Prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as biocontrol agents

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Bacterial Strains and Growth Conditions
  • Staphylococcal Strain Typing
  • Bacteriophage Enrichment and Isolation
  • Bacteriophage Host Range
  • Temperate Versus Lytic Phage Determination
  • Cross-Immunity Assays
  • Phage Temperature Stability
  • Phage Antimicrobial Activity in Milk
  • Statistical Analysis
• Results and Discussion
  • Bacteriophage Prevalence in Dairy Samples
  • Temperature Stability of Phages ΦH5 and ΦA72 in Milk
  • Absence of Cross-Immunity Between ΦH5 and ΦA72
  • Antimicrobial Activity of Phages in Milk
  • Antimicrobial Activity of Phages in Milk Under Storage and Breakdown Temperature
• Conclusions
• Acknowledgments
• References
• Copyright

Abstract 

The prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples was assessed. Fourteen Staph. aureus strains were used in enrichment cultures of 75 dairy samples. All samples grew specific Staph. aureus bacteriophages. According to the host range, 8 different phages were isolated. Three of them, phages ΦH5, ΦG7, and ΦA72, were found in 89% of the samples; all the isolated phages were temperate. Phages ΦH5 and ΦA72 were used in preliminary bacterial challenge tests against Staph. aureus in milk. A phage mixture (1:1) was more effective than each single phage, most likely by preventing the survival of lysogenized cells. Phages inhibited Staph. aureus in UHT and pasteurized whole-fat milk. However, the phages were less active in semi-skimmed raw milk and little inhibition was achieved in whole, raw milk. Killing of Staph. aureus was observed at room temperature and at 37°C, but not at refrigeration temperature.

Key words: bacteriophage, dairy product, Staphylococcus aureus, biocontrol

Back to Article Outline

Introduction 

Staphylococcus aureus is a relevant pathogen to the food processing industry because of the ability of some strains to produce heat-stable enterotoxins and other virulence factors that cause staphylococcal food poisoning (Dinges et al., 2000; Le Loir et al., 2003). In France, for instance, 25 out of 149 foodborne staphylococcal outbreaks that occurred in 1999 were attributed to the consumption of raw milk cheeses, and 3 out of 13 were also reported in Italy (WHO, 2000). A mass outbreak of staphylococcal poisoning was reported in Japan caused by the consumption of reconstituted skimmed milk (Ikeda et al., 2005).

Staphylococcus aureus is also a frequent cause of IMI in dairy cows (Gruet et al., 2001) and may consequently contaminate milk. Mastitis caused by Staph. aureus is a major concern because of its resistance to antibiotic treatment and its propensity to recur (Makovec and Ruegg, 2003). Growing concerns about antibiotic resistance have stimulated research into alternative treatment methods (Skurnik and Strauch, 2006).

Bacteriophages (viruses of bacteria) were investigated as antibacterial agents as far back as the 1920s as a means of eliminating bacteria, including staphylococci, in human infections. These efforts resulted in a wide range of phage therapy research results, which have been comprehensively reviewed (Kutter and Sulakvelidze, 2005).

Bacteriophages have been also used as bactericidal agents in foods (Hudson et al., 2005). The FDA has recently approved the use of Listeria monocytogenes phage, Listex P100 (EBI Food Safety, Wageningen, the Netherlands) as GRAS (generally recognized as safe) for all food products (FDA, 2006). In milk and dairy products, phages have been successfully applied to prevent Salmonella Enteritidis development during Cheddar cheese manufacture and storage (Modi et al., 2001), Listeria monocytogenes growth in red smear cheeses (Carlton et al., 2005), and Staph. aureus proliferation in curd manufacturing processes (García et al., 2007). Therefore, there is clearly renewed interest in the exploitation of phages as antibacterial agents, with many pathogenic bacteria being targeted (Hagens and Loessner, 2007; Hanlon, 2007).

The objective of this study was to isolate a representative collection of bacteriophages of dairy origin infecting Staph. aureus as a preliminary approach to develop phage-based antimicrobial strategies with future applications in food biopreservation. With this in mind, we have used bovine Staph. aureus strains isolated from mastitic milk samples as hosts to assess the prevalence of Staph. aureus phages in the dairy environment. Finally, preliminary assays were performed to test the effectiveness of a phage mixture to inhibit Staph. aureus growth in milk.

Back to Article Outline

Materials and Methods 

Bacterial Strains and Growth Conditions 

Bovine Staph. aureus strains used for bacteriophage isolation and determination of host range were previously isolated from milk samples of mastitic cows belonging to different farms (Table 1). Eight Staph. aureus isolates from conventional bulk tank and organic milk (laboratory collection) were also used for phage host range determination (Table 2). Staphylococcal cells were cultured in 2xYT broth (Sambrook et al., 1989) using routine methods.

Table 1. Random amplification of polymorphism DNA (RAPD) profile of isolated bovine Staphylococcus aureus strains and efficiency of plaque formation (EOP) of phages ΦH5 and ΦA72

Table 2. Staphylococcus aureus bacteriophages selected in this study along with their respective source and host range

			No. of samples1 (n=75)	Host range2
Phage	Temperate	Source		Majestic strains	Milk strains
ΦC1	+	Cabrales cheese	3	Sa1	GDC6
ΦP1	+	Peñamellera cheese	1	Sa9	—
ΦL7	+	Milk	1	Sa3	AC9, FG1, AC11, GDC6, GRA16
ΦL13	+	Milk	1	Sa1, Sa2, Sa4, Sa9	AC9, DC6
ΦA8	+	Milk	2	Sa1, Sa2, Sa9, Sa11	—
ΦH5	+	Milk	26	Sa1, Sa9	AC9, FG1, AC11, GDC6, GRA16, JFL2,
ΦG7	+	Milk	20	Sa1, Sa9, Sa11	—
ΦA72	+	Milk	21	Sa1, Sa2, Sa3, Sa4, Sa9, Sa11	AC9, AFG1, AC11, GDC3, GDC6, GRA16, JFL2, JFL6

Staphylococcal Strain Typing 

Bovine Staph. aureus strains were typed by random amplification of polymorphic DNA (RAPD)-PCR using the oligonucleotide RAPD5 from the RAPD Analysis Primer Set (Amersham Biosciences Europe GmbH, Madrid, Spain). Amplification conditions were 5min at 95°C, 35 cycles of 1min at 95°C, 1min at 32°C, 2min at 72°C, and a final 10-min extension step at 72°C. The RAPD-PCR band patterns were scanned with the Gel Doc 2000 Gel Documentation System equipped with Quantity One software (BioRad Laboratories, Hercules, CA). Sorensen's similarity coefficient was calculated as a function of the presence/absence of the different bands for each pattern, different patterns being grouped using the unweighted pair group method with arithmetic averages (Priest and Austin, 1995).

Bacteriophage Enrichment and Isolation 

Bulk tank milk from 72 collaborative farms in the Principado de Asturias (northern Spain), and 3 traditional cheeses manufactured in 3 different factories were used for bacteriophage screening purposes. Each milk (100μL) and cheese sample (500mg) was added to 2mL of a bovine Staph. aureus strain (Table 2) growing in 2xYT containing 10 mg/L CaCl₂ and 10 mg/L MgSO₄. The cultures were incubated overnight at 37°C with shaking. Samples were centrifuged at 13,000×g for 5min and filtered. The supernatants were subjected to plaque assays using each of the 14 strains as indicators. Plaques were reisolated, propagated, and stored at −80°C in SM buffer (20 mg/L Tris HCl, 10 mg/L MgSO₄, 10 mg/L CaCl₂, 100 mg/L NaCl, pH 7.5) containing 50% glycerol (vol/vol). Phages were purified by ultracentrifugation (100,000×g for 90min) followed by CsCl continuous gradient centrifugation (Sambrook et al., 1989).

Bacteriophage Host Range 

The host range of phages was determined by the plaque assay: a 0.1-mL volume of stationary-phase host culture (10⁹ cfu/mL) was mixed with several dilutions of individual phage suspensions in 3mL of molten 2xYT top agar (0.7% agar) and the mixture was poured on 2xYT agar plates. Efficiency of plaque formation (EOP) of selected phages was determined by dividing the phage titer on the test strain by the phage titer on the reference strain Staph. aureus Sa9. This strain was selected because it is infected by most of the isolated phages (see Results section, Table 2).

Temperate Versus Lytic Phage Determination 

To determine if a phage was temperate, putative lysogens (resistant to infection) were isolated from lysis plaques. For each phage, several plaques were scratched and viable cells were colony-isolated on 2xYT agar plates. Twenty isolated colonies were challenged by the plaque assay with the corresponding phage to confirm resistance to infection. Additionally, exponentially growing cultures were subsequently induced by adding mitomycin C (0.5μg/mL) to confirm prophage release. After incubation at 37°C for 4h with shaking, supernatants were filtered and tested by the plaque assay against all the staphylococcal strains to verify the host range of the phage.

Cross-Immunity Assays 

Staphylococcus aureus Sa9 was lysogenized by phages ΦH5 or ΦA72 as described (see previous section). Once it was confirmed that the phage was integrated in the chromosome, cross-immunity was assessed by the spot test. Plates were prepared by adding 100μL of an overnight culture of the lysogenic strain to 5mL of molten 2xYT agar (0.7%). A 3-μL volume of the phage stock (10⁹ pfu/mL) was spotted onto the surface. Plates were incubated at 37°C for 18h and checked for clearing zones.

Phage Temperature Stability 

Phage stocks were diluted in UHT whole-fat milk to obtain 10⁵ and 10⁷ pfu/mL. The suspensions were incubated at 4, 22, and 37°C for 8h and the phage titer was determined. Similarly, phage suspensions were incubated at 72°C and samples were removed at 15s, and 1, 3, 5, and 15min for phage titration.

Phage Antimicrobial Activity in Milk 

The effect of phage infection on Staph. aureus growth was tested in commercial UHT and pasteurized whole-fat milk, and in whole-fat and semi-skimmed raw milks (which were centrifuged at 6,000×g for 20min to remove part of the fat) supplied by a collaborating farm. Milk was inoculated with diluted overnight cultures of Staph. aureus Sa9 (10² cfu/mL) and the phages ΦH5 and ΦA72 (10⁴ to 10⁵ pfu/mL). The mixtures were incubated at 37°C without shaking. In experiments simulating a breakdown in refrigerated storage, UHT whole-fat milk was inoculated with diluted overnight cultures of Staph. aureus Sa9 (10² cfu/mL) and a mixture of phages ΦH5 and ΦA72 (10¹, 10², and 10⁴ pfu/mL). The mixtures were incubated at 4°C for 18h and then shifted to 22°C for the following 30h. In all experiments, samples were taken at different time intervals and scored for Staph. aureus on Chapman agar plates (Scharlau Chemie S.A., Barcelona, Spain) and for phages using the plaque assay. The absence of Staph. aureus in noninoculated milk was verified by direct plating.

Statistical Analysis 

Results were compared by one-way ANOVA analysis using the SPSS 11.0 software for Windows (SPSS Inc., Chicago, IL). Experiments were performed in triplicate.

Back to Article Outline

Results and Discussion 

Bacteriophage Prevalence in Dairy Samples 

We proceeded to isolate, from the dairy environment, new staphylococcal phages that might be better adapted and, therefore, more effective for biocontrol purposes in milk and dairy products. Fourteen Staph. aureus strains previously isolated from mastitic milk samples and RAPD-typed were used as hosts in enrichments assays to detect phages able to infect Staph. aureus in dairy samples. These strains were grouped into 5 clusters (A to E) with a similarity >90% within a cluster (Figure 1, Table 1). The enrichment procedure was a very efficient method for phage isolation, and phages were detected in all the dairy samples tested (72 raw milk samples and 3 cheeses). At this step, only phages able to infect the strain used in enrichment cultures were selected for further assays. In this way, any potential prophage released from the staphylococcal strains used as hosts was discarded. It is important to note that different host ranges were revealed when phages were plated on several strains of mastitic and dairy origin, but no correlation to the RAPD grouping was observed (i.e., strains belonging to the same RAPD profile showed a distinct phage susceptibility pattern). According to the host range, 8 different phages (2 from artisan cheeses and 6 from raw milk) were obtained (Table 2). A few phages were predominant in the dairy environment studied. In our screening, 89% of the samples contained one of the ΦH5, ΦG7, or ΦA72 phages, which were detected in 26, 21, and 20 dairy samples, respectively. Some of the phages were very specific, infecting only one of the panel strains, whereas others, such as ΦA72, infected a much wider range that included both mastitic and milk-derived staphylococcal strains (Table 2). The complete set of isolated phages was able to infect all our mastitic strains, and a suitable mixture of these phages would, theoretically, inhibit the major clonal staphylococcal types associated with mastitis in our region and, consequently, the main source of staphylococcal contamination in milk.

• 
  • View Large Image
  • Download to PowerPoint

• Figure 1. 

  Random amplification of polymorphic DNA (RAPD)-PCR profiles of bovine Staphylococcus aureus isolates obtained with the oligonucleotide RAPD5 and the similarity dendrogram generated by the unweighted pair group method with arithmetic averages.

However, none of the selected phages was able to generate plaques on any of 13 Staph. aureus from human clinical samples (data not shown), suggesting that the livestock and human strains could be differentiated by typing with these phages. Similar results were obtained with phages CS1 and DW2 isolated from farmyard slurry (O’Flaherty et al., 2005b). Thus, the origin of phages should also be taken into account when designing either a biopreservative or a therapeutic preparation for humans.

All these newly isolated phages were temperate, because lysogenic bacteria were easily isolated from the clearing zones (Table 2). This seems to be a general feature within phages infecting Staph. aureus (Kwan et al., 2005). Temperate phages may be involved in lysogenic conversion and horizontal transfer of undesirable traits. Thus, their genetic content, their transduction ability, as well as their putative contribution to host fitness should be carefully analyzed before their use as biocontrol agents in food. However, because of the genetic amenability of phages, undesirable traits could be changed or deleted. Based on this, their lysogenic nature per se should not immediately preclude their potential use as antimicrobials, as reviewed by Matsuzaki et al. (2005) and Mann (2008).

Two phages, ΦH5 and ΦA72, were selected to assess their antimicrobial potential in milk on the basis of their broad host range, which comprised most of the mastitic and dairy strains (Table 2). The morphology of their virions under electron microscopy showed isometric capsids and long noncontractile flexible tails typical of the Siphoviridae family as described recently (García et al., 2007). Their EOP, where the efficiency of strain Staph. aureus Sa9 is taken to be 1.0, ranged from 0 to 1.32 (Table 1).

Temperature Stability of Phages ΦH5 and ΦA72 in Milk 

The stability in milk of these phages was tested at different temperatures. Similar phage titers were maintained at 4°C for 8h, confirming that 0 to 5°C is the most suitable range for phage stability (Civerolo, 1990). Incubations at 22 and 37°C resulted in 20 to 30% phage inactivation. Both phages withstood very short exposures at higher temperatures (72°C, 15s) but the titer was reduced to below the limit of detection (<10 pfu/mL) after 1min of exposure (Table 3). It would be expected, therefore, that mixtures of these phages will not be fully inactivated by current pasteurization processes and then might inhibit Staph. aureus growth during manufacture of dairy products. Resistance of phages to pasteurization has already been described (Madera et al., 2004).

Table 3. Survival of ΦH5 and ΦA72 phage suspensions in UHT whole-fat milk during storage at different temperatures and heat treatment

	Survival2 (%)
	ΦH5		ΦA72
Treatment1	4×105 pfu/mL	3×107 pfu/mL	4×105 pfu/mL	3×107 pfu/mL
4°C, 8h	100	100	100	100
22°C, 8h	78	79	72	80
37°C, 8h	73	75	70	78
72°C, 15s	86	98	93	99
72°C, 1min	0	0	0	0
72°C, 3min	0	0	0	0
72°C, 5min	0	0	0	0
72°C, 15min	0	0	0	0

Absence of Cross-Immunity Between ΦH5 and ΦA72 

The ability of phages to lysogenize might hinder the use of phages as biocontrol agents. Lysogenized strains, whereby the resident repressor blocks the replication of the infecting phage, become immune to infection by similar phages (Ladero et al., 1998). However, the use of a complex mixture of several phages would minimize the risk. To test this, lysogenic cultures to each phage were generated (Staph. aureus Sa9 lysH5 and Staph. aureus Sa9 lysA72, respectively). Staphylococcus aureus Sa9 lysH5 was resistant to infection by phage ΦH5 but was infected and lysed by phage ΦA72. Likewise, Staph. aureus Sa9 lysA72 cells containing ΦA72 as a prophage were immune to ΦA72 but susceptible to ΦH5 infection. Based on these results, ΦA72 and ΦH5 belong to a distinct immunity group and, thus, suitable mixtures of these phages would prevent the development of lysogenic derivative strains. Alternatively, another feasible strategy would be to obtain lytic derivative phages unable to lysogenize (García et al., 2007).

Antimicrobial Activity of Phages in Milk 

Preliminary challenge trials were performed in UHT whole-fat milk to assess the ability of the phages to lyse Staph. aureus Sa9 individually or in combination. The growth of Staph. aureus was inhibited regardless of whether single phages or a combination were used (Figure 2). However, the mixture was significantly more efficient (P<0.05), most likely by preventing the selection of bacteriophage-insensitive mutants and lysogenic derivatives.

• 
  • View Large Image
  • Download to PowerPoint

• Figure 2. 

  Inhibitory activity of single phages and the phage mixture (10⁴ to 10⁵ pfu/mL) against Staphylococcus aureus Sa9 at 37°C in UHT whole-fat milk: ◊=Staph. aureus, ■=Staph. aureus + ΦA72, ▴=Staph. aureus + ΦH5, ●=Staph. aureus + ΦA72 + ΦH5.

Staphylococcus aureus Sa9 was also challenged with the phage mixture in pasteurized, semi-skimmed, and whole-fat raw milks to determine to what extent commonly used milk treatment procedures (i.e., heating and skimming) affected the ability of the phages to inhibit Staph. aureus (Figure 3). In pasteurized whole-fat milk, the Staph. aureus population was 3.6 log units lower than the control culture without phages at the end of the incubation period. Accordingly, the phage titer increased with time (Figure 3A). An earlier effect of the phage mixture on Staph. aureus growth was observed in semi-skimmed raw milk because lower staphylococcal counts were detected compared with the control culture after 4h of incubation. Differences were significant (P<0.05) throughout the incubation period (Figure 3B). In contrast, the antibacterial activity of the phage mixture was clearly reduced in whole-fat raw milk (Figure 3C). Nevertheless, significantly lower counts were observed (P<0.05) after 11h. It is worth noting that in all cases, the phage mixture kept Staph. aureus counts low enough to avoid toxin accumulation over concentrations that cause food intoxication (Waldvogel, 2001), even in raw milk. This would be of great relevance to the food industry, as it would prevent accumulation of toxins that are not easily inactivated by heat treatments.

• 
  • View Large Image
  • Download to PowerPoint

• Figure 3. 

  Antibacterial activity of phages ΦH5 and ΦA72 against Staphylococcus aureus Sa9 at 37°C in A) pasteurized whole-fat milk, B) semi-skimmed raw milk, and C) whole-fat raw milk. □=Staph. aureus, ■=Staph. aureus + ΦA72+ ΦH5,×=phage titer.

In all tested conditions, total clearance of Staph. aureus in milk was not achieved. This might be expected because of the very low phage/cell ratio used in the experiments or because of resistance development. It is widely recognized that at low cell densities, larger numbers of phages are required to ensure efficient infection (Kasman et al., 2002). The antibacterial activity of the phages could also be reduced by the slow growth of Staph. aureus in both raw milks, likely because of the antagonistic and competitive effect of milk microbiota. Furthermore, the complexity linked to untreated raw milks may hinder phage diffusion. This will clearly hinder the chance of a phage encountering its host.

We observed that phage titer decreased about 1 log unit throughout the incubation period in semi-skimmed and whole-fat raw milk assays (Figure 3B and Figure 3C). This could indicate partial inactivation of the phages and their progeny in unheated milks. The raw milk used in these assays was not homogenized, in contrast to the commercial UHT and pasteurized milks. Thus, fat globules and milk protein might aggregate phage particles and render them unable to infect the host under the assay conditions. Similar inactivation in raw milk has been described for bacteriophage K (O’Flaherty et al., 2005a; Gill et al., 2006). A more detailed analysis should be carried out to determine whether milk components protect cells from infection or the phages themselves are inactivated.

Antimicrobial Activity of Phages in Milk Under Storage and Breakdown Temperature 

Because development of Staph. aureus is usually a consequence of a breakdown in the cold chain during milk processing, we also tested how phages influence the growth of the pathogen under refrigeration conditions followed by a shift to a warmer incubation. Refrigerated milk was contaminated with Staph. aureus Sa9 (10² cfu/mL) and a phage mixture (ΦH5 + ΦA72) at 10¹, 10², and 10⁴ pfu/mL. Incubation was performed at 4°C for 18h and the temperature was then increased to 22°C for the following 30h. During cold storage of milk, nonappreciable changes in the viable counts and phage titer occurred (Figure 4). The shift to a permissive temperature resulted in exponential development of Staph. aureus in both control and phage-infected milk cultures. Nevertheless, lower pathogen growth clearly dependent on the phage concentration was observed in phage-infected cultures for the first 24h of incubation at 22°C. At the end of the incubation period, however, a 5-log-unit reduction in bacterial counts compared with the control was observed regardless of the initial phage concentration (Figure 4A). The phage titer was stable at refrigeration temperatures and increased sharply after 12h of incubation at 22°C. As previously reported (Cairns et al., 2009), phage propagation only occurred when Staph. aureus could multiply actively and reach a threshold bacterial concentration that allowed the phage titer to increase (Figure 4B). These results showed that bacteriophages might be used as an additional countermeasure against temperature breakdown.

• 
  • View Large Image
  • Download to PowerPoint

• Figure 4. 

  Inhibitory activity of a mixture of phages ΦH5 and ΦA72 against Staphylococcus aureus Sa9 in UHT whole-fat milk during a simulated breakdown in the cold chain. Incubation proceeded at 4°C for 18h, shifted to 22°C, and was maintained there for 30h. A) Growth of Staph. aureus Sa9 (♦=Staph. aureus, ■=Staph. aureus + phages ΦH5 and ΦA72 (10¹ pfu/mL), ▴=Staph. aureus + phages ΦH5 and ΦA72 (10² pfu/mL), □=Staph. aureus + phages ΦH5 and ΦA72 (10⁴ pfu/mL); B) titer of bacteriophages in samples in which phages were added at initial concentrations of 10¹ pfu/mL (■), 10² pfu/mL (▴), or 10⁴ pfu/mL (□).

Back to Article Outline

Conclusions 

Our findings suggest that the dairy environment is an extensive source of staphylococcal phages, which may be effective in certain milk types to inhibit staphylococcal strains that often cause mastitis and contaminate milk. However, all the isolated phages were temperate, a feature that may hinder their efficacy as biocontrol agents in food. The use of phage mixtures is advisable to cover a wider inhibitory spectrum and to avoid, at least to some extent, the development of resistance. Phages of dairy origin were shown to be suitable to counteract the harmful effects of temperature failure during milk processing and storage.

Back to Article Outline

Acknowledgments 

This research study was supported by grants SAF2004-0033 and AGL2006-03659/ALI from the Ministry of Science and Technology (Spain) and the FEDER Plan and by the contracts CN-05-048 and CN-05-049 from Mercadona S.A. to Juan Evaristo Suárez. Pilar García is a fellow of the Spanish Ministry of Education Ramón y Cajal Research Programme. The authors thank Julián C. Rivas-Gonzalo and the Nutrition and Bromatology Unit (University of Salamanca, Spain) for hosting Pilar García. We also thank LILA-Asturias-ALCE-Calidad (Asturias, Spain) for providing the dairy samples.

Back to Article Outline

References 

1. Cairns BJ, Timms AR, Jansen VAA, Connerton IF, Payne RJH. Quantitative models of in vitro bacteriophage-host dynamics and their application to phage therapy. PLoS Pathog. 2009;5:e1000253
   • View In Article
   • CrossRef
2. Carlton RM, Noordman WH, Biswas B, de Meester ED, Loessner MJ. Bacteriophage P100 for control of Listeria monocytogenes in foods: Genome sequence, bioinformatic analyses, oral toxicity study, and application. Regul. Toxicol. Pharmacol. 2005;43:301–312
   • View In Article
   • MEDLINE
   • CrossRef
3. Civerolo EL. Bacteriophages. In:  Klement Z,  Rudolph K,  Sands DC editor. Methods in Phytobacteriology. Budapest, Hungary: Akademiai Kiado; 1990;p. 205–213
   • View In Article
4. Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 2000;13:16–64
   • View In Article
   • MEDLINE
   • CrossRef
5. FDA. Food additives permitted for direct addition to food for human consumption; bacteriophage preparation. Fed. Regist. 2006;71:47729–47732
   • View In Article
6. García P, Madera C, Martínez B, Rodríguez A. Biocontrol of Staphylococcus aureus in curd manufacturing processes using bacteriophages. Int. Dairy J. 2007;17:1232–1239
   • View In Article
7. Gill JJ, Sabour PM, Leslie KE, Griffiths MW. Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K. J. Appl. Microbiol. 2006;101:377–386
   • View In Article
   • MEDLINE
   • CrossRef
8. Gruet P, Maicent P, Berthelot X, Kaltsatos V. Bovine mastitis and intramammary drug delivery: review and perspectives. Adv. Drug Deliv. Rev. 2001;50:245–259
   • View In Article
   • MEDLINE
   • CrossRef
9. Hagens S, Loessner MJ. Application of bacteriophages for detection and control of foodborne pathogens. Appl. Microbiol. Biotechnol. 2007;76:513–519
   • View In Article
   • CrossRef
10. Hanlon GW. Bacteriophages: An appraisal of their role in the treatment of bacterial infections. Int. J. Antimicrob. Agents. 2007;30:118–128
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (296 KB)
    • CrossRef
11. Hudson JA, Billington C, Carey-Smith C, Greening G. Bacteriophages as biocontrol agents in food. J. Food Prot. 2005;68:426–437
    • View In Article
    • MEDLINE
12. Ikeda T, Tamate N, Yamaguchi K, Makino S. Quantitative analysis of Staphylococcus aureus in skimmed milk powder by real-time PCR. J. Vet. Med. Sci. 2005;67:1037–1041
    • View In Article
    • MEDLINE
    • CrossRef
13. Kasman LM, Kasman A, Westwater C, Dolan J, Schmidt MG, Norris JS. Overcoming the phage replication threshold: A mathematical model with implications for phage therapy. J. Virol. 2002;76:5557–5564
    • View In Article
    • MEDLINE
    • CrossRef
14. Kutter E, Sulakvelidze A. Bacteriophages Biology and Applications. Boca Raton, FL: CRC Press; 2005;
    • View In Article
15. Kwan T, Liu J, Dubow M, Gros P, Pelletier J. The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc. Natl. Acad. Sci. USA. 2005;102:5174–5179
    • View In Article
16. Ladero V, García P, Bascarán V, Herrero M, Álvarez MA, Suárez JE. Identification of the repressor-encoding gene of the temperate bacteriophage A2. J. Bacteriol. 1998;180:3474–3476
    • View In Article
    • MEDLINE
17. Le Loir Y, Baron F, Gautier M. Staphylococcus aureus and food poisoning. Genet. Mol. Res. 2003;2:63–76
    • View In Article
    • MEDLINE
18. Madera C, Monjardín C, Suárez JE. Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies. Appl. Environ. Microbiol. 2004;70:7365–7371
    • View In Article
    • MEDLINE
    • CrossRef
19. Makovec JA, Ruegg PL. Antimicrobial resistance of bacteria isolated from dairy cow milk samples submitted for bacterial culture: 8,905 samples (1994–2001). J. Am. Vet. Med. Assoc. 2003;222:1582–1589
    • View In Article
    • MEDLINE
20. Mann NH. The potential of phages to prevent MRSA infections. Res. Microbiol. 2008;159:400–405
    • View In Article
    • CrossRef
21. Matsuzaki S, Rashel M, Uchiyama J, Sakurai S, Ujihara T, Kuroda M, et al. Bacteriophage therapy: A revitalized therapy against bacterial infectious diseases. J. Infect. Chemother. 2005;1:211–219
    • View In Article
22. Modi R, Hirvi Y, Hill A, Griffiths MW. Effect of phage on survival of Salmonella Enteritidis during manufacture and storage of Cheddar cheese made from raw and pasteurized milk. J. Food Prot. 2001;64:927–933
    • View In Article
    • MEDLINE
23. O’Flaherty S, Coffey A, Meaney WJ, Fitzgerald GF, Ross RP. Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk. Lett. Appl. Microbiol. 2005;41:274–279
    • View In Article
    • MEDLINE
    • CrossRef
24. O’Flaherty S, Ross RP, Flynn J, Meaney WJ, Fitzgerald GF, Coffey A. Isolation and characterization of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections. Lett. Appl. Microbiol. 2005;41:482–486
    • View In Article
    • MEDLINE
    • CrossRef
25. Priest F, Austin B. Numerical taxonomy. In:  Priest F,  Austin B editor. Modern Bacterial Taxonomy. London, UK: Chapman and Hall; 1995;p. 14–49
    • View In Article
26. Sambrook J, Maniatis T, Fritsch EF. Molecular Cloning: A Laboratory Manual. 2nd ed.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989;
    • View In Article
27. Skurnik M, Strauch E. Phage therapy: Facts and fiction. Int. J. Med. Microbiol. 2006;296:5–14
    • View In Article
    • MEDLINE
    • CrossRef
28. WHO (World Health Organization). 2000. Surveillance Report: Programme for Control of Foodborne Infections and Intoxications in Europe. 8th Report 1999–2000. http://www.bfr.bund.de/internet/8threport/8threp_fr.htm.
    • View In Article
29. Waldvogel FA. Staphylococcus aureus (including staphylococcal toxic shock). In:  Mandell JL,  Bennett JE,  Dolin R editor. Principles and Practice of Infectious Diseases. Philadelphia, PA: Churchill Livingstone; 2001;p. 2069–2092
    • View In Article