GC-MS-Based Metabolomics Study of Single- and Dual-Species Biofilms of Candida albicans and Klebsiella pneumoniae.
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ABSTRACT:
SUBMITTER: Galdiero E
PROVIDER: S-EPMC8037927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dental caries and periodontitis are the most common oral disease of all age groups, affecting billions of people worldwide. These oral diseases are mostly associated with microbial biofilms in the oral cavity. Streptococcus gordonii, an early tooth colonizing bacterium and Candida albicans, an opportunistic pathogenic fungus, are the two abundant oral microbes that form mixed biofilms with augmented virulence, affecting oral health negatively. Understanding the molecular mechanisms of the pathogen interactions and identifying non-toxic compounds that block the growth of biofilms are important steps in the development of effective therapeutic approaches. In this in vitro study we report the inhibition of mono-species or dual-species biofilms of S. gordonii and C. albicans, and decreased levels of biofilm extracellular DNA (eDNA), when biofilms were grown in the presence of gymnemic acids (GAs), a non-toxic small molecule inhibitor of fungal hyphae. Scanning electron microscopic images of biofilms on saliva-coated hydroxyapatite (sHA) surfaces revealed attachment of S. gordonii cells to C. albicans hyphae and to sHA surfaces via nanofibrils only in the untreated control, but not in the GAs-treated biofilms. Interestingly, C. albicans produced fibrillar adhesive structures from hyphae when grown with S. gordonii as a mixed biofilm; addition of GAs abrogated the nanofibrils and reduced the growth of both hyphae and the biofilm. To our knowledge, this is the first report that C. albicans produces adhesive fibrils from hyphae in response to S. gordonii mixed biofilm growth. Semi-quantitative PCR of selected genes related to biofilms from both microbes showed differential expression in control vs. treated biofilms. Further, GAs inhibited the activity of recombinant S. gordonii glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Taken together, our results suggest that S. gordonii stimulates the expression of adhesive materials in C. albicans by direct interaction and/or signaling, and the adhesive material expression can be inhibited by GAs.
| S-EPMC6797559 | biostudies-literature
Project description:Background: Streptococcus mutans (Sm) and Candida albicans (Ca) are found in biofilms of early childhood caries. Objective: To characterize in vitro dual- and single-species biofilms of Sm and Ca formed on saliva-coated hydroxyapatite discs in the presence of sucrose. Design: Evaluation of biofilms included biochemical [biomass, proteins, matrix's water-soluble (WSP) and alkali-soluble (ASP) polysaccharides, microbiological, 3D structure, gene expression, and stress tolerance analyses. Results: Biomass and proteins were higher for dual-species and lower for Ca (p = 0.001). Comparison of Sm single- and dual-species biofilms revealed no significant difference in Sm numbers or quantity of WSP (p > 0.05). Dual-species biofilms contained a higher population of Ca (p < 0.001). The quantity of ASP was higher in dual-species biofilms (vs Ca single-species biofilms; p = 0.002). The 3D structure showed larger microcolonies and distinct distribution of Sm-derived exopolysaccharides in dual-species biofilms. Compared with dual-species biofilms, expression of gtfB (ASP) and nox1 (oxidative stress) was higher for single-species of Sm whilst expression of BGL2 (matrix), PHR1 (matrix, acid tolerance) and SOD1 (oxidative stress) was higher in single-species of Ca. There was no difference for acid tolerance genes (Sm atpD and Ca PHR2), which was confirmed by acid tolerance challenge. Dual-species biofilms were more tolerant to oxidative and antimicrobial stresses (p < 0.05). Conclusions: Dual-species biofilms present greater 3D complexity, thereby, making them more resistant to stress conditions.
| S-EPMC6807867 | biostudies-literature
Project description:In recent years, the scientific community has focused on the development of new antibiotics to address the difficulties linked to biofilm-forming microorganisms and drug-resistant infections. In this respect, synthetic antimicrobial peptides (AMPs) are particularly regarded for their therapeutic potential against a broad spectrum of pathogens. In this work, the antimicrobial and antibiofilm activities of the peptide WMR-K towards single and dual species cultures of Candida albicans and Klebsiella pneumoniae were investigated. We found minimum inhibitory concentration (MIC) values for WMR-K of 10 µM for K. pneumoniae and of 200 µM for C. albicans. Furthermore, sub-MIC concentrations of peptide showed an in vitro inhibition of biofilm formation of mono and polymicrobial systems and also a good biofilm eradication even if higher concentrations of it are needed. In order to provide additional evidence for the effect of the examined peptide, a study of changes in extracellular metabolites excreted and/or uptaken from the culture medium (metabolomic footprinting) in the poly-microbial association of C. albicans and K. pneumoniae in presence and absence of WMR-K was performed. Comparing to the untreated dual species biofilm culture, the metabolomic profile of the WMR-K treated culture appears significantly altered. The differentially expressed compounds are mainly related to the primary metabolic pathways, including amino acids, trehalose, pyruvic acid, glycerol and vitamin B6.
| S-EPMC7920046 | biostudies-literature
Project description:IntroductionCombined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus-hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence.ObjectivesTo explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards.MethodsOptimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media.ResultsThe targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation.ConclusionEvaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids.
| S-EPMC5097782 | biostudies-literature
Project description:Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography-mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen.
| S-EPMC4992082 | biostudies-literature
Project description:BackgroundBiofilms are the leading cause of nosocomial infections and are hard to eradicate due to their inherent antimicrobial resistance. Candida albicans is the leading cause of nosocomial fungal infections and is frequently co-isolated with the bacterium Pseudomonas aeruginosa from biofilms in the cystic fibrosis lung and severe burn wounds. The presence of C. albicans in multispecies biofilms is associated with enhanced antibacterial resistance, which is largely mediated through fungal extracellular carbohydrates sequestering the antibiotics. However, significantly less is known regarding the impact of polymicrobial biofilms on antifungal resistance.ResultsHere we show that, in dual-species biofilms, P. aeruginosa enhances the susceptibility of C. albicans to amphotericin B, an effect that was biofilm specific. Transcriptional analysis combined with gene ontology enrichment analysis identified several C. albicans processes associated with oxidative stress to be differentially regulated in dual-species biofilms, suggesting that P. aeruginosa exerts oxidative stress on C. albicans, likely through the secretion of phenazines. However, the mitochondrial superoxide dismutase SOD2 was significantly down-regulated in the presence of P. aeruginosa. Monospecies biofilms of the sod2Δ mutant were more susceptible to amphotericin B, and the susceptibility of these biofilms was further enhanced by exogenous phenazines.ConclusionsWe propose that in dual-species biofilms, P. aeruginosa simultaneously induces mitochondrial oxidative stress, while down-regulating key detoxification enzymes, which prevents C. albicans mounting an appropriate oxidative stress response to amphotericin B, leading to fungal cell death. This work highlights the importance of understanding the impact of polymicrobial interactions on antimicrobial susceptibility.
| S-EPMC10477122 | biostudies-literature
Project description:Polymicrobial biofilms consisting of fungi and bacteria are frequently formed on endotracheal tubes and may contribute to development of ventilator associated pneumonia (VAP) in critically ill patients. This study aimed to determine the role of early Candida albicans biofilms in supporting dual-species (dual-kingdom) biofilm formation with respiratory pathogens in vitro, and investigated the effect of targeted antifungal treatment on bacterial cells within the biofilms. Dual-species biofilm formation between C. albicans and three respiratory pathogens commonly associated with VAP (Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) was studied using quantitative PCR. It was shown that early C. albicans biofilms enhanced the numbers of E. coli and S. aureus (including methicillin resistant S. aureus; MRSA) but not P. aeruginosa within dual-species biofilms. Transwell assays demonstrated that contact with C. albicans was required for the increased bacterial cell numbers observed. Total Internal Reflection Fluorescence microscopy showed that both wild type and hyphal-deficient C. albicans provided a scaffold for initial bacterial adhesion in dual species biofilms. qPCR results suggested that further maturation of the dual-species biofilm significantly increased bacterial cell numbers, except in the case of E.coli with hyphal-deficient C. albicans (Ca_gcn5Δ/Δ). A targeted preventative approach with liposomal amphotericin (AmBisome®) resulted in significantly decreased numbers of S. aureus in dual-species biofilms, as determined by propidium monoazide-modified qPCR. Similar results were observed when dual-species biofilms consisting of clinical isolates of C. albicans and MRSA were treated with liposomal amphotericin. However, reductions in E. coli numbers were not observed following liposomal amphotericin treatment. We conclude that early C. albicans biofilms have a key supporting role in dual-species biofilms by enhancing bacterial cell numbers during biofilm maturation. In the setting of increasing antibiotic resistance, an important and unexpected consequence of antifungal treatment of dual-species biofilms, is the additional benefit of decreased growth of multi-drug resistant bacteria such as MRSA, which could represent a novel future preventive strategy.
| S-EPMC8031443 | biostudies-literature
Project description:Candida albicans, the most pervasive fungal pathogen that colonizes humans, forms biofilms that are architecturally complex. They consist of a basal yeast cell polylayer and an upper region of hyphae encapsulated in extracellular matrix. However, biofilms formed in vitro vary as a result of the different conditions employed in models, the methods used to assess biofilm formation, strain differences, and, in a most dramatic fashion, the configuration of the mating type locus (MTL). Therefore, integrating data from different studies can lead to problems of interpretation if such variability is not taken into account. Here we review the conditions and factors that cause biofilm variation, with the goal of engendering awareness that more attention must be paid to the strains employed, the methods used to assess biofilm development, every aspect of the model employed, and the configuration of the MTL locus. We end by posing a set of questions that may be asked in comparing the results of different studies and developing protocols for new ones. This review should engender the notion that not all biofilms are created equal.
| S-EPMC4981664 | biostudies-other
Project description:The goals of this dual-seq experiment were to 1) identify transcriptional changes between mono-species and dual-species biofilms of Candida albicans and Pseudomonas aeruginosa and 2) identify transcriptional changes within mono- or dual-species P. aeruginosa biofilm cells in response to meropenem treatment.
2022-11-11 | GSE167137 | GEO
Project description:Candida albicans is among the most prevalent fungal species of the human microbiota and asymptomatically colonizes healthy individuals. However, it is also an opportunistic pathogen that can cause severe, and often fatal, bloodstream infections. The medical impact of C. albicans typically depends on its ability to form biofilms, which are closely packed communities of cells that attach to surfaces, such as tissues and implanted medical devices. In this Review, we provide an overview of the processes involved in the formation of C. albicans biofilms and discuss the core transcriptional network that regulates biofilm development. We also consider some of the advantages that biofilms provide to C. albicans in comparison with planktonic growth and explore polymicrobial biofilms that are formed by C. albicans and certain bacterial species.
| S-EPMC5726514 | biostudies-literature