A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius

Fibrillar and fimbriate strains of Streptococcus salivarius were compared for their ability to adhere to buccal epithelial cells and saliva-coated hydroxyapatite beads, and for their ability to coaggregate with Veillonella strains. The fibrillar Lancefield group K strains adhered statistically significantly better to both buccal epithelial cells and saliva-coated hydroxyapatite beads than the fimbriate strains, which lacked the Lancefield group K antigen. After 1 h the fibrillar strains coaggregated statistically significantly better than the fimbriate strains with V. parvula strain V1, but after 24 h, coaggregation both of fibrillar and of fimbriate strains reached approximately 90%. Freshly isolated Veillonella strains all coaggregated with the S. salivarius strains, but the percentage coaggregation varied considerably after 1 h depending on the Veillonella strain. Coaggregation was independent of the presence of Ca2+. S. salivarius strain HB-V5, a mutant of strain HB that had lost the Veillonella-binding protein, coaggregated weakly with V. parvula strain V1, but coaggregated very well with other wild-type veillonellae, suggesting the presence of an alternative mechanism for Veillonella-binding for strain HB. Fibrillar strains were, therefore, more adhesive to oral surfaces and coaggregated with veillonellae after 1 h better than the fimbriate S. salivarius strains. Both fibrillar and fimbriate strains were highly hydrophobic in the hexadecane-buffer partition assay.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology.

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Complete genome sequence of Acetohalobium arabaticum type strain (Z-7288)

Veillonella parvula (Veillon and Zuber 1898) Prévot 1933 is the type species of the genus Veillonella in the family Veillonellaceae within the order Clostridiales. The species V. parvula is of interest because it is frequently isolated from dental plaque in the human oral cavity and can cause opportunistic infections. The species is strictly anaerobic and grows as small cocci which usually occur in pairs. Veillonellae are characterized by their unusual metabolism which is centered on the activity of the enzyme methylmalonyl-CoA decarboxylase. Strain Te3(T), the type strain of the species, was isolated from the human intestinal tract. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the large clostridial family Veillonellaceae, and the 2,132,142 bp long single replicon genome with its 1,859 protein-coding and 61 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

Acetate kinase in the genus Veillonella: effect of succinate, serological cross-reactivity, and separation by electrophoresis

Acetate kinases from the genus Veillonella were divided into two types: a succinate-stimulated enzyme and a succinate-independent enzyme. Three strains, V. parvula ATCC 17743 (antigenic group II), V. parvula ATCC 17744 (V), and V. parvula ATCC 10790 (VI), contained the succinate-stimulated enzyme. Among four types strains of V. alcalescens, three strains, ATCC 17747 (I), ATCC 17746 (III), and ATCC 17748 (VII), contained the succinate-independent enzyme, whereas only one strain, ATCC 17745 (IV), contained the succinate-stimulated enzyme. Small amounts of antiserum to the purified acetate kinase from V. alcalescens ATCC 17748 completely inhibited the purified and crude enzyme activity from the strain. Classification of the enzymes on the basis of stimulation by succinate was consistent with classification based on serological reactions using the antiserum as an independent parameter. The succinate-stimulated enzyme could be separated into two classes according to the degree of sensitivity to succinate: (i) enzymes from V. parvula ATCC 17744 and V. alcalescens ATCC 17745, which could be demonstrated on gel after electrophoresis by a histochemical method to be highly stimulated by the presence of succinate in the reaction mixture, and (ii) enzymes from V. parvula ATCC 10790 and V. parvula ATCC 17743, which could be easily demonstrated without succinate. Four groups of acetate kinases from the genus Veillonella were separated by gel electrophoretic mobility. The results showed that almost all enzymes from the seven type strains were heterogeneous at the molecular level.     

Polysaccharide-lipid complexes from Veillonella parvula

Mergenhagen, Stephan E. (National Institutes of Health, Bethesda, Md.). Polysaccharide-lipid complexes from Veillonella parvula. J. Bacteriol. 90:1730-1734. 1965.-A strain of Veillonella parvula (V2) elaborates an extracellular slime when grown in a nutrient medium containing only dialyzable components. Deproteinization with chloroform-butanol of ethyl alcohol-precipitated material from the supernatant culture fluid leads to the isolation of a water-soluble lipopolysaccharide (LPS1). Another component (LPS2), showing similarity in biological and immunological properties to the endotoxic antigen (LPC) isolated from whole cells, was extracted with phenol from the insoluble emulsion remaining after chloroform-butanol extraction of slime. Analysis of polysaccharides by thin-layer chromatography demonstrated the presence of glucose and galactose in LPS1 and glucose, glucosamine, galactosamine, and a methyl pentose in LPC. LPS1 failed to give a positive epinephrine skin test after intravenous injection in rabbits and failed to kill pertussis-sensitized mice, whereas LPS2 and LPC were active in both of these bioassays. Both lipopolysaccharides (LPS1 and LPC) exhibited type-specific haptenic activity in hemagglutination tests with numerous anti-Veillonella rabbit sera. LPS1 was found in these tests to be unrelated to a heterologous strain of Veillonella possessing a related somatic antigen. These experiments reveal the presence of two chemically and immunologically distinguishable polysaccharide-lipid complexes in this strain of V. parvula.     

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 onStreptococcus oralis 34,Streptococcus oralis C104, andStreptococcus SM PK509. Fifty-nine spontaneously-occurring coaggregation-defective (Cog^–) mutants ofS. gordonii DL1 unable to coaggregate with partner streptococci were isolated. Six representative Cog^– mutants were characterized by their coaggregation properties with fourActinomyces naeslundii strains (T14V, PK947, PK606, PK984),Veillonella atypica PK1910, andPropionibacterium acnes PK93. The six representative Cog^– mutants showed altered coaggregation with their streptococcal partners,A. naeslundii PK947, andP. acnes PK93. Based on the coaggregation phenotypes of these mutants, a model for the lactose-inhibitable coaggregation betweenS. gordonii DL1 and its partner bacteria is proposed. The potential use of these mutants in studies of oral biofilms is discussed.     

Chemically defined media for growing anaerobic bacteria of the genus Veillonella

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirement for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition.No specific requirements for single amino acids were observed. A combination of l-cysteine, dl-aspartic acid, l-glutamic acid, l-serine and l-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source.Decarboxylation of l-ornithine, l-lysine and l-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, l-lysine, l-ornithine and l-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate l-ornithine while forming putrescine.     

Lactate Metabolism by Veillonella parvula

A strain of Veillonella parvula M4, which grows readily in lactate broth without a requirement for carbon dioxide, has been isolated from the oral cavity. Anaerobic, washed cells of this organism fermented sodium lactate to the following products (moles/100 moles of lactate): propionate, 66; acetate, 40; carbon dioxide, 40; and hydrogen, 14. Cells grew readily in tryptone-yeast extract broth with pyruvate, oxaloacetate, malate, and fumarate, but poorly with succinate. The fermentation of pyruvate, oxaloacetate, or lactate plus oxaloacetate by washed cells resulted in the formation of propionate and acetate in ratios significantly lower than those observed with lactate as the sole carbon source. This was primarily due to increased acetate production. Cell-free extracts were unable to degrade lactate but metabolized lactate in the presence of oxaloacetate, indicating the presence of malic-lactic transhydrogenase in this organism. Lactic dehydrogenase activity was not observed. Evidence is presented for oxaloacetate decarboxylase and malic dehydrogenase activities in extracts.     

Phospholipid biosynthesis in some anaerobic bacteria.

We have identified and characterized enzymes of phospholipid synthesis in two plasmalogen-rich anaerobes. Megasphaera elsdenii and Veillonella parvula, and one anaerobe lacking plasmalogens. Desulfovibrio vulgaris. All three species contained phosphatidate cytidylyltransferase and phosphatidylserine synthase. Phosphatidylglycerophosphate synthesis was detected only D. vulgaris extracts. Phosphatidylserine (diacyl form) was the major product of the phosphatidylserine synthase assay with particles from M. elsdenii or V. parvula. The amounts of phosphatidylethanolamine formed were very low. Only D. vulgaris particles had an active phosphatidylserine decarboxylase.     

Lipid-phase transitions of the strictly anaerobic bacteria Veillonella parvula and Anaerovibrio lipolytica.

As a basis for physicochemical studies on the membranes of the strictly anaerobic bacteria Veillonella parvula, Anaerovibrio lipolytica, and Megasphaera elsdenii, the fatty acyl and alk-1-enyl moieties on the phosphoglycerides of these organism were characterized. Uncommon is the high proportion of a heptadecenoic acyl and alk-1-enyl moiety in these three lactate-fermenting bacteria. In contrast to V. parvula and A. lipolytica, M. elsdenii contains high amounts of branched-chain acyl and alk-1-enyl moieties. Freeze-etching electron microscopy showed that the lipids of the plasma membranes of V. parvula and A. lipolytica go from the liquid crystalline to the gel state upon lowering of the temperature, indicating that the membrane lipids are predominantly in the fluid state. No lipid-protein segregation could be detected in the plasma membrane of M. elsdenii. This can be explained by the abundance of branched-chain fatty acyl and alk-1-enyl residues in the membranes of this organism which may prevent lipid-protein segregation during the lipid-phase transition.     

Putrescine and cadaverine are constituents of peptidoglycan in Veillonella alcalescens and Veillonella parvula.

Veillonella alcalescens ATCC 17745, a strictly anaerobic, gram-negative small coccus, requires putrescine or cadaverine for growth (M. B. Ritchey, and E. A. Delwiche, J. Bacteriol. 124:1213-1219, 1975). Both putrescine and cadaverine were demonstrated to be incorporated exclusively into the peptidoglycan layer of V. alcalescens ATCC 17745. V. parvula GAI 0574 also proved to contain putrescine as a component of peptidoglycan. The primary chemical structure of the peptidoglycan common to the two Veillonella species is N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid gamma-meso-diaminopimelic acid-D-alanine. Putrescine or cadaverine links covalently to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan is necessary for normal cell growth. In V. alcalescens ATCC 17745, above 40% saturation at cadaverine linked to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan is necessary for normal growth.     

Cell wall composition and incorporation of radio-labelled compounds by Veillonella alcalescens.

The cell wall of Veillonella alcalescens was shown to have a typically Gram-negative appearance and composition. The wall contains 24% lipid, 0.8% phosphorus, and 6.8% hexosamine. It is estimated to contain about 5% murein, unlike the 24% reported by other for Veillonella parvula. The amounts of 19 amino acids, including diaminopimelic acid, were determined. Though Veillonella sp. cannot metabolize sugars for energy, V. alcalescens incorporates ribose and fructose by separate, specific mechanisms and uses most of the incorporated sugar in nucleic acid synthesis. Large excesses of either sugar in the medium do not repress gluconeogenesis from the pyruvate level. We have been unable to detect phosphoglyceromutase (EC 2.7.5.3) by several assay methods but have no indication of a gluconeogenic pathway other than reverse glycolysis.     

Interaction between Streptococcus spp. and Veillonella tobetsuensis in the Early Stages of Oral Biofilm Formation

Dental plaque is a multispecies oral biofilm, the development of which is initiated by adherence of the pioneer Streptococcus spp. Oral Veillonella spp., including V. atypica, V. denticariosi, V. dispar, V. parvula, V. rogosae, and V. tobetsuensis, are known as early colonizers in oral biofilm formation. These species have been reported to coaggregate with Streptococcus spp. in a metabolic cooperation-dependent manner to form biofilms in human oral cavities, especially in the early stages of biofilm formation. However, in our previous study, Streptococcus gordonii showed biofilm formation to the greatest extent in the presence of V. tobetsuensis, without coaggregation between species. These results suggest that V. tobetsuensis produces signaling molecules that promote the proliferation of S. gordonii in biofilm formation. It is well known in many bacterial species that the quorum-sensing (QS) system regulates diverse functions such as biofilm formation. However, little is known about the QS system with autoinducers (AIs) with respect to Veillonella and Streptococcus spp. Recently, autoinducer 1 (AI-1) and AI-2 were detected and identified in the culture supernatants of V. tobetsuensis as strong signaling molecules in biofilm formation with S. gordonii. In particular, the supernatant from V. tobetsuensis showed the highest AI-2 activity among 6 oral Veillonella species, indicating that AIs, mainly AI-2, produced by V. tobetsuensis may be important factors and may facilitate biofilm formation of S. gordonii. Clarifying the mechanism that underlies the QS system between S. gordonii and V. tobetsuensis may lead to the development of novel methods for the prevention of oral infectious diseases caused by oral biofilms.     

成人牙周炎龈下套氧菌分离鉴定及药敏试验结果分析

分离并鉴定了329例成人牙周炎龈下优势厌氧菌群,并对不同病程中的菌群变迁、厌氧菌的药物敏感性进行了分析.成人牙周炎龈下标本中厌氧菌阳性检出率为97.9%,其中以牙龈紫质单胞菌检出率最高(38.5%),具核梭杆菌次之(18.9%).随着牙周病变程度的加重,牙龈紫质单胞菌、具核梭杆菌、产黑色素普氏菌、星群厌氧链球菌、厌氧消化链球菌的检出率增高(P＜0.05),小韦荣球菌的检出率下降(P＜0.01),表明前5种厌氧菌在AP发病过程中有重要作用,小韦荣球菌与之无关.替硝唑、甲硝唑和克林霉素对438株革兰氏阴性厌氧菌的MIC90分别为1～8,2～8和4～16 mg/L,对278株革兰氏阳性厌氧菌的MIC90分别为16～32,16～64和4～16 mg/L,表明替硝唑和甲硝唑体外抗革兰氏阴性厌氧菌效果优于克林霉素,抗革兰氏阳性厌氧菌作用不如克林霉素.     

Synergism between Ultrasonic Waves and Hydrogen Peroxide in the Killing of Micro-organisms

The lethal effect on different micro-organisms of ultrasonic waves and hydrogen peroxide separately and in combination was examined. Ultrasonic waves were able to disintegrate Fusobacterium nucleatum within 3 min and to kill Veillonella parvula after 15 min and Streptoccus sanguis after 20 min; 20 vols H₂O₂ (6% w/v) killed V. parvula, Strep. sanguis and Staphylococcus aureus after 5 min treatment, and Clostridium sporogenes spores after 25 min. Sonication of Cl. sporogenes spores, Bacillus cereus spores and Candida albicans in 20 vols H₂O₂, using an ultrasonic probe, was lethal to the organisms after 15, 10 and 10 min, respectively. The latter 2 organisms were not killed by 30 min exposure to either agent separately. Similar results were obtained when an ultrasonic tank was used for sonication.     

Central Role of the Early Colonizer Veillonella sp. in Establishing Multispecies Biofilm Communities with Initial,Middle, and Late Colonizers of Enamel

Human dental biofilm communities comprise several species, which can interact cooperatively or competitively. Bacterial interactions influence biofilm formation, metabolic changes, and physiological function of the community. Lactic acid, a common metabolite of oral bacteria, was measured in the flow cell effluent of one-, two- and three-species communities growing on saliva as the sole nutritional source. We investigated single-species and multispecies colonization by using known initial, early, middle, and late colonizers of enamel. Fluorescent-antibody staining and image analysis were used to quantify the biomass in saliva-fed flow cells. Of six species tested, only the initial colonizer Actinomyces oris exhibited significant growth. The initial colonizer Streptococcus oralis produced lactic acid but showed no significant growth. The early colonizer Veillonella sp. utilized lactic acid in two- and three-species biofilm communities. The biovolumes of all two-species biofilms increased when Veillonella sp. was present as one of the partners, indicating that this early colonizer promotes mutualistic community development. All three-species combinations exhibited enhanced growth except one, i.e., A. oris, Veillonella sp., and the middle colonizer Porphyromonas gingivalis, indicating specificity among three-species communities. Further specificity was seen when Fusobacterium nucleatum (a middle colonizer), Aggregatibacter actinomycetemcomitans (a late colonizer), and P. gingivalis did not grow with S. oralis in two-species biofilms, but inclusion of Veillonella sp. resulted in growth of all three-species combinations. We propose that commensal veillonellae use lactic acid for growth in saliva and that they communicate metabolically with initial, early, middle, and late colonizers to establish multispecies communities on enamel.The human oral cavity contains a widely diverse community of resident bacteria composed of several hundred species (1, 18). They organize into multispecies communities through a recurrent sequence of colonization that occurs after each oral hygiene treatment; for example, dental plaque development on enamel starts with the initial colonizers streptococci and actinomyces (7, 15), which are followed by early-colonizing veillonellae (7, 11, 14), middle-colonizing porphyromonads (7) and fusobacteria (7, 10, 11), and late-colonizing aggregatibacters (9).During the initial stage of biofilm formation, streptococci and actinomyces bind to host-derived receptors in the salivary pellicle coating of enamel. In turn, other species bind to already-adherent cells, a process called coadhesion (2). This process and coaggregation (10), defined as specific cell-to-cell recognition between genetically distinct cells, as well as growth of adherent cells contribute to dental plaque development. While it is known that pure cultures of oral bacteria metabolize dietary sugars to lactic acid, little is known about the importance of lactic acid to community growth on saliva as a sole nutrient source. Most pure cultures and many combinations of species are unable to grow on whole saliva, which is a complex nutritional source. Growth might, in fact, require spatial organization and mutualistic interactions among selected species that collectively possess a combination of metabolic properties that are capable of converting latent nutrition into usable nutrition. In succession, groups of other selected species with other combined metabolic capabilities can further process this complex nutritional source, with a resultant assembling and disassembling of constantly changing oral biofilm communities.Streptococci make up 60 to 90% of the supragingival plaque biomass in the first 24 h of colonization (12, 15). They catabolize carbohydrates to short-chain organic acids, such as lactic acid and pyruvic acid (4). Veillonellae constitute as much as 5% of the initial plaque biomass but are unable to catabolize sugars. They rely on the fermentation of organic acids such as lactic acid (6) and thus set up a convenient metabolic food chain in dental plaque.In vivo studies using gnotobiotic rats demonstrated that veillonellae were unable to establish monoinfections. Yet when a strain of Veillonella was inoculated into rats already monoinfected with a strain of Streptococcus mutans that coaggregates with that Veillonella strain, the number of veillonellae on the teeth of the coinfected animals was 1,000-fold higher than the number when a noncoaggregating Veillonella strain was used (13). Also, in gnotobiotic rats, lower caries and plaque scores were obtained for two-species biofilms than for single-species colonization by streptococci, and inclusion of veillonellae reduced caries activity and demineralization of the enamel by streptococci (13). Streptococcus-Veillonella communities containing coaggregation partners were micromanipulated from 8-h human dental plaque, providing additional evidence of the close association of these two species in vivo (3). Further, Veillonella spp. are juxtaposed with coaggregation receptor polysaccharide-bearing streptococci in early communities in vivo, and a rapid succession of veillonella phylotypes occurs in these communities (16). These reports offer broad-based evidence that veillonellae and streptococci are linked in oral biofilms.The focus of the current investigation was to explore Veillonella-based mixed-species communities in saliva-fed flow cells. The concentration of lactic acid in the effluent of flow cells containing biofilm communities was determined. We hypothesize that spatiotemporal metabolic interactions and coaggregation of Veillonella sp. with Streptococcus oralis and early, middle, and late colonizers allow these organisms to form three-species biofilm communities. We show high specificity of community partnerships among the six species examined, suggesting that successions of species in naturally recurring dental plaque in vivo are centered on metabolic and physical interactions of the community participants which support the nonrandom sequential appearance of species in the development of oral biofilms.