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.     

Proteomic tracking and analysis of a bacterial mixed culture

To improve the understanding of microbial behaviors in communities, proteomic tracking, an approach for relative quantification of species-specific population dynamics of mixed cultures, was developed. Therefore, a bacterial mixed culture was analyzed during batch cultivations with and without addition of the antibiotic Ceftazidime. The community was composed of Burkholderia cepacia, Pseudomonas aeruginosa, and Staphylococcus aureus, pathogens causing infections in cystic fibrosis patients. Gel-based proteomics and mass spectrometry were used to obtain qualitative and quantitative proteomic data. During cultivation, P. aeruginosa became dominant within the mixed culture while S. aureus was inhibited in growth. Analysis of samples - taken along cultivation - revealed about 270 differentially expressed proteins. Some of those proteins are related to bacterial interactions, response to antibiotic treatment or metabolic shifts. For instance, the enzymes PhzS(flavin-containing monooxygenase), PhzD (phenazine biosynthesis protein), and PhzG2 (pyridoxamine 5'-phosphate oxidase) indicated the production of the antibiotic pigment pyocyanine by P. aeruginosa that is related to oxidative stress and therefore, might inhibit growth of S. aureus. Overall, the strategy applied not only allows species-specific tracking of the community composition but also provides valuable insights into the behavior of mixed cultures.     

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities

AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF). METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B. cepacia. Reverse challenges were also conducted. Both B. cepacia and P. aeruginosa were able to form steady-state monoculture biofilms after 48 h growth. When steady-state biofilms of B. cepacia NTCT 10661 were challenged with planktonically grown P. aeruginosa PAO1 known to produce a B. cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B. cepacia. By contrast, established biofilms of P. aeruginosa PAO1 resisted immigration of B. cepacia 10661. Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P. aeruginosa 313113 and B. cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances. Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm. Similar observations were made for biofilms generated in CDFFs. In such instances following pan-swapping between two monoculture CDFFs, B. cepacia 313113 was able to integrate into an established P. aeruginosa 313113 biofilm to form a stable binary biofilm. CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P. aeruginosa predisposes uncolonized sections of the surface to permit B. cepacia colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species. Disinfection of a surface might well increase the probability of pathogen harbourage.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Diminution of the antibacterial activity of antibiotics in cultures and in experimental mixed infections]

We have studied interactions between Staphylococcus aureus and Pseudomonas aeruginosa in the absence and the presence of four different antimicrobials in mixed cultures and experimental infections. These two bacterial species, in addition to having different properties, are known to be opportunistic pathogens often present in human microflora. Two main aspects have been investigated and they are related to modifications in two species affecting their equilibrium in the mixed bacterial population and also their pathogenicity markers. Our results indicate that individual growth of S. aureus and P. aeruginosa is not modified in vitro in mixed cultures in the absence of antimicrobials; in vivo, in mouse peritoneal cavity, there is a synergism favorable to S. aureus. In the presence of rifamycin SV and three cell wall inhibitors, pencillin G,D-cycloserine, and vancomycin, we have observed that P. aeruginosa protected S. aureus against the inhibitory effect of these antimicrobials in vitro and in vivo. Such results were obtained in different conditions of culture, stationary, shaken, and in special apparatuses, an "Ecologen" and a "Chemostat." When any one of the antimicrobials was allowed to be in contact for 6 to 8 h with P. aeruginosa cells in a culture, we observed a decrease in their inhibitory effects against S. aureus. These results were supported by microscopical observation. It seems that the inhibitory effects of the antimicrobials have hindered the formation of toxic products of S. aureus, e.g., alpha toxin, and that it was not restored in the presence of P. aeruginosa. Conversely, P. aeruginosa remained apparently unchanged through all these experiments. Our observations may imply that the inhibitory effect of an antimicrobial towards a bacterial species may be significantly decreased in the presence of another species, sometimes present in human microflora.     

Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures

Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

Analysis of two interacting bacterial populations with opposite substrate preferences

Interactions between two bacterial species which exhibit opposite substrate preferences in response to the same environmental conditions were investigated. Klebsiella oxytoca preferentially utilizes glucose over citrate as a substrate while Pseudomonas aeruginosa utilizes citrate over glucose. Both species were grown on single as well as mixed substrates, namely, D-glucose and citrate, with various ratios of inocula and substrates. Competition is observed between the two species with citrate alone while amensalism is observed with glucose alone. In mixed cultures-mixed substrates experiments, amensalism of K. oxytoca on P. aeruginosa prevailed until the exhaustion of glucose.     

The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication.

The effect of concentrated cell-free extracellular material from stationary-phase cultures of Burkholderia cepacia 10661 and Pseudomonas aeruginosa PAO1 on virulence factor production in B. cepacia was assessed. While increasing concentrations of the B. cepacia exoproduct caused a slight increase in siderophore, lipase, and protease production in the producing organism, a significant in productivity was observed for all three virulence factors with the addition of the PAO1 exoproduct. Moreover, the addition of the exoproduct from a strain of P. aeruginosa producing reduced amounts of autoinducer caused only a slightly greater response than that of the control. Both B. cepacia 10661 and P. aeruginosa PAO1, along with two matched clinical isolates of both organisms obtained from a cystic fibrotic patient, were shown to produce variable amounts of three different types of autoinducer. The potential for interspecies signalling in microbial pathogenicity is discussed.     

Glucose Fermentation Products of Ruminococcus albus Grown in Continuous Culture with Vibrio succinogenes: Changes Caused by Interspecies Transfer of H2

The influence of a H(2)-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H(2)-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenes depended on the production of H(2) from glucose by R. albus. V. succinogenes used the H(2) produced by R. albus to obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albus alone. The only products detected in continuous cultures of R. albus alone were acetate, ethanol, and H(2). CO(2) was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H(2) was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albus alone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H(2) for R. albus alone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H(2) by R. albus. Thus, in the mixed cultures, ethanol production by R. albus is eliminated with a corresponding increase in acetate and H(2) formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albus is grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H(2) when R. albus is grown with V. succinogenes. The ecological significance of this interspecies transfer of H(2) gas and the theoretical basis for its causing changes in fermentation patterns of R. albus are discussed.     

Increase in the in vitro susceptibility of Staphylococcus aureus to antimicrobial agents in the presence of Candida albicans.

In experiments with mixed cultures of Staphylococcus aureus and Candida albicans both in the absence and in the presence of 5-fluorocytosine (5-FC), we have observed that (1) there is an inhibition of S. aureus growth in mixed cultures with C. albicans in media supplemented with 1 microgram/mL of 5-fc and that 5-FC has no effect on staphylococci in pure cultures; (2) this inhibition occurred with clinically isolated and laboratory strains and could be reversed by specific metabolites; (3) Staphylococcus aureus was inhibited by filtrates of C. albicans cultures treated with 5-FC and this seemed to be favored by some C. albicans filterable product which can affect the cell wall and the permeability of the staphylococcal cells since they become sensitive to 5-FC; (4) nine other commonly used antimicrobials showed an increased inhibitory activity against S. aureus in mixed cultures with C. albicans; and (5) there is a decrease in the number of precipitating antigens of S. aureus and of the activity of alpha toxin when this species was grown with both C. albicans and 5-FC. Our results indicate that the susceptibility of some species to antimicrobials could be significantly modified in the presence of other species. One cannot exclude that a similar phenomenon could happen in hosts under treatment with antibiotics against infection.     

Growth of mixed cultures of Actinomyces viscosus and Streptococcus mutans under dual limitation of glucose and oxygen

Abstract Streptococcus mutans and Actinomyces viscosus are among the dominant species in human dental plaque. In their natural environment, carbohydrate- and oxygen-limited conditions are likely to occur frequently. Therfore, mixed cultures of the 2 species were studied under dual limitation of glucose and oxygen. Over a wide range of oxygen-supply rates, coexistence of A. viscosus and S. mutans was observed, within this range A. viscosus increased almost linearly with oxygen supply. A mathematical model based on Monod-type type kinetics and accounting for uncompetitive inhibition of growth by oxygen was developed to simulate these mixed cultures. The model predicted coexistence over a fairly large range of aeration rates. This finding, in combination with the results of the chemostat experiments, led to the conclusion that coexistence of the two species     

On enhancing productivity of bioethanol with multiple species

The present work is initiated to investigate whether a defined culture comprising a mixture of three yeast species, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia stipitis can ferment a mixture of sugars to produce bioethanol at rates higher than those achieved by pure cultures of the same. For this purpose, we develop models of single species based on the hybrid cybernetic model framework, and simulate fermentations in the mixed culture by combining individual models. An underlying assumption is that the behavior of each species is determined only by the common environment independently of the presence and metabolism of other species. Model performance is thoroughly assessed using the experimental data available in the literature. The dynamic behavior of mixed cultures in mixed culture experiments are accurately predicted by the model reflecting faithfully the simultaneous/sequential uptake patterns of mixed substrates. This model is then used to investigate performance of various possible reactor configurations. With the foregoing species of organisms, mixed culture itself does not lead to a significant increase of bioethanol productivity. Rather, the model shows that substantial improvement is acquired by sequential use of different, properly chosen organisms during fermentation. Thus, the successive use of K. marxianus and P. stipitis is shown to increase bioethanol productivity up to about 58% in comparison to fermentation by single species alone.     

Effect of substrate on the regulation of exoprotease production by Pseudomonas aeruginosa ATCC 10145

Exoprotease production by Pseudomonas aeruginosa ATCC 10145 was growth-associated when cultures were grown on complex substrates such as proteins but it occurred during the decelerating growth phase when the organism was grown on amino acids, mixtures of amino acids or simple carbon sources. NH4Cl and simple carbon sources caused repression. Exoprotease was produced in chemostat cultures in response to growth under any of the nutrient limitations studied (carbon, nitrogen or phosphate). Furthermore, by growing at rates less than approximately 0.1 h-1, the repression of enzyme production could be overcome to a large degree. At low growth rates there was an inverse relationship between growth rate and exoprotease production. Thus, exoprotease production was depressed by available energy sources and was increased in response to any nutrient limitation.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.     

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.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.