Using metabolic networks to predict cross-feeding and competition interactions between microorganisms.

Understanding the interactions between microorganisms and their impact on bacterial behavior at the community level is a key research topic in microbiology. Different methods, relying on experimental or mathematical approaches based on the diverse properties of bacteria, are currently employed to study these interactions. Recently, the use of metabolic networks to understand the interactions between bacterial pairs has increased, highlighting the relevance of this approach in characterizing bacteria. In this study, we leverage the representation of bacteria through their metabolic networks to build a predictive model aimed at reducing the number of experimental assays required for designing bacterial consortia with specific behaviors. Our novel method for predicting cross-feeding or competition interactions between pairs of microorganisms utilizes metabolic network features. Machine learning classifiers are employed to determine the type of interaction from automatically reconstructed metabolic networks. Several algorithms were assessed and selected based on comprehensive testing and careful separation of manually compiled data sets obtained from literature sources. We used different classification algorithms, including K Nearest Neighbors, XGBoost, Support Vector Machine, and Random Forest, tested different parameter values, and implemented several data curation approaches to reduce the biological bias associated with our data set, ultimately achieving an accuracy of over 0.9. Our method holds substantial potential to advance the understanding of community behavior and contribute to the development of more effective approaches for consortia design.IMPORTANCEUnderstanding bacterial interactions at the community level is critical for microbiology, and leveraging metabolic networks presents an efficient and effective approach. The introduction of this novel method for predicting interactions through machine learning classifiers has the potential to advance the field by reducing the number of experimental assays required and contributing to the development of more effective bacterial consortia.

Exemplifying endophytes of banana (Musa paradisiaca) for their potential role in growth stimulation and management of Fusarium oxysporum f. sp cubense causing panama wilt.

In the present study, potentiality of endophytic microorganisms such as Rigidiporus vinctus AAU EF, Trichoderma reesei UH EF, and Sphingobacterium tabacisoli UH EB in the management of panama wilt and growth promotion of banana was assessed through artificial inoculation. During the study, a total of 220 bacterial and 110 fungal endophytes were isolated from root, pseudostem, and leaf samples of banana, and they were evaluated against Fusarium oxysporum f. sp cubense causing panama wilt. Out of total 330 bacterial and fungal endophytes, only five endophytes exhibited antagonism against Fusarium oxysporum f. sp cubense, out of which only three isolates, namely Trichoderma reesei UH EF, Rigidiporus vinctus AAU EF, and Sphingobacterium tabacisoli UH EB, produced indole acetic acid, siderophore, and hydrogen cyanide, except one bacterial strain Sphingobacterium tabacisoli UH EB which does not produce hydrogen cyanide. Furthermore, these three endophytes were identified through cultural and morphological characteristics as well as by the sequencing internal transcribed spacer (ITS) and 16S rRNA gene sequences analysis for bacteria, respectively. The response of host plant to endophyte inoculation was assessed by measuring the change in four growth parameters; plant height, pseudo stem girth (diameter), number of roots, and total number of leaves. The application of endophytes, irrespective of isolate and treatment type promoted the overall growth of the plant growth when compared with diseased plants with significant higher values recorded for all parameters assessed. The endophytes reported as growth promoters were found to have significant inhibition effect on Foc which can evidenced with lowest AUDPC values and epidemic rate at 99.09 units2 and 0.02 unit/day, respectively.

Dynamics of volatilomes emitted during cross-talking of plant-growth-promoting bacteria and the phytopathogen, Fusarium solani.

The dynamics of volatilomes emitted during the interaction between plant-growth-promoting bacteria (PGPB) and the phytopathogen Fusarium solani were evaluated for 5 days. The first screening was done to evaluate the antagonist activity of volatile compounds emitted by PGPB against F. solani. Volatilomes from 11 PGPB were determined individually and together with F. solani by using solid-phase microextraction coupled to gas-chromatography-mass spectrometry. Isolates of PGPB belonged to the Bacillus genus and inhibited from 18 to 24% the fungal mycelium growth. The isolates also induced morphological alterations of fungal hyphae, like small globular vesicles and the formation of chlamydospores, suggesting a stress mechanism response by the fungus. Volatilome profile showed 49 different compounds that appeared in the bacterial-fungal interaction, such as ketones, sesquiterpenes, monoterpenoids, alkanes, alkenes, carboxylic acids, and fatty acids. Some ketones and alcohols were detected in high abundance only in the interaction PGPB-fungus at 3 and 5 days. Bacillus circulans A19, Bacillus amyloliquefaciens A21, and Bacillus wiedmannii S18 shared a group of emitted alcohols and ketones when they were exposed to F. solani. F. solani produced its own volatilome profile, with the presence of sesquiterpenes, such as α-cubebene and caryophyllene, which increased significantly in co-incubation with the tested bacteria, suggesting chemical communication between them.

The contest of microbial pigeon neighbors: Interspecies competition between Serratia marcescens and the human pathogen Cryptococcus neoformans.

In nature, microorganisms often exhibit competitive behavior for nutrients and limited space, allowing them to alter the virulence determinants of pathogens. The human pathogenic yeast Cryptococcus neoformans can be found organized in biofilms, a complex community composed of an extracellular matrix which confers protection against predation. The aim of this study was to evaluate and characterize antagonistic interactions between two cohabiting microorganisms: C. neoformans and the bacteria Serratia marcescens. The interaction of S. marcescens with C. neoformans expressed a negative effect on biofilm formation, polysaccharide capsule, production of urease, and melanization of the yeast. These findings evidence that competition in mixed communities can result in dominance by one species, with direct impact on the physiological modulation of virulence determinants. Such an approach is key for understating the response of communities to the presence of competitors and, ultimately, rationally designing communities to prevent and treat certain diseases.

Negative interaction of Staphylococcus aureus on Fusarium falciforme growth ocular isolates in an in vitro mixed biofilm.

The interactions between prokaryotes and eukaryotes are abundant in nature. These microorganisms also interact in the human body. Fungal-bacteria interactions are present in many diseases. In this study, we evaluated the microbial interaction of Fusarium falciforme and Staphylococcus aureus developing mixed biofilm in vitro. When both microorganisms grew up together the mixed biofilm biomass decreased than F. falciforme monobiofilm biomass. S. aureus was able to interact and form aggregates over the mycelium and conidia surface of F. falciforme. Our results suggest that S. aureus could bind to colloidal chitin. On another hand, the supernatants from S. aureus biofilm and S. aureus-F. falciforme presented an antifungal effect over F. falciforme biofilm formation. Finally we found that the pH had an inhibitory effect over fungal biofilm formation. We concluded that S. aureus can affect the F. falciforme growth negatively in mixed biofilm involving factors like pH, supernatants compounds, anchor to chitin, and bacterial viability.

Metabolic potential of uncultured bacteria and archaea associated with petroleum seepage in deep-sea sediments.

The lack of microbial genomes and isolates from the deep seabed means that very little is known about the ecology of this vast habitat. Here, we investigate energy and carbon acquisition strategies of microbial communities from three deep seabed petroleum seeps (3 km water depth) in the Eastern Gulf of Mexico. Shotgun metagenomic analysis reveals that each sediment harbors diverse communities of chemoheterotrophs and chemolithotrophs. We recovered 82 metagenome-assembled genomes affiliated with 21 different archaeal and bacterial phyla. Multiple genomes encode enzymes for anaerobic oxidation of aliphatic and aromatic compounds, including those of candidate phyla Aerophobetes, Aminicenantes, TA06 and Bathyarchaeota. Microbial interactions are predicted to be driven by acetate and molecular hydrogen. These findings are supported by sediment geochemistry, metabolomics, and thermodynamic modelling. Overall, we infer that deep-sea sediments experiencing thermogenic hydrocarbon inputs harbor phylogenetically and functionally diverse communities potentially sustained through anaerobic hydrocarbon, acetate and hydrogen metabolism.

Quorum sensing communication: Bradyrhizobium-Azospirillum interaction via N-acyl-homoserine lactones in the promotion of soybean symbiosis.

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.

Influence of Streptococcus mitis and Streptococcus sanguinis on virulence of Candida albicans: in vitro and in vivo studies.

The aim was to evaluate in vitro possible interactions, gene expression, and biofilm formation in species of Candida albicans, Streptococcus mitis, and Streptococcus sanguinis and their in vivo pathogenicity. The in vitro analysis evaluated the effects of S. mitis and S. sanguinis on C. albicans's biofilm formation by CFU count, filamentation capacity, and adhesion (ALS1, ALS3, HWP1) and transcriptional regulatory gene (BCR1, CPH1, EFG1) expression. In vivo studies evaluated the pathogenicity of the interaction of the microorganisms on Galleria mellonella, with analyses of the CFU per milliliter count and filamentation. In vitro results indicated that there was an observed decrease in CFU (79.4-71.5%) in multi-species biofilms. The interaction with S. mitis inhibited filamentation, which seems to increase its virulence factor with over-expression of genes ALS1, ALS3, and HWP1 as well the interaction with S. sanguinis as ALS3 and HWP1. S. mitis upregulated BRC1, CPH1, and EFG1. The histological images of in vivo study indicate an increase in the filamentation of C. albicans when in interaction with the other species. It was concluded that S. mitis interaction suggests increased virulence factors of C. albicans, with periods of lower virulence and proto-cooperation in the interaction with S. sanguinis.

Chemical interaction of endophytic fungi and actinobacteria from Lychnophora ericoides in co-cultures.

Microorganisms interact chemically in natural environments; however, the compounds and mechanisms involved in this phenomenon are still poorly understood. Using the cocultivation approach, changes in metabolic profiles due to interactions between endophytic fungal and actinobacterial strains isolated from the plant Lychnophora ericoides (Asteraceae) were assessed. The production of the cytotoxic compound cytochalasin H by the fungus Phomopsis sp. FLe6 was remarkably inhibited in solid and liquid co-cultures with the actinobacteria Streptomyces albospinus RLe7. This was a consequence of the fungal growth inhibition caused by antifungal compounds produced by S. albospinus RLe7, including amphotericin B. Cytochalasin H is not toxic to S. albospinus RLe7, suggesting that this microorganism does not require a defense mechanism to prevent the potentially harmful effects of such fungal compound. By exhibiting various competitive phenotypes, these microbes can control each other's growth when sharing an environment.

Lactobacillus spp. impair the ability of Listeria monocytogenes FBUNT to adhere to and invade Caco-2 cells.

OBJECTIVES: The objective of this study was to evaluate the ability of Lactobacillus curvatus CRL705, CRL1532, and CRL1533 and Lactobacillus sakei CRL1613 to survive under simulated gastrointestinal conditions. Moreover, a microencapsulation approach was proposed to improve gastrointestinal survival. Finally, experiments were performed to demonstrate that Lactobacillus spp. can modulate the ability of Listeria monocytogenes FBUNT to adhere to and invade Caco-2 cells. RESULTS: Lactobacillus strains were encapsulated in alginate beads to enhance the survival of bacteria under in vitro gastrointestinal conditions. All strains hydrolyzed bile salts using chenodeoxycholic acid as a substrate and adhered to Caco-2 cells. Cell-free supernatants (CFSs) showed antimicrobial activity against L. monocytogenes as demonstrated by agar diffusion assays. The average percentages of L. monocytogenes adhesion decreased from 67.74 to 41.75 and 38.7% in the presence of 50 and 90% (v/v), respectively, for all CFSs tested. The highest concentrations of CFSs completely inhibited the L. monocytogenes invasion of Caco-2 cells. CONCLUSIONS: The studied Lactobacillus strains have protective effects against the adhesion and invasion of L. monocytogenes FBUNT. Alginate encapsulation of these bacteria improved gastrointestinal tolerance such that they could be further studied as potential probiotics against intestinal pathogenic bacteria.

Dual-species biofilm of Listeria monocytogenes and Escherichia coli on stainless steel surface.

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σB, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.

An ascomycota coculture in batch bioreactor is better than polycultures for cellulase production.

Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, ß-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, ß-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and ß-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.

Azospirillum brasilense Increases CO2 Fixation on Microalgae Scenedesmus obliquus, Chlorella vulgaris, and Chlamydomonas reinhardtii Cultured on High CO2 Concentrations.

Mutualism interactions of microalgae with other microorganisms are widely used in several biotechnological processes since symbiotic interaction improves biotechnological capabilities of the microorganisms involved. The interaction of the bacterium Azospirillum brasilense was assessed with three microalgae genus, Scenedesmus, Chlorella, and Chlamydomonas, during CO2 fixation under high CO2 concentrations. The results in this study have demonstrated that A. brasilense maintained a mutualistic interaction with the three microalgae assessed, supported by the metabolic exchange of indole-3-acetic acid (IAA) and tryptophan (Trp), respectively. Besides, CO2 fixation increased, as well as growth and cell compound accumulation, mainly carbohydrates, in each microalgae evaluated, interacting with the bacterium. Overall, these results propose the mutualism interaction of A. brasilense with microalgae for improving biotechnological processes based on microalgae as CO2 capture and their bio-refinery capacity.

Antifungal activity of avocado rhizobacteria against Fusarium euwallaceae and Graphium spp., associated with Euwallacea spp. nr. fornicatus, and Phytophthora cinnamomi.

Plant rhizobacteria have been successfully used as biocontrol agents against fungal phytopathogens. However, their potential to control two important avocado diseases, namely Fusarium dieback (FD) and Phytophthora root rot (PRR), has been poorly studied. FD is an emerging disease triggered by fungi associated with two ambrosia beetle species (Euwallacea fornicatus species complex), while PRR is caused by Phytophthora cinnamomi, a soil-borne oomycete. In the present work, the antifungal activity of bacteria isolated from avocado rhizosphere was tested in dual culture assays against Fusarium euwallaceae, Graphium euwallaceae and Graphium sp., causal agents of FD, and against P. cinnamomi. In 2015, rhizosphere soil samples of FD infested and non-infested avocado trees were collected from a commercial avocado orchard in Escondido, California. In an initial screening, 72 of the 168 assessed bacterial isolates reduced mycelial growth of F. euwallaceae by up to 46%. Eight bacterial isolates showing inhibition percentages larger than 40% were then selected for further antagonism assays against the other fungal pathogens. Five bacterial isolates, determined by 16S rDNA sequencing to belong to the Bacillus subtilis/Bacillus amyloliquefaciens species complex, successfully inhibited the mycelial growth of both Graphium species by up to 30%. The same isolates and an additional isolate identified as Bacillus mycoides, inhibited the growth of P. cinnamomi by up to 25%. This is the first report of avocado rhizobacteria with antifungal activity against pathogens responsible for FD and PRR in avocado.

Differential oxidative stress tolerance of Streptococcus mutans isolates affects competition in an ecological mixed-species biofilm model.

Streptococcus mutans strongly influences the development of pathogenic biofilms associated with dental caries. Our understanding of S. mutans behaviour in biofilms is based on a few well-characterized laboratory strains; however, individual isolates vary widely in genome content and virulence-associated phenotypes, such as biofilm formation and environmental stress sensitivity. Using an ecological biofilm model, we assessed the impact of co-cultivation of several S. mutans isolates with Streptococcus oralis and Actinomyces naeslundii on biofilm composition following exposure to sucrose. The laboratory reference strain S. mutans UA159 and clinical isolates Smu44 (most aciduric), Smu56 (altered biofilm formation) and Smu81 (more sensitive to oxidative stress) were used. Our data revealed S. mutans isolates varied in their ability to compete and become dominant in the biofilm after the addition of sucrose, and this difference correlated with sensitivity to H2 O2 produced by S. oralis. Smu81 was particularly sensitive to H2 O2 and could not compete with S. oralis in mixed-species biofilm, despite forming robust biofilms on its own. Thus, diminished oxidative stress tolerance in S. mutans isolates can impair their ability to compete in complex biofilms, even in the presence of sucrose, which could influence the progression of a healthy biofilm community to one capable of causing disease.

Unveiling microbial interactions in stratified mat communities from a warm saline shallow pond.

Modern phototrophic microbial mats are complex communities often used as analogs of major Precambrian ecosystems. Characterizing biotic, notably metabolic, interactions among different microbial mat members is essential to gain insights into the ecology and biogeochemistry of these systems. We applied 16S/18S rRNA metabarcoding approaches to characterize the structure of archaea, bacteria and protist communities from microbial mats collected along strong physicochemical (oxygen, salinity, temperature, depth) gradients in a shallow pond at the salar de Llamara (Chile). All mats were highly diverse, including members of virtually all known high-rank eukaryotic and prokaryotic taxa but also many novel lineages. Bacterial candidate divisions accounted for almost 50% of sequences in deeper mats, while Archaea represented up to 40% of sequences in some mat layers. Molecular phylogenetic analyses revealed six novel deeply divergent archaeal groups, along abundant and diverse Pacearchaeota and Woesearchaeota. Multivariate statistical analyses showed that local environmental conditions strongly influenced community composition. Co-occurrence network structure was markedly different between surface mats located in the oxygenated zone and mats located in transition and anoxic water layers. We identified potential biotic interactions between various high- and low-rank taxa. Notably, a strong positive correlation was observed between Lokiarchaeota and the poorly known candidate bacterial division TA06.

Inhibition of mycotoxin-producing fungi by Bacillus strains isolated from fish intestines.

Bacillus strains isolated from the aquatic environment of the Brazilian Amazon region were tested for their activity against mycotoxigenic fungi. All tested bacteria showed antifungal activity, inhibiting at least 7 indicator fungi. Four Bacillus strains showing promising antifungal results were subsequently evaluated for their activity in reducing mycelial growth rate, sporulation, spore germination percentage, and mycotoxin production. Bacillus sp. P1 and Bacillus sp. P11 had a remarkable antifungal effect on toxigenic fungi. Washed bacterial cell suspension of strains P1 and P11 (107CFU/ml) reduced by >70% the fungal colony diameters, including a complete inhibition of ochratoxin A (OTA) producing Aspergillus spp. Significant reduction of growth rate, sporulation and spore germination were also observed. The bacteria influenced the production of mycotoxins, causing a reduction around 99 and 97% in AFB1 and OTA concentration, respectively. Chromatographic analysis revealed the presence of lipopeptides (iturin A and surfactin isomers) in butanol extracts of cell-free supernatants and cell pellets of strains P1 and P11. Furthermore, antifungal activity of these extracts was confirmed against A. flavus A12 and A. carbonarius ITAL293, producers of AFB1 and OTA, respectively. These bacterial strains could be promising biocontrol agents against toxigenic fungi.

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions.

Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata and Candida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (10(7) cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a single C. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions

Abstract Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata andCandida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (107 cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a singleC. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.