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Poult Sci 2007. 86:2536-2540. doi:10.3382/ps.2007-00250
© 2007 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE

Salmonella Host Range of Bacteriophages That Infect Multiple Genera

L. Bielke, S. Higgins, A. Donoghue, D. Donoghue and B. M. Hargis1

Center of Excellence for Poultry Science, University of Arkansas, Fayetteville 72701

1 Corresponding author: bhargis{at}uark.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conventionally, bacteriophages are considered viruses capable of amplification only in a narrow range of closely related bacteria. Presently, we selected bacteriophages with the ability to infect more than 1 bacterial genus. Initially, wild-type bacteriophages were selected for ability to form plaques in Salmonella enteritidis agar overlays. For determination of host specificity, a pool of 44 bacteriophages was combined with each bacterial isolate in tryptic soy broth. This mixture was incubated with fresh bacterial culture and media for 4 sequential passes, and the resulting bacteriophage titer was determined using S. enteritidis. One Klebsiella and 3 different Escherichia isolates successfully amplified some bacteriophage(s) from the S. enteritidis-selected bacteriophage pool (experiment 1). Amplification of bacteriophages in each species was confirmed by the formation of increased plaque forming units in a tryptic soy agar overlay with the enteric (alternative host) bacteria, Klebsiella or Escherichia (experiment 2). Two selected bacteriophages, confirmed to amplify in Escherichia or Klebsiella, were further evaluated for ability to amplify in 10 different Salmonella serovars by amplification in broth culture (experiment 3). One had the ability to amplify in 6 different Salmonella serovars, and the other had the ability to amplify in 2 different Salmonella serovars. These experiments suggest that bacteriophage host range is not always genera-restricted and that selection of subpopulations of bacteriophages capable of amplification in alternative genera may provide a tool for selection of broad host-range bacteriophages for the pathogen of interest. Selection of non-pathogenic host isolates to support replication of Salmonella bacteriophages may allow improved safety for bacteriophage application to poultry because this would reduce the necessity for 100% purification of the bacteriophages(s) from resistant host bacteria.

Key Words: Salmonella • bacteriophage • host specificity • wide host range • poultry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacteriophages are viruses that infect and replicate in prokaryotic cells rather than eukaryotic cells and represent a possible alternative to antibiotic usage or therapy (Cann, 1993). Bacteriophages are ubiquitous and are commonly found in water, sewage, and soil. Smith and Huggins (1983) and Smith et al. (1987) successfully used bacteriophages to treat calves, pigs, and lambs infected with Escherichia coli. In 1998, Barrow et al. also successfully treated E. coli-infected calves with an intramuscular injection of bacteriophages. This same type of treatment also reduced morbidity and mortality in poultry in later studies. Berchieri et al. (1991) showed efficacy in treating chickens infected with Salmonella typhimurium when treatment and pathogen were administered simultaneously. This experiment also showed that bacteriophages remained present in the gastrointestinal tract and increased in numbers as long as the host bacteria were present. More recently, Huff et al. (2002) have demonstrated the ability of bacteriophages to prevent airsacculitis in chickens caused by E. coli.

Bacteriophages are host specific and often infect only 1 bacterial species or only 1 serotype within a species (Ackerman et al., 1978). Although this property of bacteriophages is important for classifying bacteria, it is a limiting factor in therapeutic treatment of bacterial infections because, currently, an effective bacteriophage must be identified for each individual disease outbreak. However, not all bacteriophages are host-specific. In 1998, Jensen et al. were able to isolate bacteriophages with the ability to lyse either Sphaerotilus natans and E. coli or Pseudomonas aeruginosa and E. coli. Similarly, Greene and Goldberg (1985) were able to isolate bacteriophages capable of lysing more than 1 species of Streptomycetes. This evidence suggests that wide-host-range (WHR) bacteriophages exist in common sources of bacteriophages (wastewater and soil). We have hypothesized that WHR bacteriophages would be easier to use for the treatment of bacterial infections or contamination because bacteriophages would not have to be isolated for individual infections. Indeed, a small library of bacteriophages could potentially treat a wide range of infections. Additionally, amplification of bacteriophages in a nonpathogenic alternative host eliminates the possibility of accidentally administering a pathogen if purification is not complete. In the following experiments, we sought to isolate WHR bacteriophages with the ability to amplify in nonpathogenic bacterial isolates of multiple genera and to evaluate their Salmonella host range.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteriophage Isolation
Bacteriophages were isolated as previously described (Higgins et al., 2005). Briefly, wastewater samples were obtained from a local municipal wastewater treatment plant and filtered through a 0.2-µm filter (Catalog No. 2004–08, Pall-Gelman Laboratory, Ann Arbor, MI). A combination of 100 µL of 107 cfu/mL Salmonella enteritidis PT 13A (SE) and 1 mL of the wastewater sample filtrate were added to 1.5 mL of tryptic soy agar (TSA, Catalog No. 211043, Becton Dickinson, Sparks, MD) and poured over a warm TSA petri plate. Plates were incubated overnight at 37°C, and those with confluent lysis of SE were then flooded with 15 mL of sterile 0.9% NaCl (saline). The fluid was then poured off the plate and filtered through a 0.2-µm filter. Serial 10-fold dilutions were made in saline. Plates were poured as described above with 1 mL of each bacteriophage dilution. Individual distinct plaques resulting from this plating were then differentiated on the basis of plaque morphology, and different plaques were sequentially passed on TSA plates at least 3 subsequent times to establish bacteriophage isolate purity.

With this technique, 44 bacteriophages were isolated from 4 wastewater samples, each collected on separate days. Although several different plaque morphologies were noted and isolates were obtained from 4 different wastewater collections, the 44 bacteriophage isolates in our library may contain unknown redundancy. For experiment 1, all 44 bacteriophage isolates were initially combined into a single pooled bacteriophage mixture.

Experiment 1
Thirty-five nonpathogenic enteric bacterial isolates of a poultry origin were used to screen the 44-pooled bacteriophage isolates for their ability to amplify in more than one species of bacteria. These bacteria were previously selected for their ability to inhibit in vitro growth of SE and were able to reduce SE recovery in poults (Bielke et al., 2003). In this previous study, each of these isolates were evaluated for morbidity, mortality, and lesion-generating potential when administered intramuscularly, intraperitoneally, intraairsac, or orally in high numbers and no indication of pathogenicity was observed. Genera used in the screening process included 20 Escherichia coli, 3 Klebsiella oxytoca, 5 Citrobacter freundii, 1 Kluyvera spp., and 6 Lactobacillus spp. isolates. The pooled bacteriophages were combined with each individual bacterial isolate at a ratio of 1 mL of bacteriophages, 3 mL of bacterial culture (turbid culture), and 5 mL of tryptic soy broth (TSB, Catalog No. 211822, Becton Dickinson, Sparks, MD). This combination was incubated for 2 h at 37°C. The culture was then filtered using a 0.2-µm syringe filter. For subsequent passage, the filtrate was recombined at a ratio of 1 mL of filtrate, 3 mL of bacterial culture, and 5 mL of TSB and reincubated. This process was repeated for a total of 4 passages. To determine final plaque-forming units (PFU) of the amplified bacteriophages, 1 mL of filtrate was combined with 100 µL of 107 cfu/mL SE and 1.5 mL TSA poured over a warm TSA petri plate (soft agar overlay plates). This experiment was repeated twice. Isolated bacteriophages were sequentially passed on soft agar overlay plates at least 3 times to ensure bacteriophage isolate purity. As previously described, each of the bacterial isolates that amplified bacteriophages had previously been typed using API 20NE test strips (Catalog No. 2004-04-10, bioMerieux, Marcy l’Etoile, France) and have a unique API number, indicating that the 4 isolates are different strains of coliforms (Bielke et al., 2003).

Experiment 2
To confirm amplification and lysis of the selected alternative host bacteria by the selected WHR bacteriophages isolated in experiment 1, a second experiment was conducted in which selected bacteriophages and the alternative host bacteria were combined for coincubation as described above for 2 passages. To confirm lysis of the alternative host bacteria, PFU were determined using soft agar overlay plates utilizing the alternative host bacteria.

Experiment 3
To evaluate the Salmonella host range of 2 of the selected WHR bacteriophages isolated from experiment 1, 10 different field isolates identified by the National Veterinary State Laboratory were utilized. The serovars included S. heidelberg, S. montevideo, S. ohio, S. hadar, S. typhimurium, S. agona, S. kentucky, S. infantis, S. minnesota, and S. seftenberg. For this experiment, each Salmonella serovar was grown to turbidity and combined with each of the 4 selected WHR bacteriophages so that each bacteriophage was tested individually against 10 different Salmonella serovars. Bacteriophages and bacteria were combined for coincubation as described in experiment 1 for 2 passages. The PFU were determined using SE soft agar overlay plates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1
Of the 35 bacterial isolates screened, bacteriophages originally selected using SE were able to amplify in 4 different isolates (Figure 1Go). In trial 1, the titer of the original 44 pooled bacteriophages was 4.1 x 108 PFU, and the estimated level of bacteriophages after passage (due to dilution alone) without amplification was 1.0 x 105 PFU. Four of the 35 bacterial isolates tested (designated 8, 10, 23, and 24) amplified at least one of the bacteriophages in the original pooled culture beyond the titer of the unamplified bacteriophage similarly diluted. In trial 2, the titer of the original 44 pooled bacteriophages was 1.22 x 109 PFU, and the estimated level of bacteriophages after passage without amplification was 1.8 x 105 PFU. All 35 bacterial isolates were screened against the pooled bacteriophages. These same 4 bacterial isolates (8, 10, 23, and 24) amplified at least one of the bacteriophages beyond the titer of the similarly diluted unamplified bacteriophage (Figure 1Go).


Figure 1
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  		Figure 1. Bacteriophages were incubated in broth using alternative host bacteria for amplification. The plaque-forming units (PFU) of bacteriophages were then determined on soft agar overlay plates. Initial PFU indicates the level of bacteriophages prior to incubation with the alternative host bacteria. Average unamplified indicates the averaged titer present in the 31 bacterial isolates that did not amplify bacteriophages, titer was at or below the estimated unamplified bacteriophages. Thirty-one bacterial isolates did not amplify any bacteriophage, with determined PFU per milliliter similar to those observed with the unamplified (diluted only) samples (data not shown).

 
Experiment 2
The selected WHR bacteriophages, numbered according to the alternative host bacteria in which they amplified, WHR 8, WHR 10, WHR 23, and WHR 24, were amplified in their respective alternative host bacteria. Initial PFU were 2.7 x 104, 1.4 x 104, 4.6 x 104, and 5.1 x 104, respectively. After 2 incubation periods, the final titer of the bacteriophages was determined on soft agar plates utilizing the alternative host bacteria. The final PFU of each bacteriophage were 2.08 x 1010/mL, 1.28 x 1010/mL, 2.04 x 1010/mL, and 9.2 x 109/mL, respectively (Figure 2Go).


Figure 2
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  		Figure 2. Wide host range bacteriophages were amplified with their respective alternative host bacteria for 2 passages. The plaque-forming units (PFU) were determined using serial dilution with alternative host bacteria on soft agar overlay plates.

 
Experiment 3
Two of the WHR bacteriophages, WHR 8 and WHR 10, were tested to determine their ability to amplify in different isolates of different Salmonella serovars and to differentiate the 2 bacteriophage isolates. The WHR 8 was able to amplify in 6 different serovars of Salmonella including S. heidelberg, S. montevideo, S. ohio, S. typhimurium, S. agona, and S. minnesota. Bacteriophage WHR 10 was able to amplify in 2 isolates, S. heidelberg and S. infantis (Figure 3Go). Bacteriophages amplified in these alternative salmonellae were also confirmed using SE overlay plates.


Figure 3
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  		Figure 3. Wide host range bacteriophages 8 and 10 were evaluated for their ability to amplify in and lyse different species of Salmonella. Bacteriophages were individually amplified in each species of Salmonella, and plaque-forming units (PFU) were determined using soft agar overlay plates.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In these experiments, WHR bacteriophages were isolated from a common bacteriophage source (wastewater). Not all bacteriophages are as host specific as the types used in bacterial typing, and these types of WHR bacteriophages are potential candidates for the treatment of bacterial infections or foods. Availability of a library of WHR bacteriophages, prescreened for virulence and drug resistance genes, would allow potential application for infection or foodstuff treatment without preevaluating sensitivity of target host cell to a given bacteriophage isolate. Just 2 of the selected bacteriophages in these experiments were able to infect and lyse 7 out of 10 Salmonella isolates, suggesting that when combined with more WHR bacteriophages, it might be possible to treat many Salmonella infections (or food contamination) without antibiotics.

Some bacteriophages can carry the genetic code for bacterial virulence genes (Stanley et al., 2000). Thus, a library of candidate bacteriophages for therapeutic or food application should be screened for known virulence and drug resistance genes (Boyd, 2005). Creation of a library of pretested bacteriophages with a known WHR could simplify the selection process.

Another limitation to the application of bacteriophages for therapeutic use or food prophylaxis is the absolute necessity of separating 100% of the host bacteria from the bacteriophage preparation. During amplification of bacteriophages in a pathogenic target host isolate, some bacteria may become bacteriophage resistant (Lowbury and Hood, 1953). Assuring 100% separation of bacteriophages from resistant surviving host cells through differential centrifugation, filtration, or chemical treatment is currently tedious or expensive, or both. Alternatively, if useful bacteriophages were able to be amplified in host cells that were completely apathogenic, this process could be greatly simplified and safety of the resulting preparation could be easily assured. Results from the present experiments describe a rapid method for identifying bacteriophages that are capable of truly amplifying in non-pathogenic hosts and are simultaneously able to lyse a wide range of Salmonella isolates. It is hypothesized that those isolates capable of crossing genera may use receptors, intermediary functions, or both, common to a wide range of bacteria.

In experiment 2, we showed that the selected WHR bacteriophages could amplify to a very high titer with just a few passages in the alternative host bacteria. Another potential advantage of WHR bacteriophages amplified in alternative host bacteria, in the case of enteric disease, is the potential to coadminister bacteria capable of colonizing the gastrointestinal tract to replace natural microflora that may have been displaced by the pathogen, WHR bacteriophages, or both. However, this potential was not presently examined.

Previous studies have shown that bacteriophages can successfully treat bacterial infections when coadministered with the pathogen in a laboratory setting (Smith and Huggins, 1983; Smith et al., 1987; Berchieri et al., 1991; Barrow et al., 1998; Huff et al., 2002). Higgins and coworkers (2005) were able to successfully treat processed turkeys contaminated with Salmonella using bacteriophages. Additionally, the United States Food and Drug Administration recently approved the application of bacteriophages to ready-to-eat foods for the prevention of foodborne illness associated with Listeria monocytogenes (Intralytix Inc., 2006). For practical field application of bacteriophages for either bacterial disease or food prophylaxis, a number of scientific and political issues remain. Creation of broad host-range bacteriophage libraries, carefully examined for known virulence and drug resistance genes, and the identification of nonpathogenic host cells capable of amplifying these bacteriophage isolates, is one approach to ameliorating some of these issues.


   ACKNOWLEDGMENTS
 
We would like to thank Amanda Wolfenden and Cheryl Lester (Department of Poultry Science, University of Arkansas) for their technical assistance throughout this project. Funding provided by the Arkansas Biosciences Institute.

Received for publication June 15, 2007. Accepted for publication August 23, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ackerman, H. W., A. Audurier, L. Berthiaume, L. A. Jones, J. A. Mayo, and A. K. Vidaver. 1978. Guidelines for bacteriophage characterization. Adv. Virus Res. 23:1–24.[Medline]

Barrow, P., M. Lovell, and A. Berchieri Jr. 1998. Use of lytic bacteriophages for control of experimental Escherichia coli septicemia and meningitis in chickens and calves. Clin. Diag. Lab. Immun. 5:294–298.[Abstract/Free Full Text]

Berchieri, A., M. A. Lovell, and P. A. Barrow. 1991. The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium. Res. Microbiol. 142:541–549.[Medline]

Bielke, L. R., A. L. Elwood, D. J. Donoghue, A. M. Donoghue, L. A. Newberry, N. K. Neighbor, and B. M. Hargis. 2003. Approach for selection individual enteric bacteria for competitive exclusion in turkey poults. Poult. Sci. 82:1378–1382.[Abstract/Free Full Text]

Boyd, E. F. 2005. Bacteriophages and virulene. Pages 223–266 in Bacteiophages: Biology and Applications. E. Kutter and A. Sulakvelidze, ed. CRC Press, Boca Raton, FL.

Cann, A. J. 1993. Principles of Molecular Virology. Academic Press, Harcourt Brace and Company Publishers, New York, NY.

Greene, J., and R. B. Goldberg. 1985. Isolation and preliminary characterization of lytic and lysogenic phages with wide host range within the Streptomycetes. J. Gen. Microbiol. 131:2459–2465.[Abstract/Free Full Text]

Higgins, J. P., S. E. Higgins, K. L. Guenther, L. A. Newberry, W. E. Huff, and B. M. Hargis. 2005. Use of a specific bacteriophage treatment to reduce Salmonella in poultry. Poult. Sci. 84:1141–1145.[Abstract/Free Full Text]

Huff, W. E., G. R. Huff, N. C. Rath, J. M. Balog, H. Xie, P. A. Moore, Jr., and A. M. Donoghue. 2002. Prevention of Escherichia coli respiratory infection in broiler chickens with bacteriophage (SPR02). Poult. Sci. 81:437–441.[Abstract/Free Full Text]

Intralytix Inc. 2006. http://www.intralytix.com/Intral_News.htm Accessed Oct. 2006.

Jensen, E. C., H. S. Schrader, B. Rieland, T. L. Thompson, K. W. Lee, K. W. Nickerson, and T. A. Kokjohn. 1998. Prevalence of broad-host-range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa. Appl. Environ. Microbiol. 64:575–580.[Abstract/Free Full Text]

Lowbury, E. J. L., and A. M. Hood. 1953. The acquired resistance of Staphylococcus aureus to bacteriophage. J. Gen. Microbiol. 9:524–535.[Medline]

Smith, H. W., and M. B. Huggins. 1983. Effectiveness of phages in treating experimental Escherichia coli diarrhea in calves, piglets, and lambs. J. Gen. Microbiol. 129:2659–2675.[Abstract/Free Full Text]

Smith, H. W., M. B. Huggins, and K. M. Shaw. 1987. The control of experimental Escherichia coli diarrhea in calves by means of bacteriophages. J. Gen. Microbiol. 133:1111–1126.[Abstract/Free Full Text]

Stanley, E., L. Walsh, A. van der Zwet, G. F. Fitzgerald, and D. van Sinderen. 2000. Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance. FEMS Microbiol. Lett. 182:271–277.[CrossRef][Web of Science][Medline]





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