The bacteria used were E. coli B and Salmonella enterica serovar Typhimurium ATCC 14028S. The lytic phages T7 and T5 were used as E. coli-specific phages; SP6 was used as the Salmonella-specific phage. Both T phages are highly virulent and form large clear plaques on our strain of E. coli. T7 binds to the heptose residues in the lipopolysaccharide (7), while T5 binds to the outer membrane transporter protein FhuA (9). Competition assays have suggested that resistance to T7 comes at a high cost to the bacterial growth rate and/or competitive ability, while resistance to T5 has little or no cost in minimal media with glucose (12). SP6, a member of the same family as phage T7, forms moderate-size, clear plaques on the Salmonella strain used. The primary binding site of SP6 is the O antigen (23), but little is known about the cost of resistance to SP6. None of the three phage used here replicates on the heterologous host in the conditions employed (data not shown). The bacteria and phages were obtained from the collection of I. J. Molineux.

Design.
Flasks were set up with one of the following combinations: (i) a monoculture of one bacterium, (ii) a two-species culture of E. coli and Salmonella, (iii) a monoculture of one bacterium plus a phage specific to it, or (iv) a two-species culture of E. coli and Salmonella plus one phage.

Bacteria were grown in 125-ml flasks containing 10 ml of M9 minimal medium with 0.2% glucose and no amino acids (19). At the beginning of a serial passage line, the day 1 flask was inoculated with approximately 106 E. coli and/or Salmonella cells from stocks grown overnight in minimal medium. The cultures were grown with aeration in a shaking incubator at 37°C and 175 rpm for 24 h. After 24 h, 1 µl was transferred to a new flask that contained 10 ml of fresh medium. For trials in which E. coli was challenged with phage, 103 PFU of T7 or T5 was added to the day 1 flask after 11 h of bacterial growth. The delay in phage addition was necessary to keep E. coli from becoming extinct. For trials in which Salmonella was challenged, 106 PFU of SP6 was added to the first flask at the same time as the bacteria (no delay was necessary). All trials lasted 5 days (four transfers) or until extinction occurred.

When T7 was used to challenge E. coli in the presence of Salmonella, the E. coli density dropped profoundly. This drop made it difficult to maintain T7 at densities sufficient for transfer into the next flask. In one trial, 100 µl of the previous culture (instead of the usual 1 µl) was transferred between flasks to overcome this obstacle. This increase in transfer volume did not cause any noticeable change in the population dynamics compared to the other trials. In a second trial, a supplement consisting of 106 PFU of T7 was added on the second day and every day thereafter to the flasks otherwise transferred as described above (1-µl passages). Again this caused no noticeable change in the population dynamics compared to other trials.

Bacterial density was measured at the end of each 24-h period by plating on LB agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) (40 µl of a 20-mg/ml X-Gal solution/plate; LB agar consisted of 1% NaCl, 1% Bacto tryptone, 0.5% Bacto yeast extract, and 1.5% Bacto agar). E. coli colonies turn blue in the presence of X-Gal, as the lacZ-encoded beta-galactosidase of E. coli hydrolyzes X-Gal. Salmonella colonies remain white, as they lack the enzyme. Additionally, 106 PFU of a phage specific to one bacterium was spread on plates to kill one of the bacterial species. Phage densities in the experimental flasks were determined by plating medium from a flask on a lawn of sensitive cells. For both bacteria and phages, the lower limit of detection was 103/ml; below this level an organism was unlikely to be transferred to the next flask. The densities reported below are the final densities reached by the bacteria or phages each day after 24 h of growth.

Bacterial resistance assays.
Streak tests were used to test for phage resistance. Approximately 107 phage were spread in a line across the center of an LB agar plate and dried. A bacterial colony was then touched with a sterile loop and streaked orthogonally across the phage deposit. A control consisting of sensitive bacteria was also streaked on each plate. Colonies were scored as sensitive if the line of cell growth stopped at the line of phage and were scored as resistant if the line of cells showed no change as it crossed the line of phage.

Adsorption tests were used to assay partial phage resistance in some bacteria that were sensitive in the streak tests. Cells from a single colony were grown in a 10-ml culture under the same conditions that were used in the experimental trials. The density of cells was monitored with a Klett-Summerson photoelectric colorimeter. Once a flask reached a turbidity of 60 (~107 cells/ml), a sample was plated to determine the cell density (C), and 106 phage were added to the flask. Five minutes after the phage were added, a sample was mixed in top agar with sensitive cells and plated to obtain a measure of the total number of phage (N0). A 1-ml sample was also centrifuged for 1 min at 10,000 rpm to pellet the bacteria and adsorbed phage; the supernatant was plated on a lawn of bacteria to determine the number of unadsorbed phage (NU). The adsorption rate (k) was estimated from the equation k = –ln(NU/N0)/Ct, where t is 5 min. Adsorption assays were performed for one isolate of E. coli from each of the three trials in which E. coli was maintained in a two-species culture with T7, as well as for two isolates of the original E. coli stock.

Resistant cell competition assay.
To determine the cost of resistance to T7 and T5, resistant E. coli cells were competed against Salmonella in the absence of phage. The resistant E. coli cells were obtained as widely separated colony isolates from monocultures grown in the presence of phage. Colonies were screened to ensure that they remained resistant and were free of phage. Competition trials were initiated with 104 cells of each bacterium, and then the passaging protocol described above was used.

Analysis.
Standard t tests were used to compare cell densities in different treatments. Analyses that treated days as independent data points were compared to analyses that treated days as nonindependent data points. Under the assumption of independence daily values were used for analysis, while under the assumption of nonindependence 5-day means were used. The two methods agreed qualitatively (although not quantitatively) concerning significance in all but one case. Both P values are reported below; the day-independent P values are given first.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Without phage.
In monocultures the bacteria attained densities of 108 to 109 bacteria/ml within the first day and then returned to these densities again each day for the duration of the trial (Fig. 1). The grand mean log10 density of E. coli reached 9.12 ± 0.04 bacteria/ml over six trials (Table 1). The grand mean log10 density of Salmonella reached 8.81 ± 0.07 bacteria/ml over three trials (Table 1).



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  		FIG. 1. Mean bacterial density by day in the absence of phage. The data are the log densities reached after 24 h of growth. (A) E. coli densities in monocultures (n = 6) ({square}) and in two-species cultures (n = 5) ({blacksquare}). (B) Salmonella densities in monocultures (n = 3) ({square}) and two-species cultures (n = 4) ({blacksquare}). The error bars indicate standard errors.

 

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  		TABLE 1. Grand mean log densities of E. coli and Salmonella

 
In communities containing both bacteria and no phage, the bacterial densities followed roughly the same pattern that they followed in monocultures (Fig. 1). Within the first day the log density of each bacterial species reached 8 to 9 bacteria/ml, and then the density rose to this value every day thereafter. The grand mean log density of E. coli was 8.45 ± 0.06 bacteria/ml over five trials (Table 1). The grand mean log density of Salmonella was 9.00 ± 0.08 bacteria/ml over four trials (Table 1). In a two-species culture, therefore, E. coli attained approximately one-fifth the mean density that it attained in a monoculture, a significant reduction (for days independent, P < 0.001; for days dependent, P < 0.001). The Salmonella density, in contrast, was slightly higher in a two-species culture than in a monoculture; the increase is marginally significant (P = 0.04) if days are treated as independent and insignificant (P = 0.18) if days are treated as nonindependent.

In view of the finding that the density of Salmonella was not reduced in the presence of high E. coli densities, we tested whether either bacterium was feeding on a metabolite of the other species. Monocultures of each bacterium were maintained at saturation for 1 day in the original medium. The spent medium was filtered and inoculated with the same bacterium or the other bacterium (filters were rinsed in sterile water first to remove soluble chemicals that might have affected growth). No substantial difference was observed between the growth rates when the bacteria were inoculated into their own spent medium or spent medium of the other species (data not shown). The same outcome was obtained with media from bacteria maintained at saturation for 3 days before filtering. It should be noted that this assay tests for metabolites of bacteria in the stationary phase, not metabolites that are limited to bacteria during rapid growth.

Additionally, we tested the ability of each bacterium to invade a population of the other species. Two-species flasks were set up in which the initial density of one bacterium was 4 orders of magnitude lower than that of its competitor. When Salmonella was rare initially, the mean log density of this organism rose to 8.03 bacteria/ml for 5 days (one trial). When E. coli was rare initially, in one trial it attained a mean log density of 7.03 bacteria/ml for 5 days. However, in two later trials, the density of E. coli remained less than 105 cells/ml and the organism was driven to extinction on the fifth day. These data suggest that Salmonella is able to invade a population even when it is rare but that E. coli is sometimes unable to enter a population if it is at a numerical disadvantage.

E. coli challenged with T7.
In monoculture, phage T7 had little long-term effect, and the E. coli density increased to high values (Fig. 2, upper panel). The grand mean log density of E. coli reached 8.19 ± 0.20 bacteria/ml every day in five trials (Table 1), which was less than 1 order of magnitude less than the monoculture density in the absence of phage, although the difference is highly significant (P < 0.001 and P = 0.007). Ten colonies from day 2 in each of two trials all tested as T7 resistant. However, sensitive cells must also have been present as T7 was maintained for 5 days at a grand mean log density of 8.03 ± 0.25 particles/ml.



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  		FIG. 2. E. coli mean densities when organisms were cultured with phage T7. The data are the log densities reached after 24 h of growth. (A) E. coli densities in monocultures with T7 (n = 5) (•). (B) E. coli densities in two-species cultures with T7 (•); values are the means of the two trials in which E. coli and T7 were maintained. Each panel also shows bacterial densities in controls in which phage were absent (from Fig. 1) ({circ}). The error bars indicate standard errors.

 
In contrast, when competing with Salmonella, E. coli was not able to reach a high density when it was challenged with T7. E. coli became extinct in three of six trials, twice on the first day and once on the second day. In one trial T7 became extinct on day 2, but even in the subsequent absence of the phage, the log density of E. coli did not reach a value higher than 5.0 bacteria/ml in the remaining 3 days. In one trial each, E. coli and phage were maintained for 5 days by passaging 100 µl daily or adding phage daily (as described in Materials and Methods). In these two trials the levels of E. coli remained low, with a grand mean log density of only 4.37 ±0.26 bacteria/ml (Table 1 and Fig. 2, lower panel). In the trial in which 100 µl was passaged, T7 was lost on the sixth day, but the E. coli levels stayed low until the trial was ended on day 7. Cells remained sensitive to T7 for the three trials in which E. coli was maintained (10 of 10 colonies from day 2 of each trial were sensitive as determined by the streak test). Adsorption tests with an isolate from each of these three trials failed to detect any reduction in phage cell attachment rates that could serve as an alternative to complete resistance.

T7-resistant E. coli showed a competitive cost initially; however, this cost was compensated with time. When resistant E. coli obtained after 8 h of exposure to T7 was competed against Salmonella in the absence of phage, E. coli was driven to extinction after 3 days. In a similar trial with E. coli first exposed to T7 for 24 h, E. coli survived for 5 days in mixtures with Salmonella, but the density never got above 105 cells/ml. The density of E. coli that was grown with T7 for 5 days rose to 107 cells/ml against Salmonella within the first day.

E. coli challenged with T5.
In monoculture, T5 had little effect on E. coli densities, as observed for T7 (Fig. 3, upper panel). The grand mean log density of E. coli with T5 reached 8.36 ± 0.24 cells/ml, which was less than 1 order of magnitude less than the density of E. coli in the absence of phage, although again the difference is significant (P = 0.003 and P < 0.001). In a resistance test 90% (27/30) of the E. coli colonies were resistant to T5 on day 3. The T5 densities peaked on day 2, and then T5 became extinct on day 4 in all trials.



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  		FIG. 3. E. coli mean densities when organisms were cultured with phage T5. The data are the log densities reached after 24 h of growth. (A) E. coli densities in monocultures with T5 (n = 3) (•). (B) E. coli densities in two-species cultures with T5 (•). E (T5) in a two-species culture is from the three trials in which E. coli was maintained for 5 days. Each panel also shows the bacterial densities in controls in which phage were absent (from Fig. 1) ({circ}). The error bars indicate standard errors.

 
T5 had a strong impact on E. coli densities in the E. coli-Salmonella two-species culture. In three trials, E. coli was driven to extinction, once on day 2, once on day 4, and once on day 5. In these trials T5 was maintained as long as E. coli was present. In three other trials, E. coli was maintained at low levels, with a grand mean log density of 6.34 ± 1.56 cells/ml. This density is significantly lower (P = 0.008) than the density of E. coli in a two-species culture without phage (Fig. 3, lower panel). T5 was lost once on day 4 and twice on day 5. In all of these cases the E. coli density remained low even in the absence of phage. Surprisingly, cells from day 3 of each trial all tested sensitive to T5 (10 of 10 colonies per trial).

To ensure that our two-species culture results were not an artifact of T5 resistance never appearing in the population, we ran two further trials. In these trials, E. coli was allowed to evolve resistance to T5 for 3 h before Salmonella was added. E. coli was maintained for 5 days at a grand mean log density of 5.88 ± 0.83 cells/ml, which was not significantly different from the values in the other two-species T5 culture trials (P = 0.23). One-half of the colonies on day 3 tested resistant.

E. coli showed no competitive cost to T5 resistance in the absence of phage. In a trial initiated with E. coli that had grown with phage for 8 h, the density of E. coli rose to 107 cells/ml within 1 day, and this level was maintained for 5 days. At the end of the 5 days the E. coli still tested resistant to T5. If resistance to T5 had a cost, one would expect the resistant E. coli cells to be replaced by sensitive cells in the absence of phage.

Salmonella challenged with SP6.
In monoculture Salmonella reached high densities whether SP6 was present or absent (Fig. 4, upper panel). In the presence of the phage, the grand mean log density of Salmonella was 8.58 ± 0.13 cells/ml in three trials (Table 1), which was not significantly different from the mean log density of Salmonella alone (P = 0.07 and P = 0.18). In resistance tests on day 3 of the three trials, all 30 colonies tested resistant. However, sensitive cells must also have been present as SP6 was maintained in all trials at a mean log density of 6.56 ± 0.45 particles/ml.



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  		FIG. 4. Salmonella mean densities when organisms were cultured with phage SP6. The data are the log densities reached after 24 h of growth. (A) Salmonella densities in monocultures with SP6 (n = 3) (•). (B) Salmonella densities in two-species cultures with SP6 (n = 3) (•). Each panel also shows the bacterial densities in controls in which phage were absent (from Fig. 1) ({circ}). The error bars indicate standard errors.

 
Salmonella reached a high density in the presence of SP6 even in competition with E. coli (Fig. 4, lower panel). In competition SP6 reduced the density of Salmonella for 2 days, but by the third day the density of Salmonella was back to densities equivalent to those observed in the absence of phage. The grand mean log density of Salmonella with SP6 was 8.14 ± 0.19 cells/ml in three trials, which was significantly lower than the density of Salmonella in the absence of phage (P < 0.001 and P = 0.01) but was a reduction of less than 1 order of magnitude. In resistance tests on day 3 of the three trials, all 30 colonies tested resistant. However, some sensitive cells must have been present as SP6 was maintained in two trials at a grand mean log density of 8.03 ± 0.27 particles/ml. In one trial, phage were lost on the third day and added back on the fourth day. On the fifth day of this trial, the log density of the phage reached 6.49 particles/ml, and there was no obvious change in the pattern of Salmonella density.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study addressed the use of phages to control bacterial densities in the external environment (phage "prophylaxis") (15). Previous work did not reveal a long-term effect of phages on bacterial densities, but in that work monocultures (single strains of bacteria) were used (2, 12, 13, 20). We studied phages in experimental environments with two bacteria, only one of which was susceptible to the phage. Our rationale was that competition between bacterial species may enhance the effectiveness of phage prophylaxis because bacterial resistance to phages can decrease the competitive ability of bacteria (12, 13). Our results suggest that the cost of resistance sometimes allows phages to suppress and maintain low densities of a susceptible bacterium when a resistant competitor is present.

In the two-bacterium cultures, phages were far more effective at maintaining low densities of E. coli than at maintaining low densities of Salmonella. The basis for this difference remains unclear. One explanation is that only E. coli truly experienced competition; the density of Salmonella was actually higher in the presence of E. coli than in a monoculture. Thus, any cost of resistance would have had a larger impact on E. coli densities than on Salmonella densities in competition. Alternatively, the difference between E. coli and the Salmonella control may have stemmed from the particular phages used and the types of bacterial resistance that they selected.

In contrast to previous work, in which single bacterial species were used, our results suggest that phages have the potential to reduce bacterial density in the environment. However, our study provides only a modest extension of previous work toward the overwhelming complexity that comprises real bacterial communities, and elaborations of our design are desirable as well. A straightforward extension of our work is to use communities consisting of three or more bacterial species. As communities are expanded to include more species, the equilibrium densities of individual species may drop to such low values that they no longer maintain phage populations, a problem that could be surmounted by periodic addition of high phage densities. Further realism would be added by allowing biofilm growth on surfaces, which adds the complexities of spatial population structure, a semisolid matrix through which phages must diffuse, and highly variable physiological states of bacteria. Work on phages in biofilms already indicates that biofilms present special challenges to phage growth and maintenance (27). Thus, the addition of greater complexity to these experiments, while increasing the realism, will also pose greater challenges to understanding.

Our results pertain to the use of phage to prevent, rather than treat, bacterial infection. When infections are treated, bacterial resistance is of little concern if the immune system is able to eliminate bacteria at levels below a threshold density (15). Thus, phage enable the infection to be controlled by killing enough cells to allow the immune response to eliminate the other cells; results of several experimental studies suggest that phage can be effectively used to treat bacterial infections (1, 3, 14, 16, 17, 21, 24, 25). The dynamics of environmental phage prophylaxis are thus vastly different than the dynamics of phage therapy (14). However, due to the medical practice regulations, phage may have more immediate application in the prevention of infection.


   ACKNOWLEDGMENTS
 
We thank S. Abedon, H. Brussow, A. Dean, D. Dykhuizen, members of the Dykhuizen lab, and two anonymous reviewers for helpful comments and suggestions.

The work was funded by a Houston Livestock Show and Rodeo Fellowship to W.R.H. and by the NIH (grant GM57756 to J.J.B.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Integrative Biology, University of Texas CO930, Austin, TX 78712. Phone: (512) 232-6283. Fax: (512) 471-3878. E-mail: harcombe{at}mail.utexas.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, September 2005, p. 5254-5259, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5254-5259.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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