Vol. 20, No. 1
INFECTION AND) IMMUNITY, Apr. 1978, p. 303-306
0019-9567/78/0020-0303$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
NOTES Isolation of a Bacteriophage for Actinomyces viscosus A. L. DELISLE,* R. K. NAUMAN, and G. E. MINAH Department of Microbiology, University ofMaryland School of Dentistry, Baltimore, Maryland 21201
Received for publication 25 August 1977
A lytic phage which produces clear plaques on a human isolate of Actinomyces viscosus was isolated from a sample of raw domestic sewage.
Actinomyces viscosus and A. naeslundii are presently suspected of being the primary etiological agents of root surface dental caries and gingivitis in man (7-11, 13, 14, 16). Fluorescentantibody techniques have recently come into widespread use and have resulted in the separation of A. viscosus and A. naeslundii into two and four serological groups, respectively (12), but no satisfactory methods of identifying specific strains of oral actinomycetes are presently available. Such methods are needed to identify and more precisely define the etiological roles of specific bacteria in the development of human periodontal diseases. Since bacteriophages can be very useful for such purposes, we attempted to isolate phages for human oral strains of A. viscosus. In this communication, we report the first successful isolation of such a virus. (Presented 12 May 1977 at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La.) All laboratory strains of A. viscosus and A. naeslundii used in this study were obtained from J. London, National Institute of Dental Research, NIH, Bethesda, Md., and included the following: A. viscosus strains M100, W1053, W859, W1528, W1628, W1537, A828, T6-1600, R28, and 626; and A. naeslundii strains W826, W1096, W752, W1527, WVU45, WVU820, WVU852, WVU398, N16, and 1544. The phage host strain of A. viscosus, MG-1, was isolated in this laboratory as follows. A sample of human gingival plaque from a patient with mild gingivitis (Navy periodontal disease index score of 3) (6) was removed with a sterile explorer and sonically treated for 3 s in 1 ml of sterile reduced transport fluid (15). Dilutions of this suspension were then spread on MM10 sucrose blood agar (15) and incubated in a vinyl anaerobic chamber under an atmosphere of 85% N2-10% H2-5% CO2 (2). One well-isolated A. viscosus-like colony was picked and purified by repeated restreaking on CNAC-20 agar (5).
This organism was found to be a gram-positive, catalase-positive, diphtheroid-shaped rod which grew well, both aerobically and anaerobically, on Trypticase soy agar (BBL Laboratories) and CNAC-20 media. The morphology and biochemical properties of this organism, designated MG-1, were then compared with the wellcharacterized A. viscosus laboratory strains M100 and W1053. The morphology of MG-1 (Fig. 1) was identical to that of the other strains of this species, and all three strains fermented sucrose, glycerol, raffinose, and inositol (12). To further confirm the identity of strain MG-1, fluorescent-antibody tests were performed, with the result that MG-1 gave a 4+ staining reaction only with labeled antiserum specific for serotype 2 of A. viscosus. These tests were kindly performed by J. London and P. Marucha, National Institute of Dental Research, NIH. All cultures were grown in the lactose-based, high-calcium, glycerol phosphate-buffered M17 medium of Terzaghi and Sandine (17), and 5 ml of a late-log-phase culture was added to 25 ml of freshly collected raw domestic sewage. Additional enrichment cultures were prepared with saliva and dental plaque as potential sources of phage. After 24 h at 37°C, the enrichment cultures were centrifuged for 5 min at 10,000 x g, and the supernatant fluids were sterile filtered. Top agar overlays of each culture (1) were prepared, using M17 medium for both top and bottom agar layers, and, after solidifying, were spotted with a drop from the respective enrichment filtrate. After incubation for 24 h in a candle jar, a large area of lysis was observed in a lawn of MG-1 spotted with one sewage enrichment filtrate. A small piece of top agar in this clear zone was picked and suspended in 1 ml of M17 broth, and serial dilutions were plated with MG-1 as the host. Isolated plaques were obtained, which were then picked and purified by replating three times. The phage thus obtained was designated Av-1. 303
304
INFECT. IMMUN.
NOTES
1 FIG. 1. Electron micrograph of host MG-i, negatively stained with sodium phosphotungstate. Bar = 0.5
gum.
HO U R S
FIG. 2. Plaque production by phage Av-i viscosus MG-I.
on
A.
Phage Av-1 produces clear plaques, 2 to 4 mm in diameter, on host strain MG-1 (Fig. 2). Subsequent to its isolation, the phage was found to plate with high efficiency in Trypticase soy top agar; the complex M17 medium is thus not necessary for phage propagation. Plaques were also produced in brain heart infusion medium (which is frequently used to cultivate Actinomyces), but with a reduced efficiency (efficiency of plating = 0.1). Growth of phage Av-1 in liquid culture (Fig. 3) routinely yielded lysates with phage titers of
FIG. 3. Growth of phage Av-i in liquid culture. Host strain MG-1 was grown in Trypticase soy broth to an optical density at 660 nm of 0.4 (corresponding, to a viable cell count of 1.6 x 108 colony-forming units per ml) and then infected (arrow) with phage Av-i at a multiplicity of infection of approximately 10. Circles = infected culture; squares = noninfected control culture.
1010 to 101 plaque-forming units per ml. Electron micrographs of phage-infected MG-1 cells, showed an intracellular, predominantly polar accumulation of complete phage particles within 2 h of infection (Fig. 4); at later times (Fig. 5), virus particles appeared to be released from only' one end of each infected cell. Electron microscopic observations of negatively stained preparations of CsCl-purified Av-1 showed that it belonged to Bradley's (3) morphological group C (Fig. 6). The phage had a small polyhedral
VOL. 20, 1978
NOTES
305
4 FIG. 4. Electron micrograph of host MG-i, 2 hpostinfection with phage Av-l. Arrow indicates intracellular phage. Negatively stained with sodium phosphotungstate. Bar = 0.5 ,m.
FIG. 5. Electron micrograph of an MG-i cell releasing phage Av-1. Negatively stained with sodium phosphotungstate. Bar = 0.5 um. head measuring 40 nm in diameter and a short different media were used. The host range of phage Av-1 thus appeared to be quite narrow. tail 26 nm in length. To the best of our knowledge, this commuSerial dilutions of phage Av-1 were plated with 10 other strains of A. viscosus and 10 strains nication is the first report of a phage infectious of A. naeslundii, but in no case was evidence of for any Actinomyces species. The extremely narlysis or growth inhibition obtained, even when row host range of phage Av-1 suggests that
306
INFECT. IMMUN.
NOTES i *S~f'ie.
.
.
7IN
6 FIG. 6. Electron micrograph of a mixture of purified suspensions of bacteriophage A and Av- 1. Arrows indicate phage Av-1. Negatively stained with sodium phosphotungstate. Bar = 0.1 ,mn.
identification of specific strains of Actinomyces by phage typing may be possible, providing other phages can be isolated. Whether bacteriophages such as Av-1 play any in vivo role in the establishment or maintenance of actinomycetes in periodontal tissues remains to be determined. LITERATURE CITED 1. Adams, M. H. 1959. Bacteriophages. Interscience Publishers, New York. 2. Aranki, A., S. A. Syed, E. B. Kenny, and R. Freter. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Apple. Microbiol. 17:568-576. 3. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 4. Ellen, R. P. 1976. Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity. Infect. Immun. 14:1119-1124. 5. Ellen, R. P., and I. B. Balcerzek-Raczkowski. 1975. Differential medium for detecting dental plaque bacteria resembling Actinomyces viscosus and Actinomyces naeslundii. J. Clin. Microbiol. 2:305-310. 6. Grossman, F. D., and P. F. Fedi. 1974. The Navy periodontal screening examination. J. Am. Soc. Prev. Dent. 3(6):41-47. 7. Howell, A., Jr. 1963. A filamentous organism isolated from periodontal plaque in hamsters. I. Isolation, morphology and general cultural characteristics. Sabouraudia 3:81-92. 8. Jordan, H. V., and B. F. Hammond. 1972. Filamentous
bacteria isolated from human root surface caries. Arch. Oral Biol. 17:1333-1342. 9. Jordan, H. V., and P. H. Keyes. 1964. Aerobic, grampositive, filamentous bacteria as etiological agents of experimental periodontal disease in hamsters. Arch. Oral Biol. 9:401-414. 10. Jordan, H. V., P. H. Keyes, and S. Bellack. 1972. Periodontal lesions in hamsters and gnotobiotic rats infected with Actinomyces of human origin. J. Periodont. Res. 7:21-28. 11. Keyes, P. H., and H. V. Jordan. 1964. Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Arch. Oral Biol. 9:377-400. 12. Slack, J. M., and M. A. Gerencser. 1975. Actinomyces, filamentous bacteria: biology and pathogenicity. Burgess Publishing Co., Minneapolis, Minn. 13. Socransky, S. S., C. Hubersak, and D. Propar. 1970. Induction of periodontal destruction in gnotobiotic rats by a human oral strain of Actinomyces naeslundii. Arch. Oral. Biol. 15:993-995. 14. Sumney, D. L., and H. V. Jordan. 1974. Characterization of bacteria isolated from human root surface carious lesions. J. Dent. Res. 53:343-351. 15. Syed, S. A., and W. J. Loesche. 1972. Survival of human dental plaque flora in various transport media. Appl. Microbiol. 24:638-644. 16. Syed, S. A., W. J. Loesche, H. L. Pape, Jr., and E. Greiner. 1975. Predominant cultivable flora isolated from human root surface caries plaque. Infect. Immun. 11:727-731. 17. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813.
INFECTION AND) IMMUNITY, Apr. 1978, p. 303-306
0019-9567/78/0020-0303$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
NOTES Isolation of a Bacteriophage for Actinomyces viscosus A. L. DELISLE,* R. K. NAUMAN, and G. E. MINAH Department of Microbiology, University ofMaryland School of Dentistry, Baltimore, Maryland 21201
Received for publication 25 August 1977
A lytic phage which produces clear plaques on a human isolate of Actinomyces viscosus was isolated from a sample of raw domestic sewage.
Actinomyces viscosus and A. naeslundii are presently suspected of being the primary etiological agents of root surface dental caries and gingivitis in man (7-11, 13, 14, 16). Fluorescentantibody techniques have recently come into widespread use and have resulted in the separation of A. viscosus and A. naeslundii into two and four serological groups, respectively (12), but no satisfactory methods of identifying specific strains of oral actinomycetes are presently available. Such methods are needed to identify and more precisely define the etiological roles of specific bacteria in the development of human periodontal diseases. Since bacteriophages can be very useful for such purposes, we attempted to isolate phages for human oral strains of A. viscosus. In this communication, we report the first successful isolation of such a virus. (Presented 12 May 1977 at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La.) All laboratory strains of A. viscosus and A. naeslundii used in this study were obtained from J. London, National Institute of Dental Research, NIH, Bethesda, Md., and included the following: A. viscosus strains M100, W1053, W859, W1528, W1628, W1537, A828, T6-1600, R28, and 626; and A. naeslundii strains W826, W1096, W752, W1527, WVU45, WVU820, WVU852, WVU398, N16, and 1544. The phage host strain of A. viscosus, MG-1, was isolated in this laboratory as follows. A sample of human gingival plaque from a patient with mild gingivitis (Navy periodontal disease index score of 3) (6) was removed with a sterile explorer and sonically treated for 3 s in 1 ml of sterile reduced transport fluid (15). Dilutions of this suspension were then spread on MM10 sucrose blood agar (15) and incubated in a vinyl anaerobic chamber under an atmosphere of 85% N2-10% H2-5% CO2 (2). One well-isolated A. viscosus-like colony was picked and purified by repeated restreaking on CNAC-20 agar (5).
This organism was found to be a gram-positive, catalase-positive, diphtheroid-shaped rod which grew well, both aerobically and anaerobically, on Trypticase soy agar (BBL Laboratories) and CNAC-20 media. The morphology and biochemical properties of this organism, designated MG-1, were then compared with the wellcharacterized A. viscosus laboratory strains M100 and W1053. The morphology of MG-1 (Fig. 1) was identical to that of the other strains of this species, and all three strains fermented sucrose, glycerol, raffinose, and inositol (12). To further confirm the identity of strain MG-1, fluorescent-antibody tests were performed, with the result that MG-1 gave a 4+ staining reaction only with labeled antiserum specific for serotype 2 of A. viscosus. These tests were kindly performed by J. London and P. Marucha, National Institute of Dental Research, NIH. All cultures were grown in the lactose-based, high-calcium, glycerol phosphate-buffered M17 medium of Terzaghi and Sandine (17), and 5 ml of a late-log-phase culture was added to 25 ml of freshly collected raw domestic sewage. Additional enrichment cultures were prepared with saliva and dental plaque as potential sources of phage. After 24 h at 37°C, the enrichment cultures were centrifuged for 5 min at 10,000 x g, and the supernatant fluids were sterile filtered. Top agar overlays of each culture (1) were prepared, using M17 medium for both top and bottom agar layers, and, after solidifying, were spotted with a drop from the respective enrichment filtrate. After incubation for 24 h in a candle jar, a large area of lysis was observed in a lawn of MG-1 spotted with one sewage enrichment filtrate. A small piece of top agar in this clear zone was picked and suspended in 1 ml of M17 broth, and serial dilutions were plated with MG-1 as the host. Isolated plaques were obtained, which were then picked and purified by replating three times. The phage thus obtained was designated Av-1. 303
304
INFECT. IMMUN.
NOTES
1 FIG. 1. Electron micrograph of host MG-i, negatively stained with sodium phosphotungstate. Bar = 0.5
gum.
HO U R S
FIG. 2. Plaque production by phage Av-i viscosus MG-I.
on
A.
Phage Av-1 produces clear plaques, 2 to 4 mm in diameter, on host strain MG-1 (Fig. 2). Subsequent to its isolation, the phage was found to plate with high efficiency in Trypticase soy top agar; the complex M17 medium is thus not necessary for phage propagation. Plaques were also produced in brain heart infusion medium (which is frequently used to cultivate Actinomyces), but with a reduced efficiency (efficiency of plating = 0.1). Growth of phage Av-1 in liquid culture (Fig. 3) routinely yielded lysates with phage titers of
FIG. 3. Growth of phage Av-i in liquid culture. Host strain MG-1 was grown in Trypticase soy broth to an optical density at 660 nm of 0.4 (corresponding, to a viable cell count of 1.6 x 108 colony-forming units per ml) and then infected (arrow) with phage Av-i at a multiplicity of infection of approximately 10. Circles = infected culture; squares = noninfected control culture.
1010 to 101 plaque-forming units per ml. Electron micrographs of phage-infected MG-1 cells, showed an intracellular, predominantly polar accumulation of complete phage particles within 2 h of infection (Fig. 4); at later times (Fig. 5), virus particles appeared to be released from only' one end of each infected cell. Electron microscopic observations of negatively stained preparations of CsCl-purified Av-1 showed that it belonged to Bradley's (3) morphological group C (Fig. 6). The phage had a small polyhedral
VOL. 20, 1978
NOTES
305
4 FIG. 4. Electron micrograph of host MG-i, 2 hpostinfection with phage Av-l. Arrow indicates intracellular phage. Negatively stained with sodium phosphotungstate. Bar = 0.5 ,m.
FIG. 5. Electron micrograph of an MG-i cell releasing phage Av-1. Negatively stained with sodium phosphotungstate. Bar = 0.5 um. head measuring 40 nm in diameter and a short different media were used. The host range of phage Av-1 thus appeared to be quite narrow. tail 26 nm in length. To the best of our knowledge, this commuSerial dilutions of phage Av-1 were plated with 10 other strains of A. viscosus and 10 strains nication is the first report of a phage infectious of A. naeslundii, but in no case was evidence of for any Actinomyces species. The extremely narlysis or growth inhibition obtained, even when row host range of phage Av-1 suggests that
306
INFECT. IMMUN.
NOTES i *S~f'ie.
.
.
7IN
6 FIG. 6. Electron micrograph of a mixture of purified suspensions of bacteriophage A and Av- 1. Arrows indicate phage Av-1. Negatively stained with sodium phosphotungstate. Bar = 0.1 ,mn.
identification of specific strains of Actinomyces by phage typing may be possible, providing other phages can be isolated. Whether bacteriophages such as Av-1 play any in vivo role in the establishment or maintenance of actinomycetes in periodontal tissues remains to be determined. LITERATURE CITED 1. Adams, M. H. 1959. Bacteriophages. Interscience Publishers, New York. 2. Aranki, A., S. A. Syed, E. B. Kenny, and R. Freter. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Apple. Microbiol. 17:568-576. 3. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 4. Ellen, R. P. 1976. Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity. Infect. Immun. 14:1119-1124. 5. Ellen, R. P., and I. B. Balcerzek-Raczkowski. 1975. Differential medium for detecting dental plaque bacteria resembling Actinomyces viscosus and Actinomyces naeslundii. J. Clin. Microbiol. 2:305-310. 6. Grossman, F. D., and P. F. Fedi. 1974. The Navy periodontal screening examination. J. Am. Soc. Prev. Dent. 3(6):41-47. 7. Howell, A., Jr. 1963. A filamentous organism isolated from periodontal plaque in hamsters. I. Isolation, morphology and general cultural characteristics. Sabouraudia 3:81-92. 8. Jordan, H. V., and B. F. Hammond. 1972. Filamentous
bacteria isolated from human root surface caries. Arch. Oral Biol. 17:1333-1342. 9. Jordan, H. V., and P. H. Keyes. 1964. Aerobic, grampositive, filamentous bacteria as etiological agents of experimental periodontal disease in hamsters. Arch. Oral Biol. 9:401-414. 10. Jordan, H. V., P. H. Keyes, and S. Bellack. 1972. Periodontal lesions in hamsters and gnotobiotic rats infected with Actinomyces of human origin. J. Periodont. Res. 7:21-28. 11. Keyes, P. H., and H. V. Jordan. 1964. Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Arch. Oral Biol. 9:377-400. 12. Slack, J. M., and M. A. Gerencser. 1975. Actinomyces, filamentous bacteria: biology and pathogenicity. Burgess Publishing Co., Minneapolis, Minn. 13. Socransky, S. S., C. Hubersak, and D. Propar. 1970. Induction of periodontal destruction in gnotobiotic rats by a human oral strain of Actinomyces naeslundii. Arch. Oral. Biol. 15:993-995. 14. Sumney, D. L., and H. V. Jordan. 1974. Characterization of bacteria isolated from human root surface carious lesions. J. Dent. Res. 53:343-351. 15. Syed, S. A., and W. J. Loesche. 1972. Survival of human dental plaque flora in various transport media. Appl. Microbiol. 24:638-644. 16. Syed, S. A., W. J. Loesche, H. L. Pape, Jr., and E. Greiner. 1975. Predominant cultivable flora isolated from human root surface caries plaque. Infect. Immun. 11:727-731. 17. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813.
