Protocol for live imaging of bacteria-cell interactions in genetically modified mouse small intestinal organoids.

Here, we present a protocol for microinjection of bacteria into mouse small intestinal organoids that recapitulates the natural route of infection of intestinal epithelial cells from the intestinal lumen. We describe steps for visualizing bacteria-cell interactions by live imaging of infected organoids using light sheet microscopy. We then detail procedures for generating doxycycline-inducible expression of mutant proteins in organoids to study essential gene functions. The different techniques described in this protocol can be used independently as required. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).1.

Refractory Pseudomonas aeruginosa Bronchopulmonary Infection After Lung Transplantation for Common Variable Immunodeficiency Despite Maximal Treatment Including IgM/IgA-Enriched Immunoglobulins and Bacteriophage Therapy.

Recipients transplanted for bronchiectasis in the context of a primary immune deficiency, such as common variable immunodeficiency, are at a high risk of severe infection in post-transplantation leading to poorer long-term outcomes than other transplant indications. In this report, we present a fatal case due to chronic Pseudomonas aeruginosa bronchopulmonary infection in a lung transplant recipient with common variable immunodeficiency despite successful eradication of an extensively drug-resistant (XDR) strain with IgM/IgA-enriched immunoglobulins and bacteriophage therapy. The fatal evolution despite a drastic adaptation of the immunosuppressive regimen and the maximal antibiotic therapy strategy raises the question of the contraindication of lung transplantation in such a context of primary immunodeficiency.

Administration of Bacteriophages via Nebulization during Mechanical Ventilation: In Vitro Study and Lung Deposition in Macaques.

Bacteriophages have been identified as a potential treatment option to treat lung infection in the context of antibiotic resistance. We performed a preclinical study to predict the efficacy of delivery of bacteriophages against Pseudomonas aeruginosa (PA) when administered via nebulization during mechanical ventilation (MV). We selected a mix of four anti-PA phages containing two Podoviridae and two Myoviridae, with a coverage of 87.8% (36/41) on an international PA reference panel. When administered via nebulization, a loss of 0.30-0.65 log of infective phage titers was measured. No difference between jet, ultrasonic and mesh nebulizers was observed in terms of loss of phage viability, but a higher output was measured with the mesh nebulizer. Interestingly, Myoviridae are significantly more sensitive to nebulization than Podoviridae since their long tail is much more prone to damage. Phage nebulization has been measured as compatible with humidified ventilation. Based on in vitro measurement, the lung deposition prediction of viable phage particles ranges from 6% to 26% of the phages loaded in the nebulizer. Further, 8% to 15% of lung deposition was measured by scintigraphy in three macaques. A phage dose of 1 × 109 PFU/mL nebulized by the mesh nebulizer during MV predicts an efficient dose in the lung against PA, comparable with the dose chosen to define the susceptibility of the strain.

Case Report: Arthroscopic "Debridement Antibiotics and Implant Retention" With Local Injection of Personalized Phage Therapy to Salvage a Relapsing Pseudomonas Aeruginosa Prosthetic Knee Infection.

Bacteriophages are viruses that specifically target bacteria. They are considered to have a high potential in patients with prosthetic joint infection (PJI), as they have a synergistic anti-biofilm activity with antibiotics. We report here the case of an 88-year-old man (63 kg) with relapsing Pseudomonas aeruginosa prosthetic knee infection. The patient had severe alteration of the general status and was bedridden with congestive heart failure. As prosthesis explantation and/or exchange was not feasible, we proposed to this patient the use of phage therapy to try to control the disease in accordance with the local ethics committee and the French National Agency for Medicines and Health Products Safety (ANSM). Three phages, targeting P. aeruginosa, were selected based on their lytic activity on the patient's strain (phagogram). Hospital pharmacist mixed extemporaneously the active phages (initial concentration 1 ml of 1 × 1010 PFU/ml for each phage) to obtain a cocktail of phages in a suspension form (final dilution 1 × 109 PFU/ml for both phages). Conventional arthroscopy was performed and 30 cc of the magistral preparation was injected through the arthroscope (PhagoDAIR procedure). The patient received intravenous ceftazidime and then oral ciprofloxacin as suppressive antimicrobial therapy. Under this treatment, the patient rapidly improved with disappearance of signs of heart failure and pain of the left knee. During the follow-up of 1 year, the local status of the left knee was normal, and its motion and walking were unpainful. The present case suggests that the PhagoDAIR procedure by arthroscopy has the potential to be used as salvage therapy for patients with P. aeruginosa relapsing PJI, in combination with suppressive antimicrobial therapy. A Phase II clinical study deserves to be performed to confirm this hypothesis.

Inhaled bacteriophage therapy in a porcine model of pneumonia caused by Pseudomonas aeruginosa during mechanical ventilation.

BACKGROUND AND PURPOSE 255: Pseudomonas aeruginosa is a main cause of ventilator-associated pneumonia (VAP) with drug-resistant bacteria. Bacteriophage therapy has experienced resurgence to compensate for the limited development of novel antibiotics. However, phage therapy is limited to a compassionate use so far, resulting from lack of adequate studies in relevant pharmacological models. We used a pig model of pneumonia caused by P. aeruginosa that recapitulates essential features of human disease to study the antimicrobial efficacy of nebulized-phage therapy. EXPERIMENTAL APPROACH: (i) Lysis kinetic assays were performed to evaluate in vitro phage antibacterial efficacy against P. aeruginosa and select relevant combinations of lytic phages. (ii) The efficacy of the phage combinations was investigated in vivo (murine model of P. aeruginosa lung infection). (iii) We determined the optimal conditions to ensure efficient phage delivery by aerosol during mechanical ventilation. (iv) Lung antimicrobial efficacy of inhaled-phage therapy was evaluated in pigs, which were anaesthetized, mechanically ventilated and infected with P. aeruginosa. KEY RESULTS: By selecting an active phage cocktail and optimizing aerosol delivery conditions, we were able to deliver high phage concentrations in the lungs, which resulted in a rapid and marked reduction in P. aeruginosa density (1.5-log reduction, p < .001). No infective phage was detected in the sera and urines throughout the experiment. CONCLUSION AND IMPLICATIONS: Our findings demonstrated (i) the feasibility of delivering large amounts of active phages by nebulization during mechanical ventilation and (ii) rapid control of in situ infection by inhaled bacteriophage in an experimental model of P. aeruginosa pneumonia with high translational value.

Live Imaging Reveals Listeria Hijacking of E-Cadherin Recycling as It Crosses the Intestinal Barrier.

Listeria monocytogenes is a foodborne bacterial pathogen that causes human listeriosis, a severe systemic infection.1 Its translocation across the intestinal epithelium is mediated by the interaction of internalin (InlA), a Listeria surface protein, with its host-species-specific receptor E-cadherin (Ecad).2-5 It occurs through goblet cells, on which Ecad is luminally accessible,6 via an unknown mechanism. In the absence of cell lines recapitulating this phenotype in vitro, we developed an ex vivo experimental system, based on the intraluminal microinjection of Listeria in untreated, pharmacologically treated, and genetically modified intestinal organoids. Using both live light-sheet microscopy and confocal imaging, we show that Listeria translocates through goblet cells within a membrane vacuole in an InlA- and microtubule-dependent manner. As Ecad undergoes constant apical-basal recycling,7,8 we hypothesized that Lm may transit through goblet cells by hijacking Ecad recycling pathway. Indeed, Listeria is stuck at goblet cell apex when Ecad endocytosis is blocked and remains trapped intracellularly at the basolateral pole of goblet cells when Rab11-dependent Ecad recycling is compromised. Together, these results show that Listeria, upon docking onto its luminally accessible receptor Ecad, hijacks its recycling pathway to be transferred by transcytosis across goblet cells. Live imaging of host-pathogen interactions in organoids is a promising approach to dissect their underlying cell and molecular biology.

Phage Therapy as Adjuvant to Conservative Surgery and Antibiotics to Salvage Patients With Relapsing S. aureus Prosthetic Knee Infection.

Objectives: To report the management of three consecutive patients with relapsing Staphylococcus aureus prosthetic knee infection (PKI) for whom explantation was not feasible who received a phage therapy during a "Debridement Antibiotics and Implant Retention" (DAIR) procedure followed by suppressive antimicrobial therapy. Methods: Each case was discussed individually in our reference center and with the French National Agency (ANSM). The lytic activity of three phages targeting S. aureus, which was produced with a controlled and reproducible process, was assessed before surgery (phagogram). A hospital pharmacist extemporaneously assembled the phage cocktail (1 ml of 1 × 1010 PFU/ml for each phage) as "magistral" preparation (final dilution 1 × 109 PFU/ml), which was administered by the surgeon directly into the joint, after the DAIR procedure and joint closure (PhagoDAIR procedure). Results: Three elderly patients were treated with the PhagoDAIR procedure. Phagograms revealed a high susceptibility to at least two of the three phages. During surgery, all patients had poor local conditions including pus in contact to the implant. After a prolonged follow-up, mild discharge of synovial fluid persisted in two patients, for whom a subsequent DAIR was performed showing only mild synovial inflammation without bacterial persistence or super-infection. The outcome was finally favorable with a significant and impressive clinical improvement of the function. Conclusions: The PhagoDAIR procedure has the potential to be used as salvage for patients with relapsing S. aureus PKI, in combination with suppressive antibiotics to avoid considerable loss of function. This report provides preliminary data supporting the setup of a prospective multicentric clinical trial.

The Potential Innovative Use of Bacteriophages Within the DAC® Hydrogel to Treat Patients With Knee Megaprosthesis Infection Requiring "Debridement Antibiotics and Implant Retention" and Soft Tissue Coverage as Salvage Therapy.

Infection is the most dramatic complication in patients with knee megaprosthesis. Its management is more complex in comparison with patients with primary arthroplasty, with a high risk of relapse. Lytic bacteriophages are considered to have a high potential in patients with prosthetic joint infection as it has been demonstrated that they have a synergistic anti-biofilm activity with antibiotics. The Defensive Antibacterial Coating (DAC®) hydrogel is a hydrogel available in the market that has been designed to prevent the adherence of bacteria on a prosthetic joint and to have the ability to transport and release anti-bacterial substances such as antibiotics. We report here the case of a patient with a catastrophic relapsing Staphylococcus aureus knee megaprosthesis infection without prosthesis loosening. We firstly perform phage susceptibility testing of the patient's strain to select an active cocktail, under the supervision of the French health authority. Then, we performed, as salvage therapy, a debridement and implant retention procedure with application of a selected cocktail of bacteriophages that was prepared extemporaneously within the DAC® hydrogel. A free flap for soft tissue coverage was required and empirical antibiotic treatment was started immediately after the surgery. Unfortunately, at 5 days after the surgery, while the local aspect of the surgical site was favorable, the patient developed myocardial infarction which required emergency stenting and dual antiplatelet therapy that were rapidly associated with bleeding at the surgical site, leading to a new prosthesis exposition. As a consequence, a transfemoral amputation was finally performed several months later. We also evaluated in vitro the impact of DAC® hydrogel on bacteriophage activity and showed that the selected phages were released very rapidly from the DAC® hydrogel, and then their titers were stable for at least 6 h. This case demonstrated the feasibility of the use of bacteriophages within a hydrogel to treat patients for knee megaprosthesis infection during a debridement procedure. The implementation requires identification of the pathogen before the debridement in order to perform phage susceptibility testing of the patient's strain and to identify a hospital pharmacist who will accept to do the preparation and to take the responsibility of the magistral preparation.

Evaluation of the Activity of a Combination of Three Bacteriophages Alone or in Association with Antibiotics on Staphylococcus aureus Embedded in Biofilm or Internalized in Osteoblasts.

Staphylococcus aureus is responsible for difficult-to-treat bone and joint infections (BJIs). This is related to its ability to form biofilm and to be internalized and persist inside osteoblasts. Recently, bacteriophage therapy has emerged as a promising option to improve treatment of such infections, but data on its activity against the specific bacterial lifestyles presented above remain scarce. We evaluated the activity of a combination of three bacteriophages, recently used for compassionate treatment in France, against S. aureus HG001 in a model of staphylococcal biofilm and a model of osteoblasts infection, alone or in association with vancomycin or rifampin. The activity of bacteriophages against biofilm-embedded S. aureus was dose dependent. In addition, synergistic effects were observed when bacteriophages were combined with antibiotics used at the lowest concentrations. Phage penetration into osteoblasts was observed only when the cells were infected, suggesting a S. aureus-dependent Trojan horse mechanism for internalization. The intracellular bacterial count of bacteria in infected osteoblasts treated with bacteriophages as well as with vancomycin was significantly higher than in cells treated with lysostaphin, used as a control condition, owing to the absence of intracellular activity and the rapid killing of bacteria released after the death of infected cells. These results suggest that bacteriophages are both inactive in the intracellular compartment after being internalized in infected osteoblasts and present a delayed killing effect on bacteria released after cell lysis into the extracellular compartment, which avoids preventing them from infecting other osteoblasts. The combination of bacteriophages tested was highly active against S. aureus embedded in biofilm but showed no activity against intracellular bacteria in the cell model used.

Intestinal Organoids as a Novel Tool to Study Microbes-Epithelium Interactions.

The gut, particularly the colon, is the host of approximately 1000 bacterial species, the so-called gut microbiota. The relationship between the gut microbiota and the host is symbiotic and mutualistic, influencing many aspects of the biology of the host. This homeostatic balance can be disrupted by enteric pathogens, such as Shigella flexneri or Listeria monocytogenes, which are able to invade the epithelial layer and consequently subvert physiological functions. To study the host-microbe interactions in vitro, the crypt culture model, known as intestinal organoids, is a powerful tool. Intestinal organoids provide a model in which to examine the response of the epithelium, particularly the response of intestinal stem cells, to the presence of bacteria. Furthermore, the organoid model enables the study of pathogens during the early steps of enteric pathogen invasion.Here, we describe methods that we have established to study the cellular microbiology of symbiosis between the gut microbiota and host intestinal surface and secondly the disruption of host homeostasis due to an enteric pathogen.

Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial.

BACKGROUND: Wound infections are the main cause of sepsis in patients with burns and increase burn-related morbidity and mortality. Bacteriophages, natural bacterial viruses, are being considered as an alternative therapy to treat infections caused by multidrug-resistant bacteria. We aimed to compare the efficacy and tolerability of a cocktail of lytic anti-Pseudomonas aeruginosa bacteriophages with standard of care for patients with burns. METHODS: In this randomised phase 1/2 trial, patients with a confirmed burn wound infection were recruited from nine burn centres in hospitals in France and Belgium. Patients were eligible if they were aged 18 years or older and had a burn wound clinically infected with P aeruginosa. Eligible participants were randomly assigned (1:1) by use of an interactive web response system to a cocktail of 12 natural lytic anti-P aeruginosa bacteriophages (PP1131; 1 × 106 plaque-forming units [PFU] per mL) or standard of care (1% sulfadiazine silver emulsion cream), both given as a daily topical treatment for 7 days, with 14 days of follow-up. Masking of treatment from clinicians was not possible because of the appearance of the two treatments (standard of care a thick cream, PP1131 a clear liquid applied via a dressing), but assignments were masked from microbiologists who analysed the samples and patients (treatment applied while patients were under general anaesthetic). The primary endpoint was median time to sustained reduction in bacterial burden by at least two quadrants via a four-quadrant method, assessed by use of daily swabs in all participants with a microbiologically documented infection at day 0 who were given at least one sulfadiazine silver or phage dressing (modified intention-to-treat population). Safety was assessed in all participants who received at least one dressing according to protocol. Ancillary studies were done in the per-protocol population (all PP1131 participants who completed 7 days of treatment) to assess the reasons for success or failure of phage therapy. This trial is registered with the European Clinical Trials database, number 2014-000714-65, and ClinicalTrials.gov, number NCT02116010, and is now closed. FINDINGS: Between July 22, 2015, and Jan 2, 2017, across two recruitment periods spanning 13 months, 27 patients were recruited and randomly assigned to receive phage therapy (n=13) or standard of care (n=14). One patient in the standard of care group was not exposed to treatment, giving a safety population of 26 patients (PP1131 n=13, standard of care n=13), and one patient in the PP1131 group did not have an infection at day 0, giving an efficacy population of 25 patients (PP1131 n=12, standard of care n=13). The trial was stopped on Jan 2, 2017, because of the insufficient efficacy of PP1131. The primary endpoint was reached in a median of 144 h (95% CI 48-not reached) in the PP1131 group versus a median of 47 h (23-122) in the standard of care group (hazard ratio 0·29, 95% CI 0·10-0·79; p=0·018). In the PP1131 group, six (50%) of 12 analysable participants had a maximal bacterial burden versus two (15%) of 13 in the standard of care group. PP1131 titre decreased after manufacturing and participants were given a lower concentration of phages than expected (1 × 102 PFU/mL per daily dose). In the PP1131 group, three (23%) of 13 analysable participants had adverse events versus seven (54%) of 13 in the standard of care group. One participant in each group died after follow-up and the deaths were determined to not be related to treatment. The ancillary study showed that the bacteria isolated from patients with failed PP1131 treatment were resistant to low phage doses. INTERPRETATION: At very low concentrations, PP1131 decreased bacterial burden in burn wounds at a slower pace than standard of care. Further studies using increased phage concentrations and phagograms in a larger sample of participants are warranted. FUNDING: European Commission: Framework Programme 7.

Salvage Debridement, Antibiotics and Implant Retention ("DAIR") With Local Injection of a Selected Cocktail of Bacteriophages: Is It an Option for an Elderly Patient With Relapsing Staphylococcus aureus Prosthetic-Joint Infection?

Local injection of a bacteriophages mix during debridement, antibiotics and implant retention ("DAIR") was performed to treat a relapsing Staphylococcus aureus chronic prosthetic joint infection (PJI). This salvage treatment was safe and associated with a clinical success. Scientific evaluation of the potential clinical benefit of bacteriophages as antibiofilm treatment in PJI is now feasible and required.

Peyer's patch myeloid cells infection by Listeria signals through gp38+ stromal cells and locks intestinal villus invasion.

The foodborne pathogen Listeria monocytogenes (Lm) crosses the intestinal villus epithelium via goblet cells (GCs) upon the interaction of Lm surface protein InlA with its receptor E-cadherin. Here, we show that Lm infection accelerates intestinal villus epithelium renewal while decreasing the number of GCs expressing luminally accessible E-cadherin, thereby locking Lm portal of entry. This novel innate immune response to an enteropathogen is triggered by the infection of Peyer's patch CX3CR1+ cells and the ensuing production of IL-23. It requires STAT3 phosphorylation in epithelial cells in response to IL-22 and IL-11 expressed by lamina propria gp38+ stromal cells. Lm-induced IFN-γ signaling and STAT1 phosphorylation in epithelial cells is also critical for Lm-associated intestinal epithelium response. GC depletion also leads to a decrease in colon mucus barrier thickness, thereby increasing host susceptibility to colitis. This study unveils a novel innate immune response to an enteropathogen, which implicates gp38+ stromal cells and locks intestinal villus invasion, but favors colitis.

Rhinoscleroma pathogenesis: The type K3 capsule of Klebsiella rhinoscleromatis is a virulence factor not involved in Mikulicz cells formation.

Rhinoscleroma is a human specific chronic granulomatous infection of the nose and upper airways caused by the Gram-negative bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. Although considered a rare disease, it is endemic in low-income countries where hygienic conditions are poor. A hallmark of this pathology is the appearance of atypical foamy monocytes called Mikulicz cells. However, the pathogenesis of rhinoscleroma remains poorly investigated. Capsule polysaccharide (CPS) is a prominent virulence factor in bacteria. All K. rhinoscleromatis strains are of K3 serotype, suggesting that CPS can be an important driver of rhinoscleroma disease. In this study, we describe the creation of the first mutant of K. rhinoscleromatis, inactivated in its capsule export machinery. Using a murine model recapitulating the formation of Mikulicz cells in lungs, we observed that a K. rhinoscleromatis CPS mutant (KR cps-) is strongly attenuated and that mice infected with a high dose of KR cps- are still able to induce Mikulicz cells formation, unlike a K. pneumoniae capsule mutant, and to partially recapitulate the characteristic strong production of IL-10. Altogether, the results of this study show that CPS is a virulence factor of K. rhinoscleromatis not involved in the specific appearance of Mikulicz cells.

Immune evasion by a staphylococcal inhibitor of myeloperoxidase.

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Listeria monocytogenes-associated biliary tract infections: a study of 12 consecutive cases and review.

At present, little is known regarding Listeria monocytogenes-associated biliary tract infection, a rare form of listeriosis.In this article, we will study 12 culture-proven cases reported to the French National Reference Center for Listeria from 1996 to 2013 and review the 8 previously published cases.Twenty cases were studied: 17 cholecystitis, 2 cholangitis, and 1 biliary cyst infection. Half were men with a median age of 69 years (32-85). Comorbidities were present in 80%, including cirrhosis, rheumatoid arthritis, and diabetes. Five patients received immunosuppressive therapy, including corticosteroids and anti-tumor necrosis factor biotherapies. Half were afebrile. Blood cultures were positive in 60% (3/5). Gallbladder histological lesions were analyzed in 3 patients and evidenced acute, chronic, or necrotic exacerbation of chronic infection. Genoserogroup of the 12 available strains were IVb (n=6), IIb (n=5), and IIa (n=1). Their survival in the bile was not enhanced when compared with isolates from other listeriosis cases. Adverse outcome was reported in 33% (5/15): 3 deaths, 1 recurrence; 75% of the patients with adverse outcome received inadequate antimicrobial therapy (P=0.033).Biliary tract listeriosis is a severe infection associated with high mortality in patients not treated with appropriate therapy. This study provides medical relevance to in vitro and animal studies that had shown Listeria monocytogenes ability to survive in bile and induce overt biliary infections.

Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display.

In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and characterization of these proteins leads to a better understanding of the pathological processes underlying infectious and inflammatory diseases and is essential in the development of new strategies for their prevention and treatment. Current techniques to functionally characterize these proteins are laborious and inefficient. Here we describe a high-throughput functional selection strategy using phage display in order to identify immune evasion proteins. Using this technique we identified two previously uncharacterized proteins secreted by Staphylococcus aureus, SElX and SSL6 that bind to neutrophil surface receptors. SElX binds PSGL-1 on neutrophils and thereby inhibits the interaction between PSGL-1 and P-selectin, a crucial step in the recruitment of neutrophils to the site of infection. SSL6 is the first bacterial protein identified that binds CD47, a widely expressed cell surface protein recently described as an interesting target in anti-cancer therapy. Our findings provide new insights into the pathogenesis of S. aureus infections and support phage display as an efficient method to identify bacterial secretome proteins interacting with humoral or cellular immune components.