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INTRODUCTION: Intestinal fibrosis is a common and serious complication of inflammatory bowel diseases (IBD) driving stricture formation in Crohn's disease patients and leading to submucosal damage in ulcerative colitis. Recent studies provided novel insights into the role of immune and nonimmune components in the pathogenesis of intestinal fibrosis. Those new findings may accelerate the development of anti-fibrotic treatment in IBD patients. AREAS COVERED: This review is designed to cover the recent progress in mechanistic research and therapeutic developments on intestinal fibrosis in IBD patients, including new cell clusters, cytokines, proteins, microbiota, creeping fat, and anti-fibrotic therapies. EXPERT OPINION: Due to the previously existing major obstacle of missing consensus on stricture definitions and the absence of clinical trial endpoints, testing of drugs with an anti-fibrotic mechanism is just starting in stricturing Crohn's disease (CD). A biomarker to stratify CD patients at diagnosis without any complications into at-risk populations for future strictures would be highly desirable. Further investigations are needed to identify novel mechanisms of fibrogenesis in the intestine that are targetable and ideally gut specific.
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Fibrose , Doenças Inflamatórias Intestinais , Humanos , Animais , Doenças Inflamatórias Intestinais/imunologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Intestinos/imunologia , Intestinos/patologia , Biomarcadores , Microbioma Gastrointestinal/imunologia , Citocinas/metabolismo , Citocinas/imunologiaRESUMO
OBJECTIVE: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvß5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.
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Antígenos de Superfície , Doença de Crohn , Matriz Extracelular , Fibrose , Proteínas do Leite , Humanos , Animais , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Proteínas do Leite/metabolismo , Proteínas do Leite/farmacologia , Antígenos de Superfície/metabolismo , Matriz Extracelular/metabolismo , Miofibroblastos/metabolismo , Modelos Animais de Doenças , Camundongos , RatosRESUMO
BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.
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Colite , Doença de Crohn , Animais , Doença de Crohn/genética , Doença de Crohn/patologia , Constrição Patológica , Intestinos/patologia , Colite/patologia , Fibroblastos/patologiaRESUMO
Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.
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OBJECTIVE: Inflammatory bowel diseases (IBD) cause chronic intestinal damage and extracellular matrix (ECM) remodeling. The ECM may play an active role in inflammation by modulating immune cell functions, including cell adhesion, but this hypothesis has not been tested in IBD. DESIGN: Primary human intestinal myofibroblast (HIMF)-derived ECM from IBD and controls, 3D decellularized colon or ECM molecule-coated scaffolds were tested for their adhesiveness for T cells. Matrisome was analysed via proteomics. Functional integrin blockade was used to investigate the underlying mechanism. Analysis of the pediatric Crohn's disease (CD) RISK inception cohort was used to explore an altered ECM gene expression as a potential predictor for a future complicated disease course. RESULTS: HIMF-derived ECM and 3D decellularized colonic ECM from IBD bound more T cells compared to control. Control HIMFs exposed to the pro-inflammatory cytokines Iinterleukin-1ß (IL-1ß) and tumor necrosis factor (TNF) increased, and to transforming growth factor-ß1 (TGF-ß1) decreased ECM adhesiveness to T cells. Matrisome analysis of the HIMF-derived ECM revealed collagen VI as a major culprit for differences in T cell adhesion. Collagen VI knockdown in HIMF reduced adhesion T cell as did the blockage of integrin αvß1. Elevated gene expression of collagen VI in biopsies of pediatric CD patients was linked to risk for future stricturing disease. CONCLUSION: HIMF-derived ECM in IBD binds a remarkably enhanced number of T cells, which is dependent on Collagen VI and integrin αvß1. Collagen VI expression is a risk factor for a future complicated CD course. Blocking immune cells retention may represent a novel approach to treatment in IBD.
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Doenças Inflamatórias Intestinais , Miofibroblastos , Criança , Humanos , Miofibroblastos/metabolismo , Adesividade , Linfócitos T/patologia , Colágeno/metabolismo , Inflamação/metabolismoRESUMO
OBJECTIVE: Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn's disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. DESIGN: Tissues from normal, UC, non-strictured and strictured Crohn's disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals. RESULTS: Crohn's disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-ß1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5ß1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix. CONCLUSION: Crohn's disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.
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Adipócitos/patologia , Movimento Celular , Doença de Crohn/patologia , Intestinos/patologia , Músculo Liso/patologia , Adipogenia/fisiologia , Tecido Adiposo/patologia , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/patologia , Fibronectinas/metabolismo , Humanos , Alicerces TeciduaisRESUMO
Mouse models are essential for investigation of underlying disease mechanisms that drive intestinal fibrosis, as well as assessment of potential therapeutic approaches to either prevent or resolve fibrosis. Here we describe several common mouse models of intestinal inflammation and fibrosis, including chemically driven colitis models, a bacterially triggered colitis model, and spontaneous intestinal inflammation in genetically susceptible mouse strains. Detailed protocols are provided for dextran sodium sulfate (DSS) colitis, 2,4,6-trinitro-benzene sulfonic acid (TNBS) colitis, adherent-invasive Escherichia coli (AIEC)-triggered colitis, the interleukin-10 knockout (IL-10KO) mouse model of spontaneous colitis, and the SAMP/YitFc model of spontaneous ileocolitis.
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Colite/etiologia , Colite/patologia , Interleucina-10/genética , Animais , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Escherichia coli/patogenicidade , Humanos , Masculino , Camundongos , Camundongos Knockout , Ácido Trinitrobenzenossulfônico/efeitos adversosRESUMO
Dysbiosis of the gut microbiome has been implicated in inflammatory bowel diseases. We have shown that levels of Candida tropicalis, along with those of Escherichia coli and Serratia marcescens, are significantly elevated in Crohn's disease (CD) patients. Here, we evaluated the ability of a novel probiotic to prevent and treat polymicrobial biofilms (PMB) formed by C. tropicalis with E. coli and S. marcescens Since Candida albicans has been reported to be elevated in CD patients, we investigated the interactions of C. albicans with these bacterial species in biofilm formation. We determined whether the interaction between Candida spp. and bacteria is specific by using Trichosporon inkin and Saccharomyces fibuligera as comparators. Additionally, the effects of probiotics on C. albicans germination and biofilm formation were determined. To determine the ability of the probiotic to prevent or treat mature biofilms, probiotic filtrate was added to the PMB at early (prevention) and mature (treatment) phases. Biofilm thickness and architecture were assessed by confocal scanning laser microscopy. The effects of the probiotic on germination were evaluated in the presence of serum. Exposure of C. tropicalis PMB to probiotic filtrate reduced biofilm matrix, decreased thickness, and inhibited hyphal formation. We showed that C. albicans or C. tropicalis formed significantly thicker PMB than control biofilms, indicating that this interaction is Candida specific. Treatment with probiotic filtrate inhibited C. albicans germination and prevented/treated C. albicans PMB. The designed probiotic may have utility in the management of biofilm-associated gastrointestinal diseases such as Crohn's and colorectal cancer.IMPORTANCE The effects of diversity of the gut microbiome on inflammation have centered mainly on bacterial flora. Recent research has implicated fungal species and their interactions with other organisms in the inflammatory process. New ways to restore microbial balance in the gut are being explored. Our goal was to identify beneficial probiotic strains that would antagonize these fungal and bacterial pathogens that are elevated in the inflamed gut, and which also have antibiofilm activity. Fungus-bacterium correlation analysis allowed us to identify candidate probiotic species that can antagonize microbial pathogens, which we subsequently incorporated into a novel probiotic formulation. Amylase, which is known to have some antibiofilm activity, was also added to the probiotic mixture. This novel probiotic may have utility for the management of inflammatory bowel diseases by disrupting polymicrobial biofilm formation.
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Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Probióticos/farmacologia , Serratia marcescens/crescimento & desenvolvimento , Antibiose , Candida tropicalis/crescimento & desenvolvimento , Interações MicrobianasRESUMO
Crohn's disease (CD) is a chronic inflammatory disease, which could affect any part of the gastrointestinal tract. A severe complication of CD is fibrosis-associated strictures, which can cause bowel obstruction. Unfortunately, there is no specific antifibrotic therapy available. More than 80% of the patients with CD will have to undergo at least 1 surgery in their life and recurrence of strictures after surgery is common. Investigations on the mechanism of fibrostenosing CD have revealed that fibrosis is mainly driven by expansion of mesenchymal cells including fibroblasts, myofibroblasts, and smooth muscle cells. Being exposed to a pro-fibrotic milieu, these cells increase the secretion of extracellular matrix, as well as crosslinking enzymes, which drive tissue stiffness and remodeling. Fibrogenesis can become independent of inflammation in later stages of disease, which offers unique therapeutic potential. Exciting new evidence suggests smooth muscle cell hyperplasia as a strong contributor to luminal narrowing in fibrostenotic CD. Approval of new drugs in other fibrotic diseases, such as idiopathic pulmonary fibrosis, as well as new targets associated with fibrosis found in CD, such as cadherins or specific integrins, shed light on the development of novel antifibrotic approaches in CD.
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Doença de Crohn/etiologia , Doença de Crohn/patologia , Animais , Constrição Patológica , Doença de Crohn/microbiologia , Modelos Animais de Doenças , Fibrose , Humanos , Inflamação/patologia , MicrobiotaRESUMO
INTRODUCTION: Atopic dermatitis (AD) is associated with changes in skin bacterial microbiome. Emollient treatment induces change in bacterial microbiome in AD, but its effect on fungal microbiome ("mycobiome") and their inter-kingdom correlations is unknown. We used Ion-Torrent sequencing to characterize the mycobiome of AD patients in response to emollient treatment. METHODS: Skin swabs were collected from lesional and non-lesional skin of AD patients suffering from moderate AD, after informed consent and according to GCP guidelines. Genomic DNA was extracted from each swab using the MoBio PowerSoil DNA Isolation kit and used for mycobiome sequencing analyses as described in our earlier publications. Principal coordinates analyses (PCoA), diversity, abundance, and correlations analyses were conducted in R and relevant packages using non-parametric tests (P less than .05 was significant). RESULTS: Swab samples from 10 patients (7 females, 3 males; mean age, 10.5 years) were analyzed. Emollient treatment induced a significant reduction of Scoring Atopic Dermatitis (SCORAD) score (P less than .001). PCoA showed pre-treatment and post-treatment samples clustered differently at all taxa levels. Six genera were detected in only non-lesional samples, while four were detected in only lesional samples. In non-lesional samples, Shannon diversity index was significantly increased after emollient treatment (P less than equal to .04), while lesional skin exhibited non-significant decrease. Ascomycota was the most abundant phylum and Dothideomycetes was the most abundant Class in most samples. Eight fungal species were either significantly different (P less than .05) or showed a strong trend (P less than .1) between pre- and post-treatment samples of lesional and non-lesional skin. In lesional skin, Gram-negative Pseudomonas spp. correlated significantly with pathogenic fungal species (Aspergillus, Candida spp.) in pre-treatment samples; these correlations were not detected in post-treatment samples. Moreover, lesional skin exhibited significant correlations between Gram-positive bacteria (Corynebacterium kroppenstedtiian and Staphylococcus pettenkoferi) and pathogenic Candida species in pre-treatment samples, but not in post- treated samples. DISCUSSION: Emollient treatment may induce beneficial microbial changes in the mycobiome and augment host-microbe balance on skin in AD. Clinical relevance of these results need to be investigated. J Drugs Dermatol. 2018;17(10):1039-1048.
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Dermatite Atópica/tratamento farmacológico , Emolientes/uso terapêutico , Microbiota/efeitos dos fármacos , Administração Tópica , Aspergillus/isolamento & purificação , Candida/isolamento & purificação , Criança , Dermatite Atópica/microbiologia , Emolientes/farmacologia , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Masculino , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The effect of smoking on microbial dysbiosis and the potential consequence of such shift on markers of HIV disease is unknown. Here we assessed the relationship of microbial dysbiosis with smoking and markers of HIV disease. METHODS: Oral wash was collected from: (1) HIV-infected smokers (HIV-SM, n = 48), (2) HIV-infected non-smokers (HIV-NS, n = 24), or (3) HIV-uninfected smokers (UI-SM, n = 24). Microbial DNA was extracted and their bacterial and fungal microbiota (bacteriome and mycobiome, respectively) were characterized using Ion-Torrent sequencing platform. Sequencing data were compared using clustering, diversity, abundance and inter-kingdom correlations analyses. RESULTS: Bacteriome was more widely dispersed than mycobiome, there was no noticeable difference in clustering between groups. Richness of oral bacteriome in HIV-SM was significantly lower than that of UI-SM (P ≤ .03). Diversity of HIV-NS was significantly lower than that of HIV-SM or UI-SM at phylum level (P ≤ .02). Abundance of Phylum Firmicutes was significantly decreased in HIV-NS compared to HIV-SM and UI-SM (P = .007 and .027, respectively), while abundance of Proteobacteria was significantly increased in HIV-NS compared to HIV-SM and UI-SM (P = .0005 and .011, respectively). Fungal phyla did not differ significantly between the three cohorts. Cumulative smoking was positively correlated with Facklamia but negatively with Enhydrobacter, and current alcohol use was negatively correlated with Geniculata. Bacteria Facklamia exhibited weakly positive correlation with longer PI duration (r = 0.094, P = 0.012), and a negative correlation with nadir CD4 count (r = -0.345; P = 0.004), while Granulicatella was negatively correlated with nadir CD4 count (r = -0.329; P = 0.007). Fungus Stemphylium correlated negatively with nadir CD4 (r = -0.323; P = 0.008). CONCLUSIONS: Dysbiosis of the oral microbiota is associated with clinical and immunologic variables in HIV-infected patients.
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Disbiose/microbiologia , Infecções por HIV/microbiologia , Microbiota , Boca/microbiologia , Micobioma , Adulto , Contagem de Linfócito CD4 , DNA Bacteriano/genética , DNA Fúngico/genética , Disbiose/imunologia , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Microbiota/genética , Pessoa de Meia-Idade , Micobioma/genética , Análise de Sequência de DNA , Fumar/efeitos adversosAssuntos
Anti-Infecciosos Locais/administração & dosagem , Etanol/farmacologia , Desinfecção das Mãos/métodos , Controle de Infecções/métodos , Microbiota/efeitos dos fármacos , Transplante de Células-Tronco , Adulto , Estudos de Coortes , Feminino , Humanos , Pacientes Internados/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Valores de Referência , Sensibilidade e Especificidade , TransplantadosRESUMO
Currently available echinocandins are generally effective against Candida biofilms, but the recent emergence of resistance has underscored the importance of developing new antifungal agents that are effective against biofilms. CD101 is a long-acting novel echinocandin with distinctive pharmacokinetic properties and improved stability and safety relative to other drugs in the same class. CD101 is currently being evaluated as a once-weekly intravenous (i.v.) infusion for the treatment of candidemia and invasive candidiasis. In this study, we determined (i) the effect of CD101 against early and mature phase biofilms formed by C. albicansin vitro and (ii) the temporal effect of CD101 on the formation of biofilms using time-lapse microscopy (TLM). Early- or mature-phase biofilms were formed on silicone elastomer discs and were exposed to the test compounds for 24 h and quantified by measuring their metabolic activity. Separate batches were observed under a confocal microscope or used to capture TLM images from 0 to 16 h. Measurements of their metabolic activity showed that CD101 (0.25 or 1 µg/ml) significantly prevented adhesion-phase cells from developing into mature biofilms (P = 0.0062 or 0.0064, respectively) and eradicated preformed mature biofilms (P = 0.04 or 0.01, respectively) compared to those of untreated controls. Confocal microscopy showed significant reductions in biofilm thicknesses for both early and mature phases (P < 0.05). TLM showed that CD101 stopped the growth of adhesion- and early-phase biofilms within minutes. CD101-treated hyphae failed to grow into mature biofilms. These results suggest that CD101 may be effective in the prevention and treatment of biofilm-associated nosocomial infections.
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Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Equinocandinas/farmacologia , Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Candidíase Invasiva/tratamento farmacológico , Candidíase Invasiva/microbiologia , Farmacorresistência Fúngica/efeitos dos fármacosRESUMO
Candidaauris, a new multidrug-resistant Candida spp. which is associated with invasive infection and high rates of mortality, has recently emerged. Here, we determined the virulence factors (germination, adherence, biofilm formation, phospholipase and proteinase production) of 16 C. auris isolates and their susceptibilities to 11 drugs belonging to different antifungal classes, including a novel orally bioavailable 1,3-ß-d-glucan synthesis inhibitor (SCY-078). We also examined the effect of SCY-078 on the growth, ultrastructure, and biofilm-forming abilities of C. auris Our data showed that while the tested strains did not germinate, they did produce phospholipase and proteinase in a strain-dependent manner and had a significantly reduced ability to adhere and form biofilms compared to that of Candida albicans (P = 0.01). C. auris isolates demonstrated reduced susceptibility to fluconazole and amphotericin B, while, in general, they were susceptible to the remaining drugs tested. SCY-078 had an MIC90 of 1 mg/liter against C. auris and caused complete inhibition of the growth of C. auris and C. albicans Scanning electron microscopy analysis showed that SCY-078 interrupted C. auris cell division, with the organism forming abnormal fused fungal cells. Additionally, SCY-078 possessed potent antibiofilm activity, wherein treated biofilms demonstrated significantly reduced metabolic activity and a significantly reduced thickness compared to the untreated control (P < 0.05 for both comparisons). Our study shows that C. auris expresses several virulence determinants (albeit to a lesser extent than C. albicans) and is resistant to fluconazole and amphotericin B. SCY-078, the new orally bioavailable antifungal, had potent antifungal/antibiofilm activity against C. auris, indicating that further evaluation of this antifungal is warranted.
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Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Candida/patogenicidade , Glicosídeos/farmacologia , Triterpenos/farmacologia , Anfotericina B/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida/crescimento & desenvolvimento , Candida/isolamento & purificação , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Adesão Celular , Divisão Celular/efeitos dos fármacos , Farmacorresistência Fúngica Múltipla , Fluconazol/farmacologia , Glucanos/biossíntese , Humanos , Testes de Sensibilidade Microbiana , Peptídeo Hidrolases/biossíntese , Fosfolipases/biossíntese , Fatores de VirulênciaRESUMO
A prospective exploratory study was conducted to characterize the oral mycobiome at baseline and determine whether changes occur after admission to the intensive care unit (ICU). We found that ICU admission is associated with alterations in the oral mycobiome, including an overall increase in Candida albicans.
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Candida albicans/isolamento & purificação , Candidíase Bucal/transmissão , Infecção Hospitalar/transmissão , Unidades de Terapia Intensiva , Micobioma/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Candidíase Bucal/microbiologia , Candidíase Bucal/prevenção & controle , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Técnicas de Tipagem Micológica , Estudos Prospectivos , Fatores de Risco , Estados Unidos/epidemiologia , Adulto JovemRESUMO
Organisms residing in the oral cavity (oral microbiota) contribute to health and disease, and influence diseases like gingivitis, periodontitis, and oral candidiasis (the most common oral complication of HIV-infection). These organisms are also associated with cancer and other systemic diseases including upper respiratory infections. There is limited knowledge regarding how oral microbes interact together and influence the host immune system. Characterizing the oral microbial community (oral microbiota) in health and disease represents a critical step in gaining insight into various members of this community. While most of the studies characterizing oral microbiota have focused on bacterial community, there are few encouraging studies characterizing the oral mycobiome (the fungal component of the oral microbiota). Our group recently characterized the oral mycobiome in health and disease focusing on HIV. In this chapter we will describe the methods used by our group for characterization of the oral mycobiome.
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Fungos , Microbiota , Boca/microbiologia , DNA Fúngico , DNA Intergênico , Fungos/classificação , Fungos/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da PolimeraseRESUMO
Intravascular device-related infections are often associated with biofilms (microbial communities encased within a polysaccharide-rich extracellular matrix) formed by pathogens on the surfaces of these devices. Candida species are the most common fungi isolated from catheter-, denture-, and voice prosthesis-associated infections and also are commonly isolated from contact lens-related infections (e.g., fungal keratitis). These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Recent technological advances have facilitated the development of novel approaches to investigate the formation of biofilms and identify specific markers for biofilms. These studies have provided extensive knowledge of the effect of different variables, including growth time, nutrients, and physiological conditions, on biofilm formation, morphology, and architecture. In this article, we will focus on fungal biofilms (mainly Candida biofilms) and provide an update on the development, architecture, and resistance mechanisms of biofilms.
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Biofilmes , Candida/efeitos dos fármacos , Candida/fisiologia , Candidíase/microbiologia , Farmacorresistência Fúngica , Animais , Antifúngicos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida/genética , Candida/crescimento & desenvolvimento , Candidíase/tratamento farmacológico , HumanosRESUMO
PURPOSE: There is a burgeoning recognition and interest in the potential of probiotics in the treatment and prevention of oral candidiasis associated with the use of dentures. Our aim was to investigate if commercially available over-the-counter probiotics can influence the ability of Candida albicans to form biofilms, which is considered a hallmark of the initiation and progression of oral candidiasis. METHODS: We conducted a 2×5 factorial in vitro study to culture C. albicans on denture strips and challenge with one of the following four commercially available probiotics in bacterial or cell-free supernatant form: Accuflora®, Align®, Culturelle® and Sustenex®. C. albicans biofilm formation was studied in triplicates in all factorial combinations of the study and assessed qualitatively with fluorescence microscopy and quantitatively with tetrazolium salt (XTT) reduction assay. Quality control measures included determination of coefficient of variation, Bland Altman plots and Pittman's test. Results were analyzed using two-way analysis of variance (ANOVA) with pairwise post-hoc Scheffe's tests. RESULTS: Our experimental conditions passed the quality control checks. Two-way ANOVA results indicated that cell-free supernatants provided a stronger and significant inhibitory effect on biofilm formation than their bacterial counterparts (2-way ANOVA p=3.8×10(-6)). Further, Lactobacillus-containing probiotic formulations (Accuflora® and Culturelle®) significantly reduced biofilm formation especially in supernatant form. CONCLUSION: Commercially available probiotics that contain Lactobacilli species interfere with the in vitro ability of C. albicans to form biofilms on dentures. The mechanistic and clinical implications of our results need to be addressed by larger in vivo studies.
Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Materiais Dentários/química , Bases de Dentadura/microbiologia , Medicamentos sem Prescrição/farmacologia , Probióticos/farmacologia , Bacillus/fisiologia , Bifidobacterium/fisiologia , Humanos , Indicadores e Reagentes , Lactobacillus/fisiologia , Lactobacillus acidophilus/fisiologia , Lacticaseibacillus rhamnosus/fisiologia , Teste de Materiais , Interações Microbianas , Microscopia de Fluorescência , Polimetil Metacrilato/química , Streptococcus thermophilus/fisiologia , Sais de TetrazólioRESUMO
Microbial biofilms play an essential role in several infectious diseases and are defined as extensive communities of sessile organisms irreversibly associated with a surface, encased within a polysaccharide-rich extracellular matrix (ECM), and exhibiting enhanced resistance to antimicrobial drugs. Forming a biofilm provides the microbes protection from environmental stresses due to contaminants, nutritional depletion, or imbalances, but is dangerous to human health due to their inherent robustness and elevated resistance.The use of indwelling medical devices (e.g., central venous catheters, CVCs) in current therapeutic practice is associated with 80-90 % of hospital-acquired bloodstream and deep tissue infections. Most cases of catheter-related bloodstream infections (CRBSIs) involve colonization of microorganisms on catheter surfaces where they form a biofilm. Additionally, Fusarium solani and F. oxysporum were the causative organisms of the 2005/2006 outbreak of contact lens-associated fungal keratitis in the United States, Europe, the UK, and Singapore, and these infections involved formation of biofilms on contact lens. Fungal biofilm formation is studied using a number of techniques, involving the use of a wide variety of substrates and growth conditions. In vitro techniques involving the use of confocal scanning laser/scanning electron microscopy, metabolic activity assay, dry weight measurements, and antifungal susceptibility assays are increasingly used by investigators to quantify and evaluate biofilm morphology. However, there are not many in vivo models used to validate biofilm-associated infections. In this protocol, we describe a clinically relevant rabbit model of C. albicans biofilm-associated catheter infection to evaluate the morphology, topography, and architecture of fungal biofilms. We also describe a murine model of contact lens-associated Fusarium keratitis.Evaluation of the formation of fungal biofilms on catheters in vivo, their analysis using scanning electron microscopy (SEM) and quantitative catheter culture (QCC), and treatment of biofilms using antimicrobial lock therapy can be completed in ~20-25 days using the described methods. The rabbit model has utility in evaluating the efficacy of lock solutions. In addition, the murine model of contact lens-associated Fusarium keratitis enables characterizing/comparing the formation of Fusarium biofilms on contact lenses in vitro and determining their role in vivo.
Assuntos
Biofilmes/crescimento & desenvolvimento , Modelos Animais de Doenças , Fungos/fisiologia , Micoses/microbiologia , Animais , Catéteres/microbiologia , Ceratite/microbiologia , Camundongos , Micologia/métodos , Complicações Pós-Operatórias/microbiologia , CoelhosRESUMO
The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.