J.L. Streckfuss

Synthetic Medium for Calcification of Bacterionema matruchotii

A synthetic medium was developed for further study of intracellular calcification by Bacterionema matruchotii. The medium contained metastable calcium phosphate, six other salts, three purines, two pyrimidines, nine vitamins, pimelic and thioctic acids, vitamin-free casein hydrolysate, and glucose. The vehicle was 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer at pH 7.4.

Fluoride Resistance and Adherence of Selected Strains of Streptococcus mutans to Smooth Surfaces After Exposure to Fluoride

The fluoride resistance and smooth surface adherence characteristics of Streptococcus mutans were examined using tooth model and radioactive cell assays. Resistance to 600 ppmF by S. mutans isolated from the plaque of radiation-induced xerostomia patients receiving daily topical applications of a caries preventive 1% NaF gel was transient. Resistance induced in vitro in two strains of S. mutans by exposure to gradually increasing levels of NaF was apparently permanent. Smooth surface adherence by both fluoride-sensitive and -resistant strains of S. mutans 6715 in a tooth model system was slightly diminished by 1% NaF gel. Fluoride-resistant strains retained 89 to 93% of their adherence capability in 600 ppmF, as determined by the cell radiolabeling assay.

Effects of a Single Application of Sodium Fluoride Gel on Dental Plaque Acidogenesis

The quantities of lactate and acetate produced by human dental plaque were compared before and after five-minute exposures to 1% sodium fluoride gel. A single topical application of the gel increased plaque fluoride by about 700 ppm, 79% of which was lost after six h. Gel use significantly reduced plaque L(+)-lactate production in vitro, and the production of L(+)- and D(-)-lactate and acetate in situ.

Effect of Sodium Fluoride on the Viability and Growth of Streptococcus mutans

A fluoride-sensitive (FS) strain of Streptococcus mutans and a laboratory-induced fluoride-resistant (FR) offspring were compared for the effects of sodium fluoride on viability and growth. There was a significant fluoride-related loss of viability in resting cell suspensions of the FS strain during a 47-hour exposure to fluoride levels above 75 ppm that was not encountered with the FR strain. The addition of 300 ppmF to actively growing six-hour broth cultures almost totally arrested the growth of the FS strain, while only slightly reducing that of the FR culture. The addition of 600 ppmF immediately terminated FS growth, and greatly reduced the rate and maximum growth of FR cultures.

Calcifiability Comparison Among Selected Microorganisms

Seven reference-strain bacteria were examined for apatitic calcifiability in a chemically-defined medium and in a metastable calcium phosphate solution. The results support the belief that calcification is limited to certain microorganisms. Additionally, the findings emphasize that determining microbiologic calcifiability only in a calcification-supporting medium is too restrictive.

A calcifiable bacillary variant isolated from Bacterionema matruchotii, ATCC 14266.

Bacterionema matrncltotii, Richardson strain 13 and ATCC 14266, acquire intracellular hydroxyapatite during growth in a synthetic medium (ENNEVER, VOGEL, and STRECKFUSS, J Dent Res 50:1327-1330, 1971). Bacillary, RF7, and streptococcal, SI, variants of the Richardson strain calcify under the same conditions (ENN.EVIR, STRECKFuss, and TAKAZOE, J Deiti Res, in press). This study shows that a bacillary variant, 66R3. which was derived from aged colonies of ATCC 14266, also forms intracellular apatite. After five days of growth on brain-heart infusion (BHI) agar,* colonies of Strain 14266 were stored at 4 C for 56 days. The centers of several colonies were cannulated into Bill broth and they were incubated at 37 C. No growth appeared until the fifth day of incubation. Loop transfers to BHI agar produced colonies after one day of incubation. The colonies were composed of bacillary rather than the filamentous forms of B mnatruchotii. The bacillary form was retained as 66R3 and it has been stable for more than two years. It is morphologically and physiologically (unpublished results) similar to the RF of strain 13. L. Georg (personal communication), however, found that the two isolates were antigenically distinct. Variant 66R3 fluoresced 3+ to homologous fluorescent antibody conjugate (ATCC 14266), whereas RF7 failed to fluoresce. To test for calcification, 66R3 and a noncalcifying control, Actinoomyces naeslundii, West Virginia strain 45, each were grown in the synthetic medium of Ennever, Vogel, and Streckfuss. Incubation was at 37 C for seven days. The cells were harvested by centrifugation at 10,000 g for five minutes at 5 C and then they were washed with water. A part of each yield was air-dried and ashedt| at 55 C for two hours. The ashed residues were examined with X-ray diffraction by use of a powder camera film technique with nickel-filtered radiation at a 30

Calcification of Selected Strains of Streptococci

In 1960, Ennever reported that Bacterionema matruchotii forms intracellular calcium phosphate deposits with X-ray maxima essentially identical to those of calcium hydroxyapatite (Ennever, J Periodont 31:304, 1960). Since then, 14 other oral isolates from man and marmoset, including four enteric species, five serotypes of cariogenic Streptococcus mutans, and one strain of Streptococcus sanguis, have been found to calcify under similar experimental conditions (Ennever et al., J Dent Res 51:1483, 1972; Streckfuss et al., J Bacteriol 120:502, 1974). This study was undertaken to determine whether other oral streptococci also possess the capability of calcification (Brown et al., J Dent Res 52: 815, 1973; DHEW Pub No (NIH) 74-286, 1973). S. mitis ATCC 6249 and four laboratory stock strains of S. mitis S21, S23, S32 and S34 were examined, along with S. salivarius ATCC 13419, S. faecalis ATCC 8043 and S. bovis ATCC 9809. A non-calcifiable S. salivarius 125C was used as a control (Ennever et al., op. cit.). Each organism was grown in synthetic calcification medium for two weeks at 370C and processed for X-ray diffraction and electron microscopy as previously reported (Ennever etal., JDentRes 50:1327, 1971). The test isolates produced X-ray diffraction patterns characteristic of apatite with diffuse bands indicating small crystals. Electron micrographs confirmed the X-ray findings, and also disclosed dispersed extracellular deposits in S. mitis S21 and S. faecalis ATCC 8043, in addition to the

Effect of a Mutagenic Agent on Calcifiability of Bacterionema matruchotii

Intracellular hydroxyapatite formation by Bacterionema matruchotii is well-documented (J. ENNEVER and H. CREAMER, Calcif Tissue Res 1:87-93, 1967). The present work was undertaken in an attempt to convert the filamentous calcifiable organism to a noncalcifiable form by exposure to the mutagenic agent, acridine orange (AO). Exponential growth phase cells of B matruchotii (Richardson Strain 13) were recovered from brain-heart infusion broth* by centrifugation at 20,000 g for five minutes at 5 C. Approximately 3.6 x 108 colony-forming units were resuspended in 9 ml of brain-heart infusion broth containing 10 mg AO per milliliter and incubated in a water bath for one hour at 37 C. The treated cells were washed three times with sterile deionized water, diluted, and plated on brain-heart infusion agar. After three to five days at 37 C, single colonies composed of filaments were removed from the agar, washed three times with water, and inoculated into a synthetic medium that supports B matruchotii calcification (J. ENNEVER, J. VOGEL, and J. L. STRECKFUSS, J Dent Res 50:1327-1330, 1971). In addition, the washed cells were inoculated into heart infusion broth* that contained 1 % carbohydrate. Twelve different carbohydrates were tested. Cultivation was for seven days at 37 C. The cells for calcification were harvested by centrifugation, water-washed, and stored at -15 C. The major portion of each yield was radio-frequency-

Characterization of Calcifiable Proteolipid from Streptococcus mutans

Proteolipid is known to initiate calcification in vitro. Apoprotein and phospholipid components of proteolipid from five of 14 calcifiable S. mutans specimens were characterized. The apoproteins contained 16 amino acids with calculated percent polarities ranging from 32.0 to 45.2. The acidic phospholipids were cardiolipin, mono- and diphosphoinositides, and phosphatidylserine. Neutral lipids, phosphatidylethanolamine and phosphatidylcholine, were also found. The latter were the most abundant in all isolates. Appropriate hydrophobic proteins and acidic phospholipids in the proteolipids accounted for S. mutans calcifiability.

Characteristics of an Induced Variant of Bacterionema matruchotii

Bacterionema matruchotii, a calcifiable filamentous organism, was treatetl ultrasonically. The disrupted cells produced typical colonies that developed macroscopic globular structures. A nonfilamentous, pleomorphic variant was derived from the globules. The variant retained the calcification potential of the filament, as well as fermentation and ultrastructural similarities.