Strains Of Actinomyces Naeslundii Research Articles

Isolation of novel <i>Actinomyces oris</i> and <i>Aggregatibacter actinomycetemcomitans</i> bacteriophages and study of their biological characteristics <i>in vitro</i>

SCIENTIFIC RELEVANCE. The incidence of periodontal infections with antibiotic-resistant strains of Actinomyces oris and Aggregatibacter actinomycetemcomitans capable of forming biofilms in the oral cavity is increasing, and the effectiveness of antimicrobials against biofilms is insufficient. Therefore, the isolation of bacteriophages active against A. oris and Ag. actinomycetemcomitans is an urgent task.AIM. This study aimed to isolate bacteriophages active against A. oris and Ag. actinomycetemcomitans, study their biological properties, and select optimum culture conditions providing pure lines and high titres of bacteriophages.MATERIALS AND METHODS. Bacteriophages were isolated from biosamples of saliva, dental plaque, and periodontal pocket contents. The study determined bacteriophage characteristics, including the optimum multiplicity of infection, phage–cell system cultivation time, resistance to various temperatures and pH, and storage stability. Bacteriophage morphology was studied using transmission electron microscopy. Negative colony morphology, lytic activity, host range, and specificity were assessed using spot testing and the Gratia method. Bacterial strains were obtained from the strain collection of the Micromir research and production centre.RESULTS. The authors isolated and studied 3 novel bacteriophages active against A. oris (vB_AorP_1/G-12, vB_AorP_2/Ch-28, and vB_AorP_3/Bl-35) and 1 bacteriophage active against Ag. actinomycetemcomitans (vB_AacS_1/Dc-1). The A. oris bacteriophages were classified as podoviruses, and the vB_AacS_1/Dc-1 bacteriophage was classified as a siphovirus. The phages formed completely transparent round plaques without a halo, with a diameter ranging from 0.8±0.1 to 4.0±0.2 mm. The optimum parameters established to obtain pure phages with maximum titres included a multiplicity of infection of 0,1–10 and phage–cell system cultivation time of 8–12 hours. The study demonstrated the ability of A. oris bacteriophages to lyse Actinomyces naeslundii strains. Of the 15 A. oris bacteriophage strains studied, vB_AorP_1/G-12, vB_AorP_2/Ch-28, and vB_AorP_3/Bl-35 lysed 10, 8, and 12 bacterial strains, respectively. The vB_AacS_1/Dc-1 phage isolate exhibited lytic activity against both tested strains of Ag. actinomycetemcomitans. The studied phages demonstrated stability under abiotic stress and long-term storage conditions.CONCLUSIONS. The authors isolated 3 novel bacteriophages active against A. oris and 1 bacteriophage active against Ag. actinomycetemcomitans and studied their biological properties. The isolated bacteriophages are promising as candidates for further research using clinical strains and whole-genome sequencing.

Genomic Diversity among Actinomyces naeslundii Strains and Closely Related Species

The aim of this study was to investigate and clarify the ambiguous taxonomy of Actinomyces naeslundii and its closely related species using state-of-the-art high-throughput sequencing techniques, and, furthermore, to determine whether sub-clusters identified within Actinomyces oris and Actinomyces naeslundii in a previous study by multi locus sequence typing (MLST) using concatenation of seven housekeeping genes should either be classified as subspecies or distinct species. The strains in this study were broadly classified under Actinomyces naeslundii group as A. naeslundii genospecies I and genospecies II. Based on MLST data analysis, these were further classified as A. oris and A. naeslundii. The whole genome sequencing of selected strains of A. oris (n = 17) and A. naeslundii (n = 19) was carried out using Illumina Genome Analyzer IIxe and Roche 454 allowing paired-end and single-reads sequencing, respectively. The sequences obtained were aligned using CLC Genomic workbench version 5.1 and annotated using RAST (Rapid Annotation using Subsystem Technology) release version 59 accessible online. Additionally, genomes of seven publicly available strains of Actinomyces (k20, MG1, c505, OT175, OT171, OT170, and A. johnsonii) were also included. Comparative genomic analysis (CGA) using Mauve, Progressive Mauve, gene-by-gene, Core, and Pan Genome, and finally Digital DNA-DNA homology (DDH) analysis was carried out. DDH values were obtained using in silico genome-genome comparison. Evolutionary analysis using ClonalFrame was also undertaken. The mutation and recombination events were compared using chi-square test among A. oris and A. naeslundii isolates (analysis methods are not included in the study). CGA results were consistent with previous traditional classification using MLST. It was found that strains of Actinomyces k20, MG1, c505, and OT175 clustered in A. oris group of isolates, while OT171, OT170, and A. johnsonii appeared as separate branches. Similar clustering to MLST was observed for other isolates. The mutation and recombination events were significantly higher in A. oris than A. naeslundii, highlighting the diversity of A. oris strains in the oral cavity. These findings suggest that A. oris forms six distinct groups, whereas A. naeslundii forms three. The correct designation of isolates will help in the identification of clinical Actinomyces isolates found in dental plaque. Easily accessible online genomic sequence data will also accelerate the investigation of the biochemical characterisation and pathogenesis of this important group of micro-organisms.

Sensitivity of caries pathogens to antimicrobial peptides related to caries risk.

Antimicrobial peptides (AMPs) represent important facets of the immune system controlling infectious diseases. However, pathogens show varying susceptibilities to AMPs. This study investigates the susceptibilities of strains of Streptococcus mutans (SM), Actinomyces naeslundii (AN), and Lactobacillus spp. (LB) towards AMPs and if there are correlations between the appearance of such high-risk strains and clinical caries status. Plaque samples were collected from patients along with clinical examinations. Bacterial strains were identified via selective media, matrix-assisted laser desorption/ionization analysis-time of flight (MALDI-TOF), and arbitrary-primed-PCR (AP-PCR). Each strain was tested for susceptibility to LL-37, HBD-2, HNP-1, and HNP-3 or phosphate-buffered saline as negative control in a biofilm model on hydroxylapatite discs. Survival rates and resulting risk classification for each strain were determined. Correlations were calculated between the number of high-risk strains (all/S. mutans) appearing in patients and their clinical caries status. Forty-seven patients were included with mean DMFT values of 11.4 ± 8.7. A total of 8 different SM, 30 LB, and 47 AN strains were detected. One-way ANOVA indicated that type/concentration of AMPs had major influence on reductions of Lactobacilli and Actinomyces. Seventeen strains of AN, 2 of SM, and 6 of LB had low susceptibilities to AMPs. The number of such strains in patients showed significant positive correlations to the DMFT values (all p = 0.001; r = 0.452; S. mutans p < 0.0001, r = 0.558). The occurrence of low susceptible strains to AMPs seems to correlate with the individual caries status. The results may lead to new ways to identify individuals with increased caries risk.

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP(I) polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP(I), were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N-terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.

Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU627, and with S. oralis C104 and 34, were dependent on expression of cshA and cshB. S. gordonii DL1 cells bound to immobilized human fibronectin (Fn), but not to soluble Fn, in a dose-dependent manner, and binding was noninhibitable by heparin. S. gordonii cshA and cshB mutants were also deficient in binding to immobilized human Fn. Antibodies to an NH2-terminal nonrepetitive region (amino acid residues 42 to 886) of recombinant CshA inhibited binding of S. gordonii DL1 cells to A. naeslundii T14V and PK606 and to immobilized Fn. Conversely, antibodies to an amino acid repeat block segment of the COOH-terminal domain (amino acid residues 2026 to 2508) were not inhibitory to adherence. Assays using CshA-specific antibodies revealed that surface expression of CshA was reduced in cshB mutants. The results suggest that CshA acts as a multifunctional adhesin in S. gordonii and that major adhesion-mediating sequences are specified within the nonrepetitive NH2-terminal region of the polypeptide.

The role of physicochemical and structural surface properties in co-adhesion of microbial pairs in a parallel-plate flow chamber

Biofilm formation on solid surfaces involves interactions between planktonic organisms and the substratum as well as between flowing and already adhering organisms (“co-adhesion”). Biochemical studies have yielded information on the structural cell surface properties involved in interspecies binding between planktonic cells (“coaggregation”). No data are available, however, on the physicochemical mechanisms governing coaggregation and co-adhesion. It was the aim of this study to characterize the physicochemical surface properties of four Actinomyces naeslundii strains and three streptococcal strains, representing a selection of coaggregating and non-coaggregating oral microbial pairs, by microelectrophoresis, contact angles and X-ray photoelectron spectroscopy. Coaggregation appeared to be governed, at least partially, by electrostatic interactions. Analysis of the deposition kinetics using a parallel-plate flow chamber showed that adhering actinomyces act as local sinks for flowing streptococci. The initial local deposition rates of streptococci co-adhering to actinomyces are far higher than those calculated on the basis of convective-diffusion, due to additional convective mass transport of streptococci to the adhering actinomyces protruding in the flow.

Fimbrial-mediated colonization of murine teeth by Actinomyces naeslundii.

Groups of mice fed diets high in sucrose or glucose were orally inoculated with 10(10), 10(9) or 10(8) colony-forming units of one of the following Actinomyces naeslundii strains possessing the type 1 (T1+) and/or the type 2 (T2+) fimbriae: T14VJ1 (T1+, T2+), 5519 (T1+), 5951 (T2+), and 147 (non-fimbriated). Ninety-six hours after inoculation their upper jaws were cultured to look at the implantation of each of these strains on the teeth. In mice fed a sucrose diet, regardless of the presence or absence of fimbriae, each bacterial strain colonized 100% of the mice at the highest inoculation doses of the infecting organism. But at a dose of 10(8), T14V-J1 was the only strain which colonized 100% (12/12) of the mice, 5519 colonized 10/11, 5951 colonized 9/11 and 147 colonized 7/11. These differences were not statistically significant. When mice were fed a high-glucose diet, 100% infection was achieved with strains T14V-J1, 5519 and 5951 only at the highest dose of 10(10) colony-forming units. Strain 147 colonized in 8/9 of the mice at that dosage. At lower dosages, no bacterial strain implanted in 100% of the mice. In the glucose experiment at a dose of 10(8), strains expressing the T1 fimbriae implanted significantly better than strains without the T1 fimbriae. At a dose of 10(9) colony-forming units, the parent strain T14V-J1 implanted significantly better than strains without the T1 fimbriae. Similarly, strain 5519 (T1+) implanted significantly better than 5951 and implanted better than 147, although the difference was not significant. These results suggest that while the presence of the T1 and T2 fimbriae may confer some advantage in the establishment of these organisms in vivo, even the strains without fimbriae were able to colonize. Strains T14VJ1 and 5519 were found to bind well to hydroxyapatite treated with mouse saliva, while strains 5951 and 147 did not. Only T2 fimbriated strains T14V-J1 and 5951 exhibited a lactose-reversible coaggreation with indigenous strains of enterococci that may contribute to the elevated levels of colonization of strain 5951 in vivo.

Catabolic pathway for aerobic degradation of lactate by Actinomyces naeslundii.

The aerobic metabolism of lactate by oral Actinomyces was studied. Six of 7 strains of Actinomyces naeslundii increased their growth in the presence of lactate under aerobic conditions. Washed cells grown on lactate aerobically degraded lactate and pyruvate to acetate with a concomitant consumption of oxygen. In the presence of catalase, the molar ratios of oxygen consumed to acetate produced were 1 for lactate degradation and 0.5 for pyruvate degradation. The enzymatic activities found in cell extracts revealed that lactate could be converted to pyruvate by NAD-independent lactate dehydrogenase (iLDH) and further to acetyl CoA by pyruvate dehydrogenase (PDH). The acetyl CoA formed could be metabolized into acetate by phosphotransacetylase (PTA) and acetate kinase (AK) with the formation of ATP. These results indicate that A. naeslundii metabolizes lactate into acetate by the sequential enzymatic reactions iLDH, PDH, PTA and AK and that hydrogens produced by iLDH and PDH are transferred to oxygen. The activity of lactate degradation and oxygen consumption may modify the environmental conditions of dental plaque.

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii, Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii, Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.

Isolation and characterization of coaggregation-defective (Cog−) mutants ofStreptococcus gordonii DL1 (Challis)

Streptococcus gordonii DL1 (Challis) bears coaggregation-mediating surface adhesins which recognize galactoside-containing surface polysaccharides on Streptococcus oralis 34, Streptococcus oralis C104, and Streptococcus SM PK509. Fifty-nine spontaneously-occurring coaggregation-defective (Cog-) mutants of S. gordonii DL1 unable to coaggregate with partner streptococci were isolated. Six representative Cog- mutants were characterized by their coaggregation properties with four Actinomyces naeslundii strains (T14V, PK947, PK606, PK984), Veillonella atypica PK1910, and Propionibacterium acnes PK93. The six representative Cog- mutants showed altered coaggregation with their streptococcal partners, A. naeslundii PK947, and P. acnes PK93. Based on the coaggregation phenotypes of these mutants, a model for the lactose-inhibitable coaggregation between S. gordonii DL1 and its partner bacteria is proposed. The potential use of these mutants in studies of oral biofilms is discussed.

Carbohydrate depletion of immunoglobulin A1 by oral species of gram‐positive rods

Bacterial deglycosylation of immunoglobulin Al (IgA1), the dominant isotype of antibody in the oral cavity, probably provides both nutrition as well as protection to the oral bacterial community. Representative strains of oral gram-positive rods were tested for their ability to remove carbohydrates from IgA1. Detection of sialic acids was performed by means of high-performance liquid chromatography (HPLC) separation (Aminex HPX-87H) and ultraviolet light absorption at 190 nm, and neutral carbohydrates were measured by HPLC separation (Capcell Pak C-18 SG 120) and ultraviolet light absorption at 245 nm after derivatization. Four strains of Actinomyces naeslundii, two strains of Corynebacterium matruchotii and one of two strains of Actinomyces odontolyticus partially or totally removed sialic acid, while two strains of Propionibacterium propionicus and the other strain of A. odontolyticus did not. Complete correlation was observed between sialic acid removal, neuraminidase activity measured with fluorogenic substrate and with one exception, altered immunoelectrophoretic mobility of IgA1. Only limited removal of other carbohydrates was observed with poor correlation to exoglycosidase activities measured with chromogenic substrates. Desialylation increases the susceptibility of glycoproteins, including IgA1, to proteolysis. Therefore, the desialylation of IgA1 by oral gram-positive rods may facilitate the proteolytic activities of other oral bacteria, and the concerted action may positively influence the survival of the bacteria in the oral community.

Inactivation of the gene encoding surface protein SspA in Streptococcus gordonii DL1 affects cell interactions with human salivary agglutinin and oral actinomyces

Cell surface protein SSP-5 in the oral bacterium Streptococcus gordonii M5 binds human salivary agglutinin in a Ca(2+)-dependent reaction (D.R. Demuth, E.E. Golub, and D. Malamud, J. Biol. Chem. 265:7120-7126, 1990). The region of the gene encoding an N-terminal segment of a related polypeptide (SspA) in S. gordonii DL1 (Challis) was isolated following polymerase chain reaction amplification of genomic DNA. The sspA gene in S. gordonii DL1 was insertionally inactivated by homologous recombination of the erythromycin resistance (Emr) determinant ermAM onto the streptococcal chromosome. The SspA polypeptide (apparent molecular mass, 210 kDa) was detected on Western blots (immunoblots) of spheroplast extracts and extracellular culture medium proteins from wild-type strain DL1 but was absent from Emr mutants. One SspA- mutant (designated OB220) was not altered in rate or extent of aggregation by whole saliva or parotid saliva but showed reduced aggregation in the presence of purified salivary agglutinin. Mutant bacteria were unaffected in their ability to adhere to hydroxylapatite beads coated with whole or parotid saliva and were unaltered in cell surface hydrophobicity. However, the SspA- strain OB220 was deficient in binding salivary agglutinin and in binding to six strains of Actinomyces naeslundii. Therefore, expression of SspA polypeptide in S. gordonii is associated with both agglutinin-dependent and agglutinin-independent aggregation and adherence reactions of streptococcal cells.

Interactions of Actinomyces naeslundii strains T14V and ATCC 12104 with saliva, collagen and fibrinogen

Approximately similar numbers of actinomyces cells adhered to hydroxylapatite beads coated with saliva, collagen or fibrinogen. Adherence generally was unaffected by the presence of free saliva. Binding of cells to collagen- or fibrinogen-coated beads was reduced in the presence of either free collagen or fibrinogen. Glucan inhibited bacterial adherence only to collagen-coated hdyroxylapatite beads. It is suggested that actinomyces bind to saliva-, collagen- or fibrinogen-coated surfaces by different mechanisms, but that these mechanisms involve some common bacterial cell-surface components.

A simple method for extracting human lymphocyte stimulants from human dental plaque bacteria of the genus actinomyces

The effect of acid concentration on the hot-acid extraction of antigens from Actinomyces viscosus that stimulate human peripheral blood lymphocytes in vitro was tested. The optimum pH for release of these antigens in a soluble low molecular-weight form was pH 2.3. Preliminary purification on Sepharose 4B demonstrated that most of the lympho-stimulatory material (about 90 per cent) had a mol. wt of about 100,000. This partially-purified antigenic material was mainly protein. The pH 2.3 hot-acid extraction was also suitable for extracting antigens that stimulate cellular immune responses from three strains of Actinomyces naeslundii and 1 strain of Actinomyces israelii, but did not release lympho-stimulatory material from Streptococcus sanguis which is a poor human peripheral-blood lymphocyte-stimulant and was used to demonstrate that the hot-acid extraction is not simply extracting a mitogenic macromolecule common to all bacteria. As good yields of soluble, biologically-active antigens were obtained from all of the Actinomyces, the procedure described will facilitate the purification and characterization of these antigens.

Isolation, partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10.

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.

Interbacterial aggregation of Actinomyces naeslundii and dental plaque streptococci.

The ability of human Actinomyces naeslundii strains to aggregate with dental plaque streptococci was found to be strain specific. A. naeslundii strains aggregated strongly more often with specific strains of Streptococcus sanguis and Streptococcus mitis than with Streptococcus mutans strains. The effects of proteolytic enzymes and heat on the interbacterial affinities of A. naeslundii 398A and S. sanguis S, two strains which were found to aggregate strongly, were studied. Enzyme or heat treatment of 398 A, but not S. sanguis S, impaired aggregation. Electron microscopic examination of aggregates of untreated bacteria revealed that the attachment between cells of strains 398 A and S was mediated by short tufts of electron‐dense fuzzy components of the cell surfaces. The affinity between cells of A. naeslundii and the streptococci which readily colonize cleaned smooth tooth surfaces may account, in part, for the previously‐reported, delayed increase in A. naeslundii proportions among bacteria forming early plaque deposits.