Mixed-species Cultures Research Articles

Fluconazole susceptibility of Candida isolates from oropharyngeal candidosis.

Fifty strains of Candida isolated from 38 patients with oropharyngeal candidosis were tested in vitro for fluconazole susceptibility with a disk diffusion test and for determination of minimal inhibitory concentrations (MICs). For 25 patients treated with fluconazole, the relationship between in vitro susceptibility and clinical outcome was analysed. A good correlation between in vitro results and therapeutic efficacy was found. In only one case was treatment failure associated with a susceptible strain. Mixed cultures of Candida albicans and non-albicans Candida species were not uncommon and, more interestingly, some samples contained different strains of C. albicans with varied fluconazole susceptibilities. Good agreement was observed between the two techniques used for fluconazole susceptibility testing.

Effect of betaine on the growth performance of chicks inoculated with mixed cultures of avian Eimeria species and on invasion and development of Eimeria tenella and Eimeria acervulina in vitro and in vivo

At 7 d postinoculation (DPI) with a mixed culture of avian Eimeria species, 21-d-old chicks maintained in batteries and floor pens on a diet containing 0.15% (3 lb/ton) betaine plus 66 ppm (60 g/ton) salinomycin were significantly heavier and had significantly lower feed conversion ratios and mortality than chicks fed diets containing 0.15% betaine or 66 ppm salinomycin alone, or the control diet. At 31 DPI, when the chicks were 45 d old, the differences between the diet groups were not as great as at 7 DPI. In vitro, except at high concentrations, betaine was nontoxic to sporozoites of Eimeria tenella or Eimeria acervulina and had little effect on their invasion and development in cultured cells. In vivo, invasion by E. tenella and E. acervulina sporozoites was significantly reduced in all chicks fed diets containing betaine or salinomycin compared with that in control chicks. There was a significant interaction between betaine and salinomycin that impacted on invasion by both species. Overall development of E. tenella did not appear to be adversely affected by addition of betaine to diets containing salinomycin. Conversely, development of E. acervulina was reduced in chicks fed diets containing 0.075% (1.5 lb/ton) betaine plus 66 ppm salinomycin as compared with that in chicks fed salinomycin alone.

Influence of Arbuscular Mycorrhizal Infection on Competition between Holcus Lanatus and Dactylis Glomerata

1 Holcus lanatus and Dactylis glomerata were grown in monocultures and mixtures to determine the influence of mycorrhizal fungi on intraand interspecific competition. 2 In monoculture, shoot biomass per plant of both species was increased over all densities by mycorrhizal infection. Increased tiller and leaf numbers per plant for H. lanatus, but not for D. glomerata, suggested that enhanced biomass was a result of greater tiller and leaf production in Holcus, but to increased tiller weight in Dactylis. Increased competition reduced shoot biomass, tiller number and leaf number in both species. 3 Although the response was variable, mycorrhizal infection generally enhanced shoot biomass of both species in mixed cultures. Tiller production in D. glomerata was generally unaffected by infection but reductions in leaf number were observed in mycorrhizal stands. In contrast to its effects on monocultures, infection had no effect on tiller production when H. lanatus was in mixture. 4 Relative yield totals (R YT) calculated for grasses grown in mixture suggest that, overall, mycorrhizal infection resulted in a reduction of resource complementarity, i.e. it increased the level of competition for the same resources. This was reflected in the relative yields which showed that the shoot biomass for each species in mixture was lower than that expected from growth in monoculture. 5 Aggressivity indices suggested that H. lanatus was more aggressive than D. glomerata when present in equal or greater numbers. Mycorrhizal infection altered the degree of aggressivity in favour of the already more aggressive plant within the combination, although, at very low densities, D. glomerata was more aggressive than H. lanatus when mycorrhizal.

A method utilizing mycelial fragments to estimate the relative biomass densities of fungal species in mixed culture

A method for determining the relative biomass densities of two fungal species, Trichoderma harzianum and Fusarium culmorum, in mixed cultures, based on fragmentation of their mycelia is described. The mycelia were fragmented using a laboratory blender under standardised conditions. The mycelial fragments were plated onto a medium containing the paramorphogen, sodium deoxycholate, to inhibit colony extension, and small colonies were produced which could be distinguished and enumerated on the basis of their mature characteristics, principally the production of different pigments. A linear relationship between the number of fungal colonies produced and the biomass dry weight was observed. The method has potential applications for studying a wide range of fungal interactions, notably those which occur in plant-associated and soil ecosystems where the vegetative mycelium contributes the majority of the significant biomass present.

The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: development of probes for Ruminococcus species and evidence for bacteriocin production.

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococcus albus and Ruminococcus flavefaciens. On the basis of specificity studies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed cultures for cellobiose as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85, competition was not observed. However, R. flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when cellobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 were grown together on cellobiose medium, R. albus 8 outcompeted R. flavefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA only 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Further, when R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown together in a triculture, R. flavefaciens FD-1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. succinogenes S85 showed a similar competition pattern to that of the dicultures. The results show that the Ruminococcus probes were effective in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Predation risk of typical ovoid and ?winged? morphs of Euplotes (Protozoa, Ciliophora)

Freshwater species of the genus Euplotes (Protozoa, Ciliophora) change their morphology in the presence of some of their predators. The ciliates develop extended lateral ‘wings’ as well as dorsal and ventral projections which make engulfment by predators more difficult. In a series of laboratory experiments ingestion rates of four protozoan predators, the ciliates Lembadion bullinum, Dileptus anser, Stylonychia mytilus and Urostyla grandis, and one metazoan predator, the turbellarian Stenostomum sphagnetorum, on three species of Euplotes (E. octocarinatus, E. patella and E. aediculatus) were determined. It was calculated that the probability of rejection by a predator changed from 1:1 for ovoid morphs of Euplotes to about 2:1–20:1 for ‘winged’ morphs of Euplotes, dependent on the prey and predator species that were combined. The nutritional condition of the prey also had some influence. In mixed-species cultures of prey and predators, transformed cells of E. octocarinatus survived for several months.

Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus. Resource competition between rotifers and daphnids under toxic stress

This study examined the ability of the freshwater rotifer Brachionus calyciflorus to coexist with the large cladoceran Daphnia magna, and the effect of sublethal concentrations of copper on the competition between both species. Preliminary laboratory experiments showed that large Daphnia (> 1.2 mm) can kill and rapidly exclude the rotifer B. calyciflorus in mixed-species cultures. Brachionus populations were suppressed by Daphnia both through exploitative competition for shared, limited food resources and through mechanical interference. At a food concentration of 1 X 10 5 cells/ml of Nannochloris oculata, competition caused high mortality rates and decreased fertility in the rotifer populations (1000 ind/1), even at low Daphnia densities (40 ind/1). Daphnia removed increasingly disproportionate shares of the daily algal food supply, leaving the rotifer populations to gradually starve to extinction. In other experiments the effect of sublethal copper concentrations (2.5, 5.0, 12.0 and 20 μg/1) on the competition between both species was analysed. Short experiments (5 days) were carried out to study the effect of this toxicant on the rotifer populations with and without Daphnia. Changes in the competitive relationships between both organisms under toxic stress and the possible ecological consequences of these observations will be discussed.

Coexistence of ecologically similar colonising species III. Drosophila aldrichi and D. buzzatii: larval performance on, and adult preference for, three Opuntia cactus species

Two Drosophila species, D. buzzatti and D. aldrichi, coexist on several species of Opuntia cacti in Australia, primarily on O. tomentosa and O. streptacantha in the northern part of the cactus distribution, and on O. stricta in the south. Thorax length of field-collected adults was less, and the variance in length greater, than that for flies reared on simulated rots in the laboratory, indicating that these species are affected by crowding in nature. A larval performance index, measured on simulated cactus rots at low, moderate and high densities in single-species cultures, and at moderate and high densities in mixed-species cultures, was used to compare the relative intensity of intra- and interspecific competition at the same total larval density per 5 g necrotic cactus. Larval performance of both fly species was greatest on O. streptacantha, intermediate on O. tomentosa, and least on O. stricta in both single-species and mixed-species cultures. On O. stricta, the performances of D. aldrichi and D. buzzatii were not different when in single-species cultures, but that of D. aldrichi decreased significantly in mixed-species cultures. On the other two cactus species, the performances of D. aldrichi and D. buzzattii were not different in mixed-species cultures. The order of preferences by adult females for the cacti differed from that for larval performance, with females of both species prefering O. stricta. Analysis of microbial numbers growing on the cacti showed little difference among cacti at the rot age used for testing adult preference, but later growth was greater on O. tomentosa and O. streptacantha, the cacti that best supported larvae. Differential larval performance on O. stricta may contribute to the rare presence of D. aldrichi in the southern part of the cactus distribution, while the superior quality of O. tomentosa and O. streptacantha (larger rot size and higher microbial concentration) may reduce competition and facilitate cocxistence of the fly species in the north.

Pectin decomposition and associated nitrogen fixation by mixed cultures of Azospirillum and Bacillus species.

Cocultures of different Azospirillum species with Bacillus polymyxa or Bacillus subtilis allow the efficient utilization of pectin as carbon and energy sources for nitrogen fixation. The nitrogenase activity obtained with cocultures was as high as 30-80 nmol C2H4 h-1 mL-1, a much higher value than that obtained with pure cultures of either Azospirillum (up to 13 nmol C2H4 h-1 mL-1) or B. polymyxa (up to 2 nmol C2H4 h-1 mL-1) alone. To establish to what extent each partner contributed to nitrogenase activity, acetylene reduction was assayed as a function of time and it was also measured on Azospirillum cultivated in the cultures filtrates of the Bacillus. The results suggested that the nitrogenase activity was mostly produced by Azospirillum. The nitrogenase activity occurred at the expense of the degradation and fermentation products of the pectin. The new pectinolytic species, Azospirillum irakense, utilized both degradation and fermentation products of pectin, whereas the nonpectinolytic strains (Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense) utilized only the fermentation products of pectin, including acetic and succinic acids. These cocultures can be considered as metabolic associations, where the Bacillus produces degradation and fermentation products of pectin, which can be used by Azospirillum species.

Use of randomly cloned DMA fragments for the identification of oral spirochetes

DNA probes were produced for the detection and identification of 4 cultivable species of oral spirochetes, Treponema denticola, Treponema socranskii, Treponema vincentii and Treponema pectinovorum. To obtain probe sequences, chromosomal DNA, isolated from representative strains within each species, was cloned in Escherichia coli K-12. Cloned DNA fragments were screened for the ability to hybridize to DNA only from homologous strains. Several such fragments were identified and shown to be specific when tested against a series of DNAs from gram-negative and gram-positive oral bacteria. The selected probe sequences were semi-conserved within strains of T. denticola and T. socranskii such that restriction fragment length polymorphism (RFLP) was observed. In the case of T. socranskii, RFLP was useful in distinguishing between the 3 known subspecies. Chromosomal DNA fragments from 2 strains of T. vincentii failed to cross-hybridize, under stringent conditions, to genomic DNA from each of these strains. The hybridization probes were suitable for the identification of clinical isolates of T. denticola and could be used to detect the presence of individual Treponema species in mixed cultures. On this basis, the probes were used successfully to detect T. denticola in uncultured plaque samples.

EXTINCTION DUE TO EVOLUTION OF A COMPETITOR.

When Drosophila simulans and Drosophila funebris were cultured together in population cages over many generations, there was a prolonged period of apparently stable coexistence followed by a rapid exclusion of D. funebris. As both species maintained large population sizes in monocultures it follows that the extinctions of D. funebris in the mixed-species cultures must have been caused by D. simulans. The time to extinction of D. funebris ranged from 26 weeks in one cage, to between 40 and 48 weeks in the other five. To test the idea that an evolutionary increase in competitive ability of D. simulans had occurred during the course of its interaction with D. funebris, a single-generation experiment was set up. In this experiment the interspecific competitive ability of a population of D. simulans that had been in competition with D. funebris for 44 weeks was compared to that of a stock population that had had no previous contact with D. funebris. In this experiment both stock and precompeted populations of D. simulans increased the egg-to-adult development time of D. funebris. However, precompeted D. simulans caused a significantly greater increase in the development time of D. funebris than did stock D. simulans. Thus D. simulans had evolved an increase in competitive ability as a result of its interaction with D. funebris. Development time is important because in the population cages the resource bottles-in which the larvae reside-were replaced every three weeks. An increase in development time of D. funebris in the multigeneration experiment similar to that observed in the single-generation experiment would lead to a rapid decrease in adult population size, resulting in the extinction of this species, as was observed to happen.

Induction of mutation inaspergillus niger for conversion of cellulose into glucose

Plant wastes are very important part of biomass used and investigated for energy, chemical, and fuel production. Cellulose is the major renewable form of carbohydrate in the world, about 10{sup 11} tons of which is synthesized annually. For general use, it must be hydrolyzed first, either chemically or by cellulases derived from a few specialized microorganisms. Enzymes are acceptable environmentally but expensive to produce. Certainly, induction of mutations and selection of high cellulose microbial strains with significant adaptability to degrade cellulose to glucose is promising solutions. Induction of mutations in other fungi and Aspergillus sp. rather than Aspergillus niger was reported. Aspergillus ustus and Trichoderma harzianum were induced by gamma irradiation indicating mutants that excrete higher cellulose yields, particularly exocellobiohydrolase (Avicelase) than their respective wild types. Mutants from the celluiolytic fungus Penicillium pinophilum were induced by chemical and UV-irradiation. Enhancing the production of endo-1,4-{Beta}-D-glucanase (CMCase) and particularly {Beta}-glucosidase was obtained by gamma irradiation of Altemaria alternate. To overcome the lower activity of {beta}-glucosidase in certain fungi species rather than A. niger, mixed cultures of different species were tried. Thus, Aspergillus phonicis with Trichoderma reesei Rut 30, produced a cellulose complex that improved activity twofold over cellulose from Trichoderma alone.

Kinetics of spontaneous wine production

Substrate consumption, ethanol production, microbial growth, and temperature increase in a fermentation vessel are metabolically interrelated biological phenomena. In spontaneous wine fermentations, mixed cultures of various microbial species are involved in the fermentation process. Due to the continuously changing species contributing to the microbial population, these metabolically interrelated parameters are treated separately in the enology literature. In the present study, mathematical models are presented to interrelate these parameters, by employing the data obtained from a totally uncontrolled spontaneous wine production process. Ethanol production rates were related to biomass production with a modified Luedeking-Piret equation. In the ethanol production phase of the process, total microbial growth did not slow down with ethanol accumulation. This might be caused by the compensating effects of temperature increase. Ethanol production yield of the culture increased with ethanol accumulation in the medium, possibly due to elimination of the low-alcohol-tolerant, poor alcohol-producing species. Microbial growth was simulated with the logistic equation in the malo-lactic fermentation phase.

OXYRASETM ENZYME AND MOTILITY ENRICHMENT FUNG‐YU TUBE FOR RAPID DETECTION OF LISTERIA MONOCYTOGENES AND LISTERIA SPECIES1

ABSTRACT A rapid and simple method using a U‐shaped glass apparatus (Fung‐Yu tube) for early determination of the presence of Listeria monocytogenes and Listeria species in mixed cultures and inoculated meat samples has been developed. This system utilizes unique biochemical and physical properties of Listeria for selective enrichment. Fraser broth was used as a selective enrichment broth especially for observation of esculin hydrolysis (blackening of broth), and semisolid Modified Oxford agar was used for selective detection of motility of Listeria. When Fung‐Yu tubes containing 0.1 unit/mL of OxyraseTM (membrane fractions of Escherichia coli) were inoculated with L. monocytogenes, an enhanced early growth of L. monocytogenes occurred. A presumptive positive result for low numbers of L. monocytogenes (1–100 CFU/g) in the presence of large numbers of competitive microflora in pre‐enriched (24 h) ground beef samples using the Fung‐Yu tube method with the aid of OxyraseTMwas obtainable within 10 h. Using this system, isolation of Listeria in the presence of mixed bacterial flora (44 species), such as Bacillus, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Staphylococcus, and Streptococcus, and in inoculated ground beef was successful in 24–48 h. The Fung‐Yu tube procedure is a highly sensitive, selective, and easy‐to‐use method to separate and isolate L. monocytogenes and other Listeria spp. from other contaminating microorganisms in meats.

Effects of Density and Species Proportion on Competition between Spurred Anoda (Anoda cristata) and Velvetleaf (Abutilon theophrasti)

Spurred anoda and velvetleaf were grown for 40 days in controlled-environment chambers in monocultures at densities of 2, 4, 8, and 12 plants per 20-cm-diam pot and in mixed culture with all combinations of 2, 4, 8, and 12 plants of each species per pot. The day/night temperature was 29/23 C, and the photosynthetic photon flux density (PPFD) was 1000 μE m–2s–1. Shoot dry weights and leaf areas of the two species were similar when they were grown in monoculture. However, in mixed culture spurred anoda exceeded velvetleaf in leaf area/plant and shoot weight/plant in 15 out of 16 treatments. Multiple linear regression equations relating shoot biomass/plant to the density of both species in mixed culture were calculated. Comparison of competition coefficients from these equations indicated that the competitive impact of a single spurred anoda plant was equivalent to the impact of 2.5 velvetleaf plants. In mixed culture, spurred anoda always contributed a greater proportion of the total shoot weight and total leaf area per pot than would be predicted from its proportion of the total plant population. These results indicate spurred anoda is competitively superior to velvetleaf.

Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.

Growth and competition between potamogeton pectinatus L. and Myriophyllum exalbescens fern. in experimental ecosystems

Two submerged aquatic macrophytes, Potamogeton pectinatus L. (sago pondweed) and Myriophyllum exalbescens Fern. (water milfoil) were grown in experimental aquaria in single- and mixed-species cultures in combinations of low and high densities. Biomass of individual plant parts (roots, shoots and tubers) was determined monthly for four months. P. pectinatus root and shoot biomass was greater than M. exalbescens root and shoot biomass, throughout the experiment, at both low and high plant densities. From the relationships between plant densities and their biomass (in the presence and absence of conspecifics and individual plants of the other species), and under specific laboratory conditions, we found that: (1) intraspecific competition, as detected from the decrease in growth rate with increased plant density, was present; (2) M. exalbescens did not exert an interspecific competitive effect on P. pectinatus; (3) the interspecific competitive effect of P. pectinatus on M. exalbescens was stronger in inhibiting plant growth than that of M. exalbescens on itself; (4) increased plant density resulted in smaller P. pectinatus roots, shoots and tubers; (5) shoots of M. exalbescens, but not roots, were affected by increased plant density.

Direct use of high molecular weight polysaccharide by heterotrophic flagellates

In aquatic ecology it is assumed that the heterotrophic (non-photosynthetic) protozoa that are abundant in marine and fresh water cannot compete with smaller-sized bacteria for labile dissolved substrates of low relative molecular mass (Mr), such as simple sugars and amino acids, at the low concentrations found in situ1,2 (typically 1–100 μg litre−1) because of surface to volume considerations. Consequently dissolved organic matter (DOM) is not considered to be a source of nutrition for heterotrophic protozoa, which are thought to be predominantly phagotrophic on bacteria and other small planktonic cells2. But there is no information as to whether protozoa might be able to use DOM of higher Mr. Here I report that heterotrophic flagellates in a salt marsh estuary and in a freshwater pond were able to ingest molecules of the polysaccharide dextran of Mr> 500,000 (500K). Mixed species cultures of estuarine and pond flagellates also showed enhanced growth in the presence of 2,000K, but not 40K, dextran. Compounds with high-Mr are often a large fraction of total DOM in natural waters3,4, and the concentrations (dry weight litre−1) of some types of labile high-Mr compounds in natural waters are equal to, or greater than, those of bacterioplankton5,6. Thus DOM of high Mr may be an alternative food resource for aquatic flagellates. Direct ingestion of high Mr compounds by heterotrophic flagellates would represent a more efficient pathway for returning a portion of DOM to aquatic food webs compared with the DOM-bacteria-flagellate-ciliate microbial loop7.

Differential cell sinking as a factor influencing diatom species competition for limiting nutrients

The effect of differential cell sinking on competition between the diatoms Thalassiosira rotula Meunier (Clone 411) and Chaetoceros cf. vixvisibilis Schiller (Clone 847) for silicic acid and nitrate was examined in semicontinuous selective-loss culture in which the most rapidly sinking cells were removed preferentially. C. cf. vixvisibilis displaced T. rotula under silicic acid and nitrate limitation, regardless of whether the cultures were mixed immediately before the daily dilution (random loss) or fast-sinking cells were removed selectively (selective loss). However, the rate at which T. rotula was displaced increased in selective-loss cultures with either limiting nutrient. In addition, the daily removal of sunken cells selected against cells of both species with high C, N, and Si quotas. Differential cell sinking played only a minor rêle in the faster displacement of T. rotula in selective-loss culture, since both species had similar sinking rates in mixed-species culture. However, cell sinking did affect competition by increasing the supply rate of either limiting nutrient. Cell-removal rates exceeded the dilution rate in selective-loss culture, raising biomass-specific nutrient-supply rates above those in random-loss cultures operated at the same dilution rate. The resulting reduction in nutrient limitation led to higher steady-state growth rates, increased residual nutrient concentrations, and, in the case of N limitation, lower C : N ratios of cells from selective-loss cultures. These physiological responses enhanced the ability of C. cf. vixvisibilis to outcompete T. rotula for both Si and N. The results suggest that cell sinking may affect interspecific competition not only through the direct loss of cells from the euphotic zone, but also by altering the dynamics of nutrient competition among planktonic algae.

Differential recoveries from faecal cultures of larvae of some gastro-intestinal nematodes of cattle.

Faecal cultures were established using bovine faeces containing known numbers of eggs from either Oesophagostomum radiatum, Haemonchus placei, Cooperia pectinata or a mixture of all three. A substantially greater percentage of larvae was recovered from cultures of O. radiatum and C. pectinata than was recovered from cultures of H. placei. The same pattern was observed in mixed cultures although yields of larvae from all species in mixed cultures were significantly reduced (p less than 0.001). The lower recovery of H. placei was not associated with a lower viability of H. placei eggs. Of the three different methods of harvesting larvae, the jar and mesh recovery technique was the least effective and significantly fewer larvae were recovered with this technique than with the Baermann and inversion techniques (p less than 0.05). The results are discussed with reference to the use of faecal culture and larval differentiation in the diagnosis of mixed species nematode infections of cattle.