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.     

Impact of Early Colonizers on In Vitro Subgingival Biofilm Formation

The aim of this study was to investigate the impact of early colonizing species on the structure and the composition of the bacterial community developing in a subgingival 10-species biofilm model system. The model included Streptococcus oralis, Streptococcus anginosus, Actinomycesoris, Fusobacterium nucleatum subsp. nucleatum, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Based on literature, we considered Streptococcus oralis, Streptococcus anginosus, and Actinomyces oris as early colonizers and examined their role in the biofilms by either a delayed addition to the consortium, or by not inoculating at all the biofilms with these species. We quantitatively evaluated the resulting biofilms by real-time quantitative PCR and further compared the structures using confocal laser scanning microscopy following fluorescence in situ hybridisation. The absence of the early colonizers did not hinder biofilm formation. The biofilms reached the same total counts and developed to normal thickness. However, quantitative shifts in the abundances of individual species were observed. In the absence of streptococci, the overall biofilm structure appeared looser and more dispersed. Moreover, besides a significant increase of P. intermedia and a decrease of P. gingivalis , P. intermedia appeared to form filamented long chains that resembled streptococci. A. oris, although growing to significantly higher abundance in absence of streptococci, did not have a visible impact on the biofilms. Hence, in the absence of the early colonizers, there is a pronounced effect on P. intermedia and P. gingivalis that may cause distinct shifts in the structure of the biofilm. Streptococci possibly facilitate the establishment of P. gingivalis into subgingival biofilms, while in their absence P. intermedia became more dominant and forms elongated chains.     

Peptostreptococcus micros coaggregates with Fusobacterium nucleatum and non-encapsulated Porphyromonas gingivalis

Coaggregation is one of the potential colonization strategies of oral microorganisms, often involving fimbrial structures in the interactions. In this study, the coaggregation characteristics of the rough and smooth genotypes of the periodontal pathogen Peptostreptococcus micros were compared to investigate the role of the fibril-like structures of the rough genotype in coaggregation. Of the 11 oral species tested, only Fusobacterium nucleatum strains and non-encapsulated Porphyromonas gingivalis strains coaggregated with P. micros. No differences in coaggregation between the smooth type (Sm), the rough type (Rg) and the smooth variant of the Rg type (Rg(Sm)) of P. micros were observed. Heat-stable, periodate-sensitive structures on P. micros appeared to interact with heat- and protease-sensitive structures on F. nucleatum and P. gingivalis. These data indicate that these unimodal coaggregations are not mediated by the proteinaceous fibril-like structures of the Rg genotype, but by carbohydrates present on both genotypes of P. micros.     

Peptostreptococcus micros coaggregates with Fusobacterium nucleatum and non-encapsulated Porphyromonas gingivalis

Numerous in vitro studies have demonstrated that Staphylococcus aureus may be internalized and survive in a bovine mammary epithelial cell line. We report here the presence of internalized and living S. aureus in alveolar cells and macrophages in milk samples of bovine mastitis. We used fluorochrome labeled monoclonal antibodies, specifically recognizing surface cell markers of bovine alveolar cells and macrophages, to isolate these two types of cells using fluorescence activated cell sorting. Extracellular bacteria and DNA were previously eliminated to exclude possible contamination. In order to detect intracellular bacterial DNA inside the isolated cells, we used PCR amplification of bacterial DNA and the PCR products were analyzed by Southern blot with a specific probe for Staphylococcus. The results showed the presence of Staphylococcus DNA inside the two isolated populations of cells, confirming that S. aureus could penetrate alveolar cells and macrophages. The demonstration of the presence of intracellular living S. aureus was determined by bacteriological culture of positive samples plated onto blood agar plates and by its further identification. Our results showed for the first time that living S. aureus and its DNA are present in both alveolar cells and macrophages in chronically infected cow milk.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

Evaluation of co-aggregation among Streptococcus mitis, Fusobacterium nucleatum and Porphyromonas gingivalis

AIMS: To develop a semi-quantitative method for evaluating co-aggregation reactions among three bacterial species, and to examine the influence of Fusobacterium nucleatum on the adherence of Porphyromonas gingivalis. METHODS AND RESULTS: The method involves coating hydroxyapatite (HAP) discs with streptococcal cells and treatment with radio-labelled bacterial cell suspensions. The sensitivity of the method was estimated by comparison with a turbidometric co-aggregation assay. Results from the two methods were in close agreement. Streptococcus mitis-coated HAP discs were immersed in a 3H-labelled Fus. nucleatum cell suspension and then a 14C-labelled P. gingivalis cell suspension. The discs were then pyrolysed to recover and quantify the released 3H and 14C radioactivity. The number of Fus. nucleatum cells on the discs increased with immersion time and this, in turn, resulted in elevated adherence of P. gingivalis. CONCLUSION: The data indicate that the method closely reflects co-aggregation characters, and that Fus. nucleatum has a positive effect on the adherence of P. gingivalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present method, which is designed to mimic the oral environment, should prove useful in the semi-quantitative evaluation of co-aggregation reactions.     

Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP(+) during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.     

Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis

A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases.     

Purification and characterization of hemolysin from Porphyromonas gingivalis A7436

Porphyromonas gingivalis, a periodontal pathogen, has the ability to lyse erythrocytes. The hemolytic activity of P. gingivalis A7436 was purified as a 45-kDa protein from the culture supernatant of a 3-days old culture using nickel-nitrilotriacetic acid chromatography. Erythrocytes treated with purified P. gingivalis hemolysin showed the presence of pores and extracellular debris by scanning electron microscopy. Active immunization of mice with 15 micrograms hemolysin induced neutralizing antibodies to hemolysin. Heating at 60 degrees C and treatment with trypsin and dithiothreitol abolished hemolytic activity, while incubation with the protease inhibitor Na-p-tosyl-L-lysine chloromethyl ketone caused no effect. We report here for the first time purification of a hemolysin from P. gingivalis A7436. The amino acid sequence of an internal peptide of hemolysin showed sequence similarity with fimbrillin from P. gingivalis HG564. However, the amino acid composition of purified hemolysin was different from that of P. gingivalis fimbrillin. Also, the ability to lyse but not agglutinate erythrocytes and to bind to nickel-nitrilotriacetic acid differentiates P. gingivalis hemolysin from fimbrillin.     

Interaction of adrenomedullin and calcitonin gene-related peptide with the periodontal pathogen Porphyromonas gingivalis

The nature of the interaction between Porphyromonas gingivalis and the multifunctional peptides adrenomedullin and calcitonin gene-related peptide (CGRP) was investigated. Growth of P. gingivalis was not inhibited in the presence of either of these peptides [minimal inhibitory concentration (MIC)>250 microg mL(-1)]. The ability of the arginine- and lysine-specific proteases from P. gingivalis to breakdown these peptides was investigated. Adrenomedullin and CGRP were incubated with culture supernatants from wild-type and protease gene knockout strains. No significant effect on antimicrobial activity against the indicator organism Escherichia coli BUE55 was found (MIC=6.25 microg mL(-1) in all cases). The role of anionic components on the surface of P. gingivalis, which may alter binding of these cationic peptides, was also investigated in relation to adrenomedullin. Growth of gene knockout strains lacking surface polysaccharide and capsule components was not inhibited (MIC>250 microg mL(-1)). It is suggested that a lack of sensitivity to adrenomedullin and CGRP may enable P. gingivalis to persist in the oral cavity and cause disease.     

Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens

Abstract The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via a high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species ( P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis , is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had orginated from healthy or diseased oral sites.     

Monitoring the uptake of protein-derived peptides by Porphyromonas gingivalis with fluorophore-labeled substrates

The aim of this study was to develop a simple method to quantify peptide uptake by the periodontopathogenic bacterium Porphyromonas gingivalis. After incubation of bacterial cells with self-quenched fluorescent bovine serum albumin (DQ Green BSA), the fluorescence measured in the supernatant of the assay mixture indicated the degree of protein degradation, whereas the fluorescence associated with the lysate of washed cells indicated the amount of BSA-derived fragments incorporated by the bacteria. The optimal conditions for uptake of fluorophore-labeled albumin fragments were found to be mid-log grown cells, 150 m M NaCl in phosphate buffer, pH 7, 37 degrees C, and anaerobiosis. Among the protease inhibitors tested, 4-(2-aminoethyl)-benzene sulfonyl-fluoride hydrochloride (AEBSF) and cathepsin B inhibitor II caused a significant inhibition of the uptake of BSA-derived peptides. This assay was applicable for other commercially available fluorescent substrates. This simple method may be useful to investigate protein processing in proteolytic bacteria and for studying the effects of environmental parameters or cell treatments on the peptide uptake.