RESUMEN
The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1ß, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.
Asunto(s)
Péptidos Antimicrobianos , Mucosa Gástrica , Helicobacter pylori , Inflamación , Moco , Humanos , Moco/metabolismo , Moco/microbiología , Péptidos Antimicrobianos/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/inmunología , Inflamación/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/inmunología , Estómago/microbiología , Organoides/metabolismo , Organoides/microbiologíaRESUMEN
Helicobacter pylori infection predisposes carriers to a high risk of developing gastric cancer. The cell-of-origin of antral gastric cancer is the Lgr5+ stem cell. Here, we show that infection of antrum-derived gastric organoid cells with H. pylori increases the expression of the stem cell marker Lgr5 as determined by immunofluorescence microscopy, qRT-PCR, and Western blotting, both when cells are grown and infected as monolayers and when cells are exposed to H. pylori in 3D structures. H. pylori exposure increases stemness properties as determined by spheroid formation assay. Lgr5 expression and the acquisition of stemness depend on a functional type IV secretion system (T4SS) and at least partly on the T4SS effector CagA. The pharmacological inhibition or genetic ablation of NF-κB reverses the increase in Lgr5 and spheroid formation. Constitutively active Wnt/ß-catenin signaling because of Apc inactivation exacerbates H. pylori-induced Lgr5 expression and stemness, both of which persist even after eradication of the infection. The combined data indicate that H. pylori has stemness-inducing properties that depend on its ability to activate NF-κB signaling.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , FN-kappa B , Receptores Acoplados a Proteínas G , Neoplasias Gástricas , Vía de Señalización Wnt , Animales , Ratones , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/microbiología , FN-kappa B/metabolismo , Organoides/metabolismo , Organoides/microbiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Células Madre/metabolismo , Estómago/microbiología , Estómago/patología , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Vía de Señalización Wnt/genéticaRESUMEN
Several reports suggest that intestinal tissue may be a natural niche for Chlamydia trachomatis infection and a reservoir for persistent infections in the human body. Due to the human specificity of the pathogen and the lack of suitable host models, there is limited knowledge on this topic. In our study, we modelled the course of the chlamydial infection in human primary gastrointestinal (GI) epithelial cells originating from patient-derived organoids. We show that GI cells are resistant to apical infection and C. trachomatis needs access to the basolateral membrane to establish an infection. Transmission electron microscopy analysis reveals the presence of both normal as well as aberrant chlamydial developmental forms in the infected cells, suggesting a possible cell-type specific nature of the infection. Furthermore, we show that the plasmid-encoded Pgp3 is an important virulence factor for the infection of human GI cells. This is the first report of C. trachomatis infection in human primary intestinal epithelial cells supporting a possible niche for chlamydial infection in the human intestinal tissue.
Asunto(s)
Infecciones por Chlamydia , Chlamydia trachomatis , Organoides , Humanos , Chlamydia trachomatis/fisiología , Organoides/microbiología , Organoides/patología , Infecciones por Chlamydia/microbiología , Mucosa Intestinal/microbiología , Células Epiteliales/microbiología , Antígenos Bacterianos/metabolismo , Proteínas BacterianasRESUMEN
In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.
Asunto(s)
Diferenciación Celular , Receptores ErbB , Organoides , Infecciones por Salmonella , Salmonella typhimurium , Células Madre , Animales , Organoides/microbiología , Células Madre/metabolismo , Ratones , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Receptores ErbB/metabolismo , Receptores ErbB/genética , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Proliferación Celular , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori-induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria.
Asunto(s)
Mucosa Gástrica , Infecciones por Helicobacter , Helicobacter pylori , Ouabaína , ATPasa Intercambiadora de Sodio-Potasio , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Humanos , Animales , Ouabaína/farmacología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Mucosa Gástrica/efectos de los fármacos , Gerbillinae , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/efectos de los fármacos , Organoides/metabolismo , Organoides/microbiologíaRESUMEN
The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies.
Asunto(s)
Antituberculosos , Pulmón , Mycobacterium tuberculosis , Organoides , Humanos , Organoides/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Pulmón/microbiología , Pulmón/patología , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiología , Macrófagos/microbiología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Células Epiteliales/microbiologíaRESUMEN
Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.
Asunto(s)
Técnicas de Cocultivo , Mucosa Intestinal , Organoides , Oxígeno , Humanos , Organoides/microbiología , Organoides/metabolismo , Oxígeno/metabolismo , Técnicas de Cocultivo/métodos , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citología , Microbioma Gastrointestinal , Interacciones Microbiota-Huesped , Bacterias Anaerobias/crecimiento & desarrollo , Bacterias Anaerobias/metabolismo , Intestinos/microbiología , Intestinos/citología , Bacteroides thetaiotaomicron/metabolismoRESUMEN
Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.
Asunto(s)
Células Caliciformes , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Mucosa Respiratoria , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Células Caliciformes/microbiología , Células Caliciformes/metabolismo , Humanos , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/citología , Infecciones por Pseudomonas/microbiología , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Organoides/microbiología , Traslocación BacterianaRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.
Asunto(s)
Escherichia coli Enterohemorrágica , Íleon , Organoides , Recto , Animales , Bovinos , Íleon/microbiología , Íleon/metabolismo , Íleon/ultraestructura , Recto/microbiología , Escherichia coli Enterohemorrágica/patogenicidad , Organoides/metabolismo , Organoides/microbiología , Moco/metabolismo , Infecciones por Escherichia coli/microbiología , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructuraRESUMEN
Salmonella enterica serovar Dublin (S. Dublin) is an important enteric pathogen affecting cattle and poses increasing public health risks. Understanding the pathophysiology and host-pathogen interactions of S. Dublin infection are critical for developing effective control strategies, yet studies are hindered by the lack of physiologically relevant in vitro models. This study aimed to generate a robust ileal monolayer derived from adult bovine organoids, validate its feasibility as an in vitro infection model with S. Dublin, and evaluate the epithelial response to infection. A stable, confluent monolayer with a functional epithelial barrier was established under optimized culture conditions. The model's applicability for studying S. Dublin infection was confirmed by documenting intracellular bacterial invasion and replication, impacts on epithelial integrity, and a specific inflammatory response, providing insights into the pathogen-epithelium interactions. The study underscores the utility of organoid-derived monolayers in advancing our understanding of enteric infections in livestock and highlights implications for therapeutic strategy development and preventive measures, with potential applications extending to both veterinary and human medicine. The established bovine ileal monolayer offers a novel and physiologically relevant in vitro platform for investigating enteric pathogen-host interactions, particularly for pathogens like S. Dublin.
Asunto(s)
Interacciones Huésped-Patógeno , Íleon , Organoides , Salmonelosis Animal , Animales , Bovinos , Organoides/microbiología , Íleon/microbiología , Íleon/patología , Salmonelosis Animal/microbiología , Salmonella enterica/patogenicidad , Salmonella enterica/fisiología , Inflamación/microbiología , Inflamación/patología , Mucosa Intestinal/microbiología , Enfermedades de los Bovinos/microbiologíaRESUMEN
Recent advances in organoid and organ-on-chip (OoC) technologies offer an unprecedented level of tissue mimicry. These models can recapitulate the diversity of cellular composition, 3D organization, and mechanical stimulation. These approaches are intensively used to understand complex diseases. This review focuses on the latest advances in this field to study host-microorganism interactions.
Asunto(s)
Dispositivos Laboratorio en un Chip , Organoides , Organoides/microbiología , Humanos , Animales , Interacciones Microbiota-Huesped , Interacciones Huésped-PatógenoRESUMEN
Organoids faithfully replicate the morphological structure, physiological functions, stable phenotype of the source tissue. Recent research indicates that bacteria can significantly influence the initiation, advancement, and treatment of tumors. This article provides a comprehensive review of the applications of organoid technology in tumor research, the relationship between bacteria and the genesis and development of tumors, and the exploration of the impact of bacteria on tumors and their applications in research.
Asunto(s)
Neoplasias , Organoides , Organoides/patología , Organoides/microbiología , Humanos , Neoplasias/patología , Neoplasias/microbiología , Bacterias , Modelos Biológicos , AnimalesRESUMEN
Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt-derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.
Asunto(s)
Pollos , Salmonella typhimurium , Humanos , Animales , Salmonella typhimurium/fisiología , Intestinos , Mucosa Intestinal/microbiología , Ciego , Organoides/microbiologíaRESUMEN
Skin infections caused by drug-resistant Staphylococcus aureus occur at high rates nationwide. Mouse primary epidermal organoids (mPEOs) possess stratified histological and morphological characteristics of epidermis and are highly similar to their derived tissue at the transcriptomic and proteomic levels. Herein, the susceptibility of mPEOs to methicillin-resistant S. aureus USA300 infection was investigated. The results show that mPEOs support USA300 colonization and invasion, exhibiting swollen epithelial squamous cells with nuclear necrosis and secreting inflammatory factors such as IL-1ß. Meanwhile mPEOs beneficial to observe the process of USA300 colonization with increasing infection time, and USA300 induces mPEOs to undergo pyroptosis and autophagy. In addition, we performed a drug screen for the mPEO infection model and showed that vancomycin restores cell viability and inhibits bacterial internalization in a concentration-dependent manner. In conclusion, we establish an in vitro skin infection model that contributes to the examination of drug screening strategies and antimicrobial drug mechanisms.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Organoides , Infecciones Estafilocócicas , Animales , Ratones , Evaluación Preclínica de Medicamentos/métodos , Epidermis/metabolismo , Epidermis/microbiología , Epidermis/patología , Proteómica , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Organoides/metabolismo , Organoides/microbiologíaRESUMEN
Approaches based on animal and two-dimensional (2D) cell culture models cannot ensure reliable results in modeling novel pathogens or in drug testing in the short term; therefore, there is rising interest in platforms such as organoids. To develop a toolbox that can be used successfully to overcome current issues in modeling various infections, it is essential to provide a framework of recent achievements in applying organoids. Organoids have been used to study viruses, bacteria, and protists that cause, for example, respiratory, gastrointestinal, and liver diseases. Their future as models of infection will be associated with improvements in system complexity, including abilities to model tissue structure, a dynamic microenvironment, and coinfection. Teaser. Organoids are a flexible tool for modelling viral, bacterial and protist infections. They can provide fast and reliable information on the biology of pathogens and in drug screening, and thus have become essential in combatting emerging infectious diseases.
Asunto(s)
Evaluación Preclínica de Medicamentos , Infecciones , Organoides , Animales , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/tendencias , Infecciones/tratamiento farmacológico , Infecciones/microbiología , Modelos Animales , Organoides/efectos de los fármacos , Organoides/microbiología , Reproducibilidad de los ResultadosRESUMEN
All-time preservation of an intact mucosal barrier is crucial to ensuring intestinal homeostasis and, hence, the organism's overall health maintenance. This complex process relies on an equilibrated signaling system between the intestinal epithelium and numerous cell populations inhabiting the gut mucosa. Any perturbations of this delicate cross talk, particularly regarding the immune cell compartment and microbiota, may sustainably debilitate the intestinal barrier function. As a final joint event, a critical rise in epithelial permeability facilitates the exposure of submucosal immunity to microbial antigens, resulting in uncontrolled inflammation, collateral tissue destruction, and dysbiosis. Organoid-derived intestinal coculture models have established themselves as convenient tools to reenact such pathophysiological events, explore interactions between selected cell populations, and assess their roles with a central focus on intestinal barrier recovery and stabilization.
Asunto(s)
Mucosa Intestinal/citología , Organoides/citología , Cultivo Primario de Células/métodos , Animales , Técnicas de Cocultivo/métodos , Microbioma Gastrointestinal , Humanos , Mucosa Intestinal/microbiología , Linfocitos/citología , Macrófagos/citología , Organoides/microbiologíaRESUMEN
Infective endocarditis (IE) is a heart valve infection with high mortality rates. IE results from epithelial lesions, inducing sterile healing vegetations consisting of platelets, leucocytes, and fibrin that are susceptible for colonization by temporary bacteremia. Clinical testing of new treatments for IE is difficult and fast models sparse. The present study aimed at establishing an in vitro vegetation simulation IE model for fast screening of novel treatment strategies. A healing promoting platelet and leucocyte-rich fibrin patch was used to establish an IE organoid-like model by colonization with IE-associated bacterial isolates Staphylococcus aureus, Streptococcus spp (S. mitis group), and Enterococcus faecalis. The patch was subsequently exposed to tobramycin, ciprofloxacin, or penicillin. Bacterial colonization was evaluated by microscopy and quantitative bacteriology. We achieved stable bacterial colonization on the patch, comparable to clinical IE vegetations. Microscopy revealed uneven, biofilm-like colonization of the patch. The surface-associated bacteria displayed increased tolerance to antibiotics compared to planktonic bacteria. The present study succeeded in establishing an IE simulation model with the relevant pathogens S. aureus, S. mitis group, and E. faecalis. The findings indicate that the IE model mirrors the natural IE process and has the potential for fast screening of treatment candidates.
Asunto(s)
Endocarditis Bacteriana/microbiología , Modelos Biológicos , Antibacterianos/farmacología , Bacteriemia/microbiología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Recuento de Colonia Microbiana , Tolerancia a Medicamentos , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/patología , Humanos , Organoides/citología , Organoides/efectos de los fármacos , Organoides/microbiologíaRESUMEN
Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.
Asunto(s)
Antibacterianos/farmacología , Modelos Biológicos , Neutrófilos/patología , Organoides/microbiología , Vejiga Urinaria/microbiología , Escherichia coli Uropatógena/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Femenino , Humanos , Imagenología Tridimensional , Ratones Endogámicos C57BL , Viabilidad Microbiana/efectos de los fármacos , Movimiento , Neutrófilos/efectos de los fármacos , Organoides/efectos de los fármacos , Organoides/ultraestructura , Vejiga Urinaria/patología , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/crecimiento & desarrollo , Escherichia coli Uropatógena/ultraestructuraRESUMEN
Small intestinal organoids, or enteroids, represent a valuable model to study host-pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4+ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host-pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.