Bacteriophages induced from lysogenic root canal isolates of Enterococcus faecalis.

INTRODUCTION: Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections. METHODS: Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis. RESULTS: Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of NsiI and NdeI DNA fragments from one of the Siphoviridae phage isolates (phage phiEf11) indicated a genome size of approximately 41 kbp. CONCLUSION: This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.

Use of bacteriophage-resistant mutants to study Actinomyces viscosus cell surface receptors.

Spontaneously occurring bacteriophage-resistant mutants of Actinomyces viscosus were isolated. The mutants exhibited altered coaggregation patterns with streptococci. From analysis of the data, it appears that a cell surface structure on A. viscosus may function both as a phage receptor and as a binding site for co-aggregation with certain oral streptococci. These mutants will be valuable in the study of surface structures that mediate one type of the cell-to-cell interactions that occur between these two important groups of oral bacteria.

Pulmonary actinomycosis of periodontal origin.

A case of pulmonary actinomycosis of periodontal origin is presented. Microbiologic testing including culture and typing of multiple systemic and oral lesions revealed the presence of Actinomyces naeslundii and A viscosus in all sites. In addition, biopsies demonstrating branching filamentous forms and "sulfur granules" were found in exudates from the lungs, submandibular region and left quadrants of the oral cavity. Seeding of tooth-associated materials containing Actinomyces sp into the pulmonary field may have resulted in this case of pulmonary actinomycosis. Therapy of the pulmonary and periodontal infections was completed and the patient is maintaining satisfactory health 18 months later.

Properties of mutacin b, an antibacterial substance produced by Streptococcus mutans strain BHT.

An antibacterial substance produced by strain BHT of Streptococcus mutans (mutacin b) was found to be a small molecule (MW 3,500-6,000) with remarkable resistance to temperature, alkali and various solvents. Enzyme sensitivity tests of partially purified preparations indicated that mutacin b is a peptide. It is sensitive to several proteolytic enzymes and its lethal effects on sensitive cells can be prevented by adding trypsin to cells exposed to mutacin b. High concentrations of mutacin b inhibited the growth of producer cells, indicating that strain BHT is only partially immune to this substance.

Production of bacteriocins in a liquid medium by Streptococcus mutans.

A sterile-filtered, liquid medium composed of one-half-strength APT broth and 4% (wt/vol) yeast extract was found to support the production of bacteriocins by Streptococcus mutans strains BHT and GS-5. Culture supernatants, adjusted to pH 7.0 and sterilized by filtration, contained bacteriocin-like activity, which could be demonstrated by spotting dilutions onto top agar lawns seeded with Streptococcus pyogenes as the sensitive indicator and by adding dilutions to log-phase indicator broth cultures. A quantitative assay was developed for BHT bacteriocin, based on its lethal effects. Bacteriocin production did not occur until after the log phase of growth had ceased and was not inducible by ultraviolet irradiation or treatment with mitomycin C. Non-bacteriocinogenic clones of strain BHT occurred spontaneously at high frequency, suggesting control by a plasmid, but this frequency was not increased by treatment with the plasmid-curing agents acridine orange and ethidium bromide.

Activity of two Streptococcus mutans bacteriocins in the presence of saliva, levan, and dextran.

The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology of the oral cavity.

Inducible bacteriophages of Actinobacillus actinomycetemcomitans.

Doses of 0.1 to 1.0 micrograms/ml of mitomycin C induced cell lysis of six of eight strains of Actinobacillus actinomycetemcomitans tested. Infectious phages were induced from ATCC strains 43717, 29524, 33384, and 43719; non-plaque-forming, possibly defective phages were induced from ATCC strains 29522 and 29523. No phages were detected in strain FDC 651 or ATCC strain 43718. No correlation between lysogeny and leukotoxin production or serotype of the strains could be established. Gel electrophoresis of phage DNAs indicated that the induced phages were of three types, based on size. By electron microscopy, the phages were found to belong to either morphotype A1 or morphotype B1; no other morphotypes were observed. Curing experiments led to the isolation of nonlysogenic derivatives of two strains, which supported plaque formation by the phages they originally carried. On the basis of our results, lysogeny appears to be widespread in A. actinomycetemcomitans.

Lytic bacteriophages ofStreptococcus mutans.

Three phages ofStreptococcus mutans were obtained and partially characterized. The three phages, designated M102, e10, and f1, were found to be strictly lytic, with host ranges restricted to only serotype c, e, and f strains of this species, respectively. Phage sensitivity was not correlated with the presence of plasmids, at least in host strains of serotypes c and e. Each phage produced clear plaques in a number of standard media, even in the presence of sucrose, indicating that the extracellular glucan polysaccharides (mutan) produced by the hosts from this substrate do not prevent phage adsorption and growth. The phages were similar in size and morphology, having icosahedral heads and long (283-287 nm), flexible, noncontractile tails. The genome of each phage was found to consist of linear, double-stranded DNA, 31-35 kb in length, with a base composition of 37-38% G+C. Restricting phage DNAs with four enzymes produced fragment patterns unique to each phage, but common bands between M102 and e10 and between e10 and f1 were produced byBamHI. Labeled e10 and M102 DNAs hybridized strongly with all three phage DNAs, indicating that they share some common sequences. The three phages appear to be more similar than expected and probably evolved from a common ancestor.

Isolation of a bacteriophage for actinomyces viscosus.

A lytic phage which produces clear plaques on a human isolate of Actinomyces viscosus was isolated from a sample of raw domestic sewage.

Use of lytic bacteriophage for Actinomyces viscosus T14V as a probe for cell surface components mediating intergeneric coaggregation.

A lytic bacteriophage for Actinomyces viscosus T14V (the reference strain for actinomyces coaggregation group A) was isolated from raw sewage. This phage, designated BF307, also lysed the T14V-derived nonfimbriated mutant PK455-2 as well as A. viscosus MG-1 and T14AV but not the other serotype 2 or serotype 1 strains of this species that were tested or any of nine Actinomyces naeslundii isolates. Phages BF307 belonged to Bradley morphological group C and was similar in appearance to the A. viscosus MG-1 phages Av-1 and Av-3, which do not productively infect A. viscosus T14V. A. viscosus MG-1 mutants selected for resistance to phage BF307, Av-3, or CT7 (a human dental plaque isolate with the same host range as BF307) were coresistant to the other two phages but sensitive to Av-1. These results indicate that the receptors on A. viscosus MG-1 for phages BF307, Av-3, and CT7 are identical or share a common precursor and that the receptor for phage Av-1 is distinct. Comparison of the genomes of BF307, Av-3, and CT7 revealed that their DNAs were similar in size but distinguishable by restriction analysis. Two altered coaggregation phenotypes were identified among the phage BF307-resistant mutants of strains MG-1, T14V, T14AV, and PK455-2. Class I mutants had lost the ability to interact with coaggregation group 1 streptococci, and class II mutants did not coaggregate with either group 1 or group 2 streptococci. These results are consistent with the proposal that the phage BF307 receptor on these A. viscosus strains is related to one of the structures that mediates coaggregation with oral streptococci. A model to delineate the various coaggregation mediators on the surface of actinomyces coaggregation group A cells is presented, and the use of these phages to probe surface components of human oral actinomyces strains is discussed.

Simultaneous loss of bacteriophage receptor and coaggregation mediator activities in Actinomyces viscosus MG-1.

Actinomyces bacteriophages were used as tools to study coaggregation between actinomyces and streptococci. Four bacteriophage isolates, phages AV-1, AV-2, AV-3, and 1281, bound to coaggregation group A Actinomyces viscosus and to group E A. naeslundii. No binding to groups B, C, D, or F was observed. Only A. viscosus MG-1 was capable of supporting a productive infection by these phages. Spontaneously occurring bacteriophage-resistant mutants of A. viscosus MG-1 were isolated and were shown to fall into two classes. Class I mutants were resistant to all four phages, whereas class II mutants were resistant only to phage AV-3. In each case, strains resistant to a particular phage were unable to bind that phage, suggesting that a loss or alteration of the cell surface phage receptor had occurred. Both classes of mutants were unable to coaggregate with streptococci representing coaggregation group 1 and had also lost the ability to mediate one type of coaggregation with group 4 streptococci. Class II mutants also were unable to coaggregate with group 2 streptococci. Lactose-inhibitable interactions with other streptococci (groups 3 and 4) were unchanged in the mutants. The simultaneous loss of sensitivity to phage AV-3 and the ability to coaggregate with coaggregation group 1 streptococci suggests the possibility of a relationship between these two cell surface structures.