Plaque formation in vitro by Actinomyces viscosus in the presence of Streptococcus sanguis or Streptococcus mutans.

Actinomyces viscosus, growing on a tooth in the presence of sucrose, slowly produced a loosely-attached plaque, the pH being 6.1 after 120 h. When the tooth was inoculated simultaneously with A. viscosus and either Streptococcus sanguis or Streptococcus mutans, firmly-adherent plaque was quickly formed and the pH fell below 5 after 33 h with the former Streptococcus and 24 h with the latter. A. viscosus disappeared from each mixed plaque by 120 h.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

The role of fructans on dental biofilm formation by Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus

Dental plaque biofilm plays a pivotal role in the progression of dental diseases. Polysaccharides are of great importance in the ecology of the dental biofilm. We studied the effect of fructans, glucans and a mixture of both fructans and glucans, synthesized in situ by immobilized fructosyltransferase or glucosyltransferase, on the adhesion of Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus to hydroxyapatite beads coated with human saliva (sHA). The adhesion of A. viscosus to sHA was found to be fructan-dependent. Adhesion of both S. sobrinus and S. mutans was found to be mediated mainly by glucans, while the adhesion of S. gordonii was found to be both glucan- and fructan-dependent. Treatment with fructanase prior to A. viscosus adhesion resulted in a significant reduction in adhesion to sHA, while adhesion of S. sobrinus, S. mutans and S. gordonii was slightly influenced by fructanase treatment. Treatment with fructanase after adhesion of S. gordonii to sHA resulted in a significant reduction in their adhesion to sHA. Our results show that fructans may play a role in the adhesion and colonization of several cariogenic bacteria to sHA, thus contributing to the formation of dental plaque biofilm.     

Experimental study of interactions between purple and green sulfur bacteria in sandy sediments exposed to illumination deprived of near-infrared wavelengths

Sedimentary biofilms of the green sulfur bacterium Prosthecochloris aestuarii strain CE 2404, the purple sulfur bacterium Thiocapsa roseopersicina strain 5811, and a mixed culture of both were cultured in fine sand (100- to 300-microm grain size) within counter gradients of oxygen and sulfide. The artificial sediments were exposed to illumination deprived of near-infrared light (NIR) by filtering out the wavelengths longer than 700 nm to simulate the critical light conditions in submerged aquatic sediments. A 16 h of visible light-8 h of dark regimen was used. We studied the effects of these light conditions on the metabolisms of and interactions between both species by comparing the single species biofilms with the mixed biofilm. The photosynthesis rates of P. aestuarii were shown to be highly limited by the imposed light conditions, because the sulfide photooxidation rates were strongly stimulated when NIR was added. T. roseopersicina performed both aerobic chemosynthesis and photosynthesis, but the photosynthesis rates were low and poorly stimulated by the addition of NIR. This species decreased the penetration depth of oxygen in the sediment by about 1 mm by actively respiring oxygen. This way, the strict anaerobe P. aestuarii was able to grow closer to the surface in the mixed culture. As a result, P. aestuarii benefited from the presence of T. roseopersicina in the mixed culture, which was reflected by an increase in the biomass. In contrast, the density of the latter species was almost completely unaffected by the interaction. Both species coexisted in a layer of the same depth in the mixed culture, and the ecological and evolutionary implications of coexistence are discussed.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

Predicting stability of mixed microbial cultures from single species experiments: 1. Phenomenological model

The growth of mixed microbial cultures on mixtures of substrates is a fundamental problem of both theoretical and practical interest. On the one hand, the literature is abundant with experimental studies of mixed-substrate phenomena [T. Egli, The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates, Adv. Microbiol. Ecol. 14 (1995) 305-386]. On the other hand, a number of mathematical models of mixed-substrate growth have been analyzed in the last three decades. These models typically assume specific kinetic expressions for substrate uptake and biomass growth rates and their predictions are formulated in terms of parameters of the model. In this work, we formulate and analyze a general mathematical model of mixed microbial growth on mixtures of substitutable substrates. Using this model, we study the effect of mutual inhibition of substrate uptake rates on the stability of the equilibria of the model. Specifically, we address the following question: How much of the dynamics exhibited by two competing species can be inferred from single species data? We provide geometric criteria for stability of various types of equilibria corresponding to non-competitive exclusion, competitive exclusion, and coexistence of two competing species in terms of growth isoclines and consumption curves. A growth isocline is a curve in the plane of substrate concentrations corresponding to the zero net growth of a given species. In [G.T. Reeves, A. Narang, S.S. Pilyugin, Growth of mixed cultures on mixtures of substitutable substrates: The operating diagram for a structured model, J. Theor. Biol. 226 (2004) 143-157], we introduced consumption curves as sets of all possible combinations of substrate concentrations corresponding to balanced growth of a single microbial species. Both types of curves can be obtained in single species experiments.     

Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen-limiting conditions

Abstract Defined mixed cultures of an obligately aerobic Pseudomonas testosteroni and anaerobic Veillonella alcalescens strain were grown under oxygen and lactate limitation in chemostats with different oxygen supply rates. The aerobic and the anaerobic bacteria were shown to coexist and to complete for common substrates over a wide range of oxygen supply rates. Under similar conditions but with formate as the major substrate chemostat enrichments gave rise to undefined mixed cultures of aerobic, fermentative and methanogenic bacteria. The relevance of these observations to natural mineralization processes is discussed.     

Experimental Study of Interactions between Purple and Green Sulfur Bacteria in Sandy Sediments Exposed to Illumination Deprived of Near-Infrared Wavelengths

Sedimentary biofilms of the green sulfur bacterium Prosthecochloris aestuarii strain CE 2404, the purple sulfur bacterium Thiocapsa roseopersicina strain 5811, and a mixed culture of both were cultured in fine sand (100- to 300-μm grain size) within counter gradients of oxygen and sulfide. The artificial sediments were exposed to illumination deprived of near-infrared light (NIR) by filtering out the wavelengths longer than 700 nm to simulate the critical light conditions in submerged aquatic sediments. A 16 h of visible light-8 h of dark regimen was used. We studied the effects of these light conditions on the metabolisms of and interactions between both species by comparing the single species biofilms with the mixed biofilm. The photosynthesis rates of P. aestuarii were shown to be highly limited by the imposed light conditions, because the sulfide photooxidation rates were strongly stimulated when NIR was added. T. roseopersicina performed both aerobic chemosynthesis and photosynthesis, but the photosynthesis rates were low and poorly stimulated by the addition of NIR. This species decreased the penetration depth of oxygen in the sediment by about 1 mm by actively respiring oxygen. This way, the strict anaerobe P. aestuarii was able to grow closer to the surface in the mixed culture. As a result, P. aestuarii benefited from the presence of T. roseopersicina in the mixed culture, which was reflected by an increase in the biomass. In contrast, the density of the latter species was almost completely unaffected by the interaction. Both species coexisted in a layer of the same depth in the mixed culture, and the ecological and evolutionary implications of coexistence are discussed.     

The significance of the asymmetric effect of crowding for coexistence in a mixed temperate forest

Abstract. The coexistence of coniferous (mostly Abies homolepis) and broad-leaved tree species (mostly Fagus crenata) in a mixed temperate old-growth forest in Japan was simulated by a size-structure dynamics model incorporating the asymmetrical (one-sided) effect of shading between these two life-form guilds. The model assumes that the crowding effect due to one-sided competition for light on a tree of a given size regulates the rate of size growth and recruitment. The cumulative basal area of trees larger than a given tree in the forest is employed to express the intensity of one-sided competition on that tree. Cumulative basal areas of both guilds negatively affected the growth rate of any tree. The shading effect by conifers on the growth rate of either guild was stronger than that by broad-leaved species. Two types of model were tested for recruitment; an additive and a reciprocal model. A reciprocal model, where basal area density of conifers and broad-leaved species has a negative effect on the recruitment of its own guild but has a positive effect on that of the other guild, fit the observed data better than an additive model where total basal area of the two guilds suppresses recruitment rates. Simulations using these models showed that, within a particular range of the set of recruitment rates, the two guilds could coexist. The tendency for reciprocal replacement, incorporated in the reciprocal model, substantially widened the range of coexistence and shortened the time required for convergence.     

An empirical model of the sympatric coexistence of two strains of the endoparasitoid wasp Venturia canescens

Recent research has demonstrated that a laboratory culture of the asexual solitary endoparasitoid wasp Venturia canescens Grav. (Hymenoptera: Ichneumonidae) contains two genetically and phenotypically distinct lines, coexisting on their host the flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). The basis to the coexistence of the two lines appears to be differences in their reproductive success under single parasitism and superparasitism. Furthermore, examination of field-derived wasps from several locations has shown that the phenotypes displayed by the laboratory colonies also co-occur in field populations. Historically, the impossibility of showing that two species do not occupy separate niches has precluded any demonstration of sympatric coexistence in the field. Here we present the results of an iterative model that uses a range of experimental life history data to predict the stable composition of a mixed population of two lines displaying the laboratory phenotypes under different rates of superparasitism. The model predicts that sympatric coexistence of the two lines is possible when the overall rate of superparasitism is between 4 and 12% or greater. These values are within the rates reported for other solitary endoparasitoid wasp species in the field, and so demonstrate that the sympatric coexistence under natural conditions of two species that display the phenotypes observed in the laboratory lines is, in principle, possible.     

Competition for mixed substrates by microbial populations

A model for the growth of an organism on multiple substrates was developed, assuming that each substrate has a competitive inhibition effect on the uptake of other substrates. The model was extended to examine mixed substrates, showing that the coexistence of several species at steady state in continuous cultures is possible, even when all the organisms all strongly prefer the one substrate. The diversity of nutrient sources in a real system may be a key factor in supporting a heterogeneous microbial population.     

Lectin-like constituents of foods which react with components of serum, saliva, and Streptococcus mutans

Hot and cold aqueous extracts were prepared from 22 commonly ingested fruits, vegetables, and seeds. When tested by agar diffusion, extracts from 13 and 10 of the foods formed precipitin bands with samples of normal rabbit serum and human saliva, respectively; extracts from four of the foods also reacted with antigen extracts of strains of Streptococcus mutans. When added to rabbit antiserum, extracts from 18 of 21 foods tested inhibited reactivity with antigen extracts derived from S. mutans MT3. Extracts from 16 foods agglutinated whole S. mutans cells, whereas those from 10 foods agglutinated human erythrocytes of blood types A and B. The lectin-like activities of extracts which reacted with human saliva were studied further. Pretreatment of saliva-coated hydroxyapatite (S-HA) beads with extracts of bananas, coconuts, carrots, alfalfa, and sunflower seeds markedly reduced the subsequent adsorption of S. mutans MT3. Pretreatment of S-HA with banana extract also strongly inhibited adsorption of S. mutans H12 and S. sanguis C1, but it had little effect on attachment of Actinomyces naeslundii L13 or A. viscosus LY7. Absorption experiments indicated that the component(s) in banana extract responsible for inhibiting streptococcal adsorption to S-HA was identical to that which bound to human erythrocytes. The banana hemagglutinin exhibited highest activity between pH 7 and 8, and it was inhibited by high concentrations of glucosamine, galactosamine, and, to a lesser extent, mannosamine. Other sugars tested had no effect. The selective bacterial adsorption-inhibiting effect noted for banana extract was also observed in studies with purified lectins. Thus, pretreating S-HA with wheat germ agglutinin and concanavalin A inhibited adsorption of S. mutans MT3 cells, whereas peanut agglutinin, Ulex agglutinin, Dolichos agglutinin, and soybean agglutinin had little effect; none of these lectins affected attachment of A. viscosus LY7. Collectively, the observations suggest that many foods contain lectins which can interact with components of human saliva and S. mutans cells. Because of their potential to influence host-parasite interactions in the mouth and elsewhere in the gastrointestinal canal, these reactions warrant further study.     

A novel concept combining experimental and mathematical analysis for the identification of unknown interspecies effects in a mixed culture

Bacteria in natural habitats only occur in consortia together with various other species. Characterization of bacterial species, however, is normally done by laboratory testing of pure isolates. Any interactions that might appear during growth in mixed-culture are obviously missed by this approach. Existing experimental studies mainly focus on two-species mixed cultures with species specifically chosen for their known growth characteristics, and their anticipated interactions. Various theoretical mathematical studies dealing with mixed cultures and possible interspecies effects exist, but often models cannot be validated due to a lack of experimental data. Here, we present a concept for the identification of interspecies effects in mixed cultures with arbitrary and unknown single-species properties. Model structure and parameters were inferred from single-species experiments for the reproduction of mixed-culture experiments by simulation. A mixed culture consisting of the three-species Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus served as a model system. For species-specific enumeration a quantitative terminal restriction length polymorphism (qT-RFLP) assay was used. Based on models fitted to single-species cultivations, the outcome of mixed-culture experiments was predicted. Deviations of simulation results and experimental findings were then used to design additional single-cell experiments, to modify the corresponding growth kinetics, and to update model parameters. Eventually, the resulting mixed-culture dynamics was predicted and compared again to experimental results. During this iterative cycle, it became evident that the observed coexistence of P. aeruginosa and B. cepacia in mixed-culture chemostat experiments cannot be explained on the basis of glucose as the only substrate. After extension of growth kinetics, that is, for use of amino acids as secondary substrates, mixed-culture simulations represented the experimental findings very well. According to the model structure, as motivated by single-species experiments, the growth of P. aeruginosa and B. cepacia on glucose and amino acids could be assumed to be independent of each other. In contrast, both substrates are taken up simultaneously by S. aureus.     

Coexistence of nearly neutral species

Aims The neutral theory of biodiversity provides a powerful framework for modeling macroecological patterns and interpreting species assemblages. However, there remain several unsolved problems, including the effect of relaxing the assumption of strict neutrality to allow for empirically observed variation in vital rates and the 'problem of time'—empirically measured coexistence times are much shorter than the prediction of the strictly neutral drift model. Here, we develop a nearly neutral model that allows for differential birth and death rates of species. This model provides an approach to study species coexistence away from strict neutrality.Methods Based on Moran's neutral model, which assumes all species in a community have the same competitive ability and have identical birth and death rates, we developed a model that includes birth–death trade-off but excludes speciation. This model describes a wide range of asymmetry from strictly neutral to nearly neutral to far from neutral and is useful for analyzing the effect of drift on species coexistence. Specifically, we analyzed the effects of the birth–death trade-off on the time and probability of species coexistence and quantified the loss of biodiversity (as measured by Simpson's diversity) due to drift by varying species birth and death rates.Important findings We found (i) a birth–death trade-off operating as an equalizing force driven by demographic stochasticity promotes the coexistence of nearly neutral species. Species near demographic trade-offs (i.e. fitness equivalence) can coexist even longer than that predicted by the strictly neutral model; (ii) the effect of birth rates on species coexistence is very similar to that of death rates, but their compensatory effects are not completely symmetric; (iii) ecological drift over time produces a march to fixation. Trade-off-based neutral communities lose diversity more slowly than the strictly neutral community, while non-neutral communities lose diversity much more rapidly; and (iv) nearly neutral systems have substantially shorter time of coexistence than that of neutral systems. This reduced time provides a promising solution to the problem of time.     

The establishment of reproducible, complex communities of oral bacteria in the chemostat using defined inocula

Nine commonly isolated oral bacterial populations were inoculated into a glucose-limited and a glucose-excess (amino acid-limited) chemostat maintained at a constant pH 7.0 and a mean community generation time of 13.9 h. The bacterial populations were Streptococcus mutans ATCC 2-27351, Strep. sanguis NCTC 7865, Strep. mitior EF 186, Actinomyces viscosus WVU 627, Lactobacillus casei AC 413, Neisseria sp. A1078, Veillonella alkalescens ATCC 17745, Bacteroides intermedius T 588 and Fusobacterium nucleatum NCTC 10593. All nine populations became established in the glucose-limited chemostat although Strep. sanguis and Neisseria sp. were present only after a second and third inoculation, respectively. In contrast, even following repeated inoculations, Strep. mutans, B. intermedius and Neisseria sp. could not be maintained under glucose-excess conditions. A more extensive pattern of fermentation products and amino acid catabolism occurred under glucose-limited growth; this simultaneous utilization of mixed substrates also contributed to the higher yields (Y molar glucose) and greater species diversity of these communities. Microscopic and biochemical evidence suggested that cell-to-cell interactions and food chains were occurring among community members. To compare the reproductibility of this system, communities were established on three occasions under glucose-limitation and twice under glucose-excess conditions. The bacterial composition of the steady-state communities and their metabolic behaviour were similar when grown under identical conditions but varied in a consistent manner according to the nutrient responsible for limiting growth. Although a direct simulation of the oral cavity was not attempted, the results show that the chemostat could be used as an environmentally-related model to grow complex but reproducible communities of oral bacteria for long periods from a defined inoculum.     

Characterization of a three bacteria mixed culture in a chemostat: evaluation and application of a quantitative terminal-restriction fragment length polymorphism (T-RFLP) analysis for absolute and species specific cell enumeration

Growth dynamics of Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus in a batch and chemostat, were investigated as a laboratory model system for persistent infections in cystic fibrosis. Most species-specific enumeration methods for mixed cultures are laborious or only qualitative, and therefore impede generation of quantitative data required for validation of mathematical models. Here, a quantitative T-RFLP method was evaluated and applied for specific and absolute cell number enumerations. The method was tested to be unbiased by quantitative sample composition and allowed reproducible enumerations of mixed cultures. For assay validation, samples of defined concentration containing one, two or three species were quantified. Logarithmically transformed absolute cell numbers of single-species dilutions were linear within a lower working range of 10(4)-10(6) cfu/mL (species-dependent) and an upper working range of 10(10) cfu/mL. Quantifications of single species (10(6)-10(10) cfu/mL) spiked with one or two other species agreed well with single species controls. Differences between slopes of first order linear regression of spiked and pure dilution series were insignificant. Coefficient of variation of defined mixed replicates was maximum 4.39%, of a three-species chemostat it was maximum 1.76%. T-RFLP monitoring of pure cultures in parallel shake flasks and of a three-species mixed chemostat gave very consistent results. Coexistence of at least two species after a time period equivalent to more than 33 volume exchanges was found. This result was not predicted from pure cultures clearly indicating the need for quantitative mixed culture experiments to better understand microbial growth dynamics and for mathematical model validation.     

Competition during submerged mixed culture of Geotrichum candidum and Penicillium camembertii on glucose and threonine

Geotrichum candidum and Penicillium camembertii were cultivated in pure and mixed cultures on glucose and threonine. In pure cultures, G. candidum used glucose as a carbon and an energy source and threonine only as a nitrogen source, even after glucose exhaustion. Contrarily, when growing in isolation, P. camembertii used simultaneously threonine and glucose as carbon sources. A diauxic growth was recorded during the mixed culture of both species, which competed for glucose, the sole carbon source available for G. candidum growth, leading to higher glucose consumption rates than those recorded during pure cultures, while after glucose exhaustion, low growth was recorded in a second step, showing a 'competition' for threonine, the sole remaining carbon and nitrogen sources, confirmed by the increase of 1.0+/-0.1 log of the G. candidum Colony Forming Units. 'Competition' between G. candidum and P. camembertii for the limiting substrate was found to have a positive effect on growth, since it did not lead to the annihilation of one species, as usually observed, but in their coexistence, leading to a rather similar final number of the CFUs for the two populations. 'Competition' resulted in the absence of assimilation of the second available carbon substrate (lactate) as previously observed, or its use only as a nitrogen source, as was the case for threonine in this work.     

Growth of mixed cultures on mixtures of substitutable substrates: the operating diagram for a structured model

The growth of mixed microbial cultures on mixtures of substrates is a problem of fundamental biological interest. In the last two decades, several unstructured models of mixed-substrate growth have been studied. It is well known, however, that the growth patterns in mixed-substrate environments are dictated by the enzymes that catalyse the transport of substrates into the cell. We have shown previously that a model taking due account of transport enzymes captures and explains all the observed patterns of growth of a single species on two substitutable substrates (J. Theor. Biol. 190 (1998) 241). Here, we extend the model to study the steady states of growth of two species on two substitutable substrates. The model is analysed to determine the conditions for existence and stability of the various steady states. Simulations are performed to determine the flow rates and feed concentrations at which both species coexist. We show that if the interaction between the two species is purely competitive, then at any given flow rate, coexistence is possible only if the ratio of the two feed concentrations lies within a certain interval; excessive supply of either one of the two substrates leads to annihilation of one of the species. This result simplifies the construction of the operating diagram for purely competing species. This is because the two-dimensional surface that bounds the flow rates and feed concentrations at which both species coexist has a particularly simple geometry: It is completely determined by only two coordinates, the flow rate and the ratio of the two feed concentrations. We also study commensalistic interactions between the two species by assuming that one of the species excretes a product that can support the growth of the other species. We show that such interactions enhance the coexistence region.