Hospital Burden of All-Cause Pneumonia and Nonbacteremic Pneumococcal Pneumonia in Adults in France Between 2013 and 2019.

Background: Community-acquired pneumonia (CAP) is associated with significant morbidity and mortality. The study objective was to describe the hospital burden of pneumonia in the adult population in France. Methods: This retrospective study was conducted from the National Health Insurance Database. All hospitalizations for pneumonia (all-cause) between 2013 and 2019 were included. Different risk categories for patients were established based on pneumococcal vaccine recommendations by French health authorities. Results: A total of 2 199 240 episodes of CAP were registered over the study period (annual mean, 314 177 [standard deviation, 17 818.6]); 75% occurred in patients aged ≥65 years, among whom 47% were not classified in the moderate- or high-risk categories recommended for French pneumococcal vaccination. The incidence of CAP increased with age (117.9, 395.3, and 1916.7 per 100 000 for the age groups 18-49, 50-64, and ≥65 years, respectively, in 2019). Furthermore, being at risk of pneumococcal disease resulted in more severe outcomes, including longer episode duration (mean, 14 days in low-risk vs 17 days in high-risk patients) and higher risk of referral to critical care units (from 20% to 27%), of rehospitalization up to 180 days (from 39% to 67%), of in-hospital death (from 12% to 19%), and of 1-year mortality (from 26% to 49%). Conclusions: This study establishes the incidence of CAP in adults in France, describes the significant burden of disease, and highlights the need for better prevention policies.

Pneumonia-Associated Emergency Transfers, Functional Decline, and Mortality in Nursing Home Residents.

OBJECTIVE: To describe nursing home residents (NHRs) transferred to the emergency department (ED) with pneumonia, and investigate the association of pneumonia with functional ability and mortality. DESIGN: Case-control observational multicenter study. SETTING AND PARTICIPANTS: Participants of the FINE study, including 1037 NHRs presenting to 17 EDs in France over 4 nonconsecutive weeks (1 per season) in 2016, mean age 87.2 years ± 7.1, 68.4% women. METHODS: Activities of daily living (ADL) performance evolution between (1) 15 days before transfer and (2) within 7 days after discharge back to the nursing home was compared in NHRs with or without pneumonia. The association of pneumonia with functional evolution was investigated by a mixed-effect linear regression of ADL and mortality was compared by a χ2 test. RESULTS: NHRs with pneumonia (n = 232; 22.4%) were more likely to have a lower ADL performance than NHRs without pneumonia (n = 805, 77.6%). They presented with a more severe clinical condition, were more likely to be hospitalized after ED and to stay longer in ED and in hospital. They showed a 0.5 decline in median ADL performance after transfer and a significantly higher mortality than NHRs without pneumonia (24.1% and 8.7%, respectively). Post-ED functional evolution did not differ significantly between NHRs with or without pneumonia. CONCLUSIONS AND IMPLICATIONS: Pneumonia-associated ED transfers resulted in longer care pathways and higher mortality, but no significant difference in functional decline. This study identified a suggestive course of symptoms that could facilitate early identification of NHRs developing pneumonia and early management to prevent ED transfer.

Mediator Subunit Med15 Regulates Cell Morphology and Mating in Candida lusitaniae.

Candida lusitaniae is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few Candida species with the ability to reproduce sexually. In this study, we showed that a dpp3Δ mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type DPP3 locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in MED15, encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of MED15 was responsible for the defective phenotypes by rescuing the dpp3Δ mutant with a wild-type copy of MED15 and constructing a med15Δ knockout mutant that mimics the phenotypes of dpp3Δ in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of MED15 in the regulation of hyphal growth, cell separation and mating, and the pathways involved in C. lusitaniae.

Role of LrrkA in the Control of Phagocytosis and Cell Motility in Dictyostelium discoideum.

LrrkA is a Dictyostelium discoideum kinase with leucine-rich repeats. LrrkA stimulates Kil2 and intra-phagosomal killing of ingested bacteria in response to folate. In this study, we show that genetic inactivation of lrrkA also causes a previously unnoticed phenotype: lrrkA KO cells exhibit enhanced phagocytosis and cell motility compared to parental cells. This phenotype is cell autonomous, is reversible upon re-expression of LrrkA, and is not due to an abnormal response to inhibitory quorum-sensing factors secreted by D. discoideum in its medium. In addition, folate increases motility in parental D. discoideum cells, but not in lrrkA KO cells, suggesting that LrrkA plays a pivotal role in the cellular response to folate. On the contrary, lrrkA KO cells regulate gene transcription in response to folate in a manner indistinguishable from parental cells. Overall, based on analysis of mutant phenotypes, we identify gene products that participate in the control of intracellular killing, cell motility, and gene transcription in response to folate. These observations reveal a mechanism by which D. discoideum encountering bacterially-secreted folate can migrate, engulf, and kill bacteria more efficiently.

Burden of pneumococcal pneumonia requiring ICU admission in France: 1-year prognosis, resources use, and costs.

BACKGROUND: Community-acquired pneumonia (CAP), especially pneumococcal CAP (P-CAP), is associated with a heavy burden of illness as evidenced by high rates of intensive care unit (ICU) admission, mortality, and costs. Although well-defined acutely, determinants influencing long-term burden are less known. This study assessed determinants of 28-day and 1-year mortality and costs among P-CAP patients admitted in ICUs. METHODS: Data regarding all hospital and ICU stays in France in 2014 were extracted from the French healthcare administrative database. All patients admitted in the ICU with a pneumonia diagnosis were included, except those hospitalized for pneumonia within the previous 3 months. The pneumococcal etiology and comorbidities were captured. All hospital stays were included in the cost analysis. Comorbidities and other factors effect on the 28-day and 1-year mortality were assessed using a Cox regression model. Factors associated with increased costs were identified using log-linear regression models. RESULTS: Among 182,858 patients hospitalized for CAP in France for 1 year, 10,587 (5.8%) had a P-CAP, among whom 1665 (15.7%) required ICU admission. The in-hospital mortality reached 22.8% at day 28 and 32.3% at 1 year. The mortality risk increased with age > 54 years, malignancies (hazard ratio (HR) 1.54, 95% CI [1.23-1.94], p = 0.0002), liver diseases (HR 2.08, 95% CI [1.61-2.69], p < 0.0001), and the illness severity at ICU admission. Compared with non-ICU-admitted patients, ICU survivors remained at higher risk of 1-year mortality. Within the following year, 38.2% (516/1350) of the 28-day survivors required at least another hospital stay, mostly for respiratory diseases. The mean cost of the initial stay was €19,008 for all patients and €11,637 for subsequent hospital stays within 1 year. One-year costs were influenced by age (lower in patients > 75 years old, p = 0.008), chronic cardiac (+ 11% [0.02-0.19], p = 0.019), and respiratory diseases (+ 11% [0.03-0.18], p = 0.006). CONCLUSIONS: P-CAP in ICU-admitted patients was associated with a heavy burden of mortality and costs at one year. Older age was associated with both early and 1-year increased mortality. Malignant and chronic liver diseases were associated with increased mortality, whereas chronic cardiac failure and chronic respiratory disease with increased costs. TRIAL REGISTRATION: N/A (study on existing database).

LrrkA, a kinase with leucine-rich repeats, links folate sensing with Kil2 activity and intracellular killing.

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.

MitoNEET-dependent formation of intermitochondrial junctions.

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.

Vps13F links bacterial recognition and intracellular killing in Dictyostelium.

Bacterial sensing, ingestion, and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. The cellular mechanisms involved in intracellular killing, their relative importance, and their specificity towards different bacteria are however poorly defined. In this study, we used Dictyostelium discoideum, a phagocytic cell model amenable to genetic analysis, to identify new gene products involved in intracellular killing. A random genetic screen led us to identify the role of Vps13F in intracellular killing of Klebsiella pneumoniae. Vps13F knock-out (KO) cells exhibited a delayed intracellular killing of K. pneumoniae, although the general organization of the phagocytic and endocytic pathway appeared largely unaffected. Transcriptomic analysis revealed that vps13F KO cells may be functionally similar to previously characterized fspA KO cells, shown to be defective in folate sensing. Indeed, vps13F KO cells showed a decreased chemokinetic response to various stimulants, suggesting a direct or indirect role of Vps13F in intracellular signaling. Overstimulation with excess folate restored efficient killing in vps13F KO cells. Finally, genetic inactivation of Far1, the folate receptor, resulted in inefficient intracellular killing of K. pneumoniae. Together, these observations show that stimulation of Dictyostelium by bacterial folate is necessary for rapid intracellular killing of K. pneumoniae.

Pycnosomes: Condensed Endosomal Structures Secreted by Dictyostelium Amoebae.

Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.

Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole.

We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.

Host-pathogen interaction and signaling molecule secretion are modified in the dpp3 knockout mutant of Candida lusitaniae.

Candida lusitaniae is an emerging opportunistic yeast and an attractive model to discover new virulence factors in Candida species by reverse genetics. Our goal was to create a dpp3Δ knockout mutant and to characterize the effects of this gene inactivation on yeast in vitro and in vivo interaction with the host. The secretion of two signaling molecules in Candida species, phenethyl alcohol (PEA) and tyrosol, but not of farnesol was surprisingly altered in the dpp3Δ knockout mutant. NO and reactive oxygen species (ROS) production as well as tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) secretion were also modified in macrophages infected with this mutant. Interestingly, we found that the wild-type (WT) strain induced an increase in IL-10 secretion by zymosan-activated macrophages without the need for physical contact, whereas the dpp3Δ knockout mutant lost this ability. We further showed a striking role of PEA and tyrosol in this modulation. Last, the DPP3 gene was found to be an essential contributor to virulence in mice models, leading to an increase in TNF-α secretion and brain colonization. Although reinsertion of a WT DPP3 copy in the dpp3Δ knockout mutant was not sufficient to restore the WT phenotypes in vitro, it allowed a restoration of those observed in vivo. These data support the hypothesis that some of the phenotypes observed following DPP3 gene inactivation may be directly dependent on DPP3, while others may be the indirect consequence of another genetic modification that systematically arises when the DPP3 gene is inactivated.