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Keywords:

  • endodontic treatment;
  • failure;
  • Enterococcus faecalis;
  • oral bacteriophage;
  • phage therapy;
  • saliva

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Aims: To detect bacteriophages for Gram-positive oral pathogens in human saliva.

Methods and Results: Saliva samples from 31 donors were screened for the presence of bacteriophages for Streptococcus sobrinus, Streptococcus mutans, Streptococcus salivarius, Actinomyces viscosus and Enterococcus faecalis. Bacteriophages for Enterococcus faecalis were found in seven samples. Enterococcus faecalis phages were still present in saliva re-collected from one donor one month, and one year after initial saliva collection.

Conclusions: The presence and stability of the Enterococcus faecalis bacteriophages in human saliva suggests a possible role of these bacteriophages in the oral ecosystem.

Significance and Impact of the Study: Phage therapy as a way to control oral bacteria might be considered.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Recent reports confirm the estimation that 500 cultivable and uncultivable bacterial species inhabit the oral cavity (Moore and Moore 2000; Paster et al. 2001). While the majority of these organisms are commensals, subsets of them are believed to initiate caries and periodontal diseases. Some of the periodontal pathogens are associated with systemic diseases (Socransky et al. 1998; Beck et al. 2000; Moore and Moore 2000).

While bacteriophages have been found to have significant influence in various microbial environments, very little is known about their impact on the oral ecology. Smoking has been reported to increase a woman's risk of contracting bacterial vaginosis, by promoting phage induction which reduce the population of vaginal lactobacilli which in turn promotes the overgrowth of anaerobic bacteria (Pavlova and Tao 2000).

Bacteriophages specific for oral bacteria such as Actinobacillus actinomycetecomitans (Haubek et al. 1997) and Actinomyces viscosus (Delisle et al. 1978; Tylenda et al. 1985; Yeung and Kozelsky 1997) have been isolated from dental plaque. To date, no salivary Enterococcus faecalis phages have been found. However, Ent. faecalis isolated from dental plaques can be classified by phages extracted from sewage (Smyth et al. 1987). To the best of our knowledge this is the first report of phage presence in human saliva.

Oral bacterial strains

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Oral bacterial species used in this study are listed in Table 1. Enterococcus faecalis DJ1 is an oral isolate identified using conventional identification scheme (Rapid ID32 STREP, BioMerieux SA, Marcy l'Etoie, France). Sequencing of the Ent. faecalis DJ1 16S rDNA (GenBank accession AF447490) performed as described previously (Bachrach et al. 2001), revealed that it is identical to that of Ent. faecalis JH2-2 (McAshan et al. 1999).

Table 1.  Bacterial species used in this study. Number of saliva samples screened, and the number of those found to contain phages for each bacterial species
Bacterial speciesNumber of saliva samples testedNumber of samples containing phage
Streptococcus sobrinus 6715150
Streptococcus mutans ATCC 27351160
Streptococcus salivarius ATCC 11742310
Actinomyces viscosus ATCC 43146310
Enterococcus faecalis DJ1 (this study)317

Saliva collection

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Stimulated saliva was collected from random donors who had not received antibiotics within the previous three months. One millilitre of each saliva sample was cleared of debris and bacteria by centrifugation at 15000 g for 10 min. Supernatant fluid was collected and kept at 4 °C until used.

Phage stability in human saliva

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Phage λ was used to test the efficiency of our phage isolation procedure and to assess phage stability in human saliva. Experiments were carried out in triplicate, and were repeated with saliva collected from three different donors. First, 105 phage λ (LamdaGEM-11, Promega Biotec, Madison, WI, USA) plaque-forming units (pfu) were added to 1 ml clarified saliva. Then 100 µl of saliva–phage λ dilutions were added to 100 µl of Escherichia coli XL1 (Promega) grown overnight in Brain Heart Infusion broth (BHI, Difco Laboratories, Detroit, MI, USA). The saliva–phage λ–bacteria mixture was vortexed briefly, incubated for 20 min at 37 °C, added to 4 ml preheated (47 °C) BHI top agar (0·7% agarose) and overlaid on BHI agar plates (1·5% agar, Difco) as described previously (Sambrook et al. 1989). Plates were incubated at 37 °C in aerobic conditions for 12–16 h until lysis zones (pfu) could be detected and counted.

Phage isolation from human saliva

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Oral bacteria (listed in Table 1) were grown overnight in BHI at 37 °C under aerobic conditions. Bacterial culture (100 µl) was added to 100 µl clarified saliva. The saliva–bacteria mixture was vortexed briefly, incubated for 20 min at 37 °C, added to 4 ml preheated (47 °C) BHI top agar and overlaid on BHI agar plates (as before). Plates were incubated at 37 °C for 5 d or until lysis zones could be detected.

Electron microscopy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Transmission electron microscopy was performed on lysis zones by rapid placing of carbon-colodium coated grids on the plaque, followed by negative staining with 1% phosphotungstic acid.

Phage stability in human saliva

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Phage λ was used to determine the appropriate conditions for phage isolation from human saliva. Plating of saliva–phage λ dilutions on E. coli XL1 revealed that 70% of phage pfu could be recovered from saliva kept for one week at 4 °C or at 37 °C. Centrifugation of saliva–phage λ mixture at 15000 g for 10 min did not result in detectable loss of phage.

Phage isolation from human saliva

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Three to five days after inoculation with Ent. faecalis, uniform turbid lysis zones (approximately 2 mm in diameter) were generated by saliva samples collected from seven of 31 individuals. Saliva samples did not generate lysis zones with any other bacterial species tested (Table 1). Ent. faecalis phages were stable in BHI for up to 4 weeks when kept at 4 °C. Phages were re-isolated from one individual 4 weeks and one year after initial sample collection. This suggests that the Ent. faecalis phages are persistent, or that re-infection occurs, possibly because the individual's oral flora is more susceptible to colonization by bacteriophage.

Phage properties

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

Transmission electron microscopy performed on lysis zones generated from one saliva sample revealed uniformed, spherical, enveloped, spiked structures with a diameter of roughly 70 nm (Fig. 1). Phage tail was not detected.

image

Figure 1. Electron micrograph of Enterococcus faecalis phages

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References

The presence of bacteriophages in human saliva and the ability to re-isolate them after one year from the saliva of one of the individuals tested, suggests that phages might play an active and an important role in the oral environment. Previous studies found that approximately 10% of the fresh dental plaque samples harboured phages able to infect Actinomyces strains (Yeung and Kozelsky 1997). In aquatic communities bacteriophages can limit bacterial abundance to several orders of magnitude below the resource-limited level (Lenski 1988). It is predicted that phages are responsible for about 10–50% of the total bacterial mortality in surface waters, and 50–100% in environments that are unfriendly to protists (single-cell eukaryote, some of which graze on bacteria), such as low-oxygen lake waters (Fuhrman 1999).

Oral infection associated with Enterococci are common but are restricted to the root canal system of the teeth. Although they make up only a small proportion of the initial flora, Enterococci (Ent. faecalis specifically) are frequently isolated as a pure culture or as a major component of the flora from root canals where the endodontic treatment has failed (Peciuliene et al. 2000; Hancock et al. 2001). This is partially explained by the ability of Ent. faecalis to survive under unusual environmental stress, its extreme resistance to medications and irrigants used during endodontic treatment (Siqueira et al. 2000), and its ability to invade radicular dentinal tubules and remain viable (Love 2001). The relative high incidence (22%) of Ent. faecalis phages found in human saliva screened in this study suggests that phages might exhibit control over the oral Ent. faecalis population and restrict its outbreaks to the relatively isolated and protected location of the tooth root system.

Considering the increase in bacterial drug resistance (Ent. faecalis Jh2–2 included) (Fines et al. 1999), and the relatively easy access to the oral cavity, phage therapy (Nakai et al. 1999; Payne et al. 2000) as a way to prevent endodontic treatment failure might be considered.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Oral bacterial strains
  6. Saliva collection
  7. Phage stability in human saliva
  8. Phage isolation from human saliva
  9. Electron microscopy
  10. Results
  11. Phage stability in human saliva
  12. Phage isolation from human saliva
  13. Phage properties
  14. Discussion
  15. Acknowledgements
  16. References
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