Invasive candidiasis is a fungal infection characterized by a high mortality rate. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family receptors play a crucial role in regulating innate responses of both leukocytes and epithelia. Human CEACAM3, CEACAM5 and CEACAM6 receptors recognize Candida albicans and are expressed in transgenic CEABAC10 mice. In a murine C. albicans infection model, CEABAC10 mice exhibited a shortened survival period attributed to an early cytokine storm, an exacerbated acute phase response, and heightened systemic inflammation compared to their wild-type littermates. The livers and kidneys of CEABAC10 mice displayed intensified purulent necrotizing inflammation, accompanied by increased infiltration of neutrophils and macrophages. Our in vivo and in vitro data indicated that the expression of CEACAM6 on monocytes of CEABAC10 mice caused the elevated cytokine levels and the subsequent exacerbation of the acute phase response upon C. albicans infection, resulting in decreased survival.

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG , which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆ mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

Community-acquired pneumonia (CAP) is a significant health threat for adults. Although conjugate vaccines have reduced pneumococcal CAP incidence in children, Streptococcus pneumoniae-related CAP remains prevalent among older adults. The nasopharynx acts as a reservoir for S. pneumoniae, yet the interplay between this pathogen and the nasopharyngeal microbiome during and after pneumonia remains poorly understood. This study included 61 adult patients diagnosed with pneumococcal CAP and 61 matched healthy controls. An S. pneumoniae-specific PCR, urine antigen tests and bacterial cultures were performed. Nasopharyngeal swabs collected at admission and three months post-infection were analyzed for microbiome dynamics through 16 S rRNA gene amplicon sequencing. 16 S rRNA gene amplicon sequencing revealed Streptococcus spp. in the majority of all nasopharyngeal samples during infection compared to the other diagnostic test performed. While overall bacterial biomass did not differ between groups, patients exhibited higher alpha diversity (p = 0.012) and lower microbiome stability post-infection. Beta diversity analysis distinguished infection from healthy status (p = 0.002). Taxonomic analysis showed similar core microbiota across groups, but Streptococcus spp. was significantly more abundant during infection, particularly in those patients with viral co-infections. Notably, unique significant bacterial interactions were identified both during and after infection, as well as in healthy states. A negative correlation was observed between Corynebacterium and Streptococcus spp. in infected patients, suggesting a potential antagonistic interaction between these taxa. The nasopharyngeal microbiome in patients with pneumococcal CAP demonstrates persistent disruption post-infection, characterized by lower resilience three months after acute illness. Additionally, we identified specific bacterial interplays during and after infection that differed from those in healthy donors. These bacterial dynamics might play critical roles in pathogen colonization resistance and infection prevention. Thus, our findings highlight the need for further investigation into microbial interactions and potential microbiome-based therapies for respiratory infections, particularly in vulnerable populations.

PURPOSE:Clostridioides difficile is a major cause of healthcare-associated infections, contributing to significant morbidity and mortality. This study aimed to investigate the genomic characteristics, antimicrobial resistance (AMR) profiles, and temporal dynamics of C. difficile strains isolated from hospitalized patients in a German tertiary hospital over nearly two decades (1997-2015). METHODS:Whole-genome sequencing was performed on 46 toxigenic C. difficile isolates to determine sequence types (STs) and phylogenetic relationships and these were compared to national surveillance data on C. dificile. AMR profiling was conducted to identify key resistance determinants at genetic level while epsilometer minimum inhibitory concentration (MIC) analyses were used to correlate genetic resistance markers with phenotypic resistance. Longitudinal antibiotic usage data were analysed to assess potential associations with resistance profiles and strains evolution. RESULTS:Five predominant STs were identified: ST1 (30%), ST54 (24%), ST3 (22%), ST11 (11%), and ST37 (4%). Phylogenetic analysis showed that ST1 (ribotype 027) emerged as the dominant and persistent lineage, replacing ST11 and ST54 over time. AMR profiling detected several resistance genetic markers such as CDD-1/CDD-2 (carbapenem resistance), ErmB (macrolide-lincosamide-streptogramin B resistance/MLS resistance), and mutations in gyrA (fluoroquinolone resistance) and rpoB (rifampicin resistance). MIC analyses confirmed high resistance rates to moxifloxacin (87%) and rifampicin (59%), while susceptibility to fidaxomicin, metronidazole, and vancomycin remained. The tetM gene, associated with doxycycline resistance, declined as ST11 and ST54 frequencies decreased. Longitudinal analysis revealed a reduction in moxifloxacin resistance following its decreased use, whereas increased doxycycline use paradoxically correlated with reduced resistance. CONCLUSION:This study highlights the dynamic strain evolution of C. difficile, reflecting national trends in strain evolution. The findings emphasize the strong correlation between epsilometer MIC values and molecular resistance markers. This observation reinforces the integration of genetic surveillance with antibiotic stewardship in the clinical routine to effectively mitigate CDI recurrence. Further research is needed to better understand the complex interactions between antibiotic exposure and strain evolution in hospital environments.

Patients with cystic fibrosis (CF) are frequently exposed to antibiotic treatments, which can alter the fungal communities (mycobiome) across their mucosal sites. This pilot study investigated the impact of antibiotic exposure on the mycobiome by analyzing fungal community dynamics in the upper respiratory- (nasal lavage) and gastrointestinal- (stool samples) tracts of 12 patients with CF following (a) long-term antibiotic treatment over a three-year period and (b) short-term antibiotic therapy during acute pulmonary exacerbations. Mycobiome profiles of the samples obtained from 38 healthy individuals were also analyzed and used for comparison purposes. The ITS1 region of the fungal rRNA gene cluster was sequenced to characterize and quantify the fungal community composition in both cohorts. Compared to healthy controls, samples from the patients with CF who had undergone long-term antibiotic treatment revealed a significantly increased fungal biomass in both sino-nasal and stool samples. Moreover, diversity metrics revealed significant differences in nasal lavage samples, whereas the stool samples showed no significant variation. Candida spp. was significantly enriched in both nasal and stool samples from CF patients. Further analyses demonstrated a strong positive correlation between the relative abundance of Candida spp. and the cumulative antibiotic intake over the three-year period in sino-nasal samples, but not in stool samples. Acute antibiotic treatment during a pulmonary exacerbation episode also led to a marked increase in the abundance of Candida spp. in sino-nasal samples. These findings highlight the increased sensitivity of the sino-nasal mycobiome to both chronic and acute antibiotic exposure in CF patients, as characterized by a site-specific fungal overgrowth, particularly of Candida spp.

PURPOSE:The accuracy of oral microbiome research depends significantly on specimen sampling protocols, as well as their storage and preservation. Traditional methods, such as freezing, may not only involve logistical hurdles but can also impact the quality of microbial data, leading to difficulties in the comparability between different studies. This study evaluates the effectiveness of the room temperature nucleic acid preservation protocol using DNA/RNA Shield buffer as compared to standard freezing in preserving oral microbial communities over the course of 7 days. RESULTS:Comparative analyses based on 16S rRNA gene sequencing revealed a high overall consistency between the microbiome data retrieved by both preservation strategies. In terms of DNA yield, qPCR analysis showed a significant increase in bacterial DNA recovered from tongue swabs, dental pockets and saliva samples when using DNA/RNA Shield. In the taxonomic analyses, the Shield buffer preserved a higher abundance of Veillonella as compared to freezing. However, the compositional data was highly comparable between both protocols for all other taxonomic groups. While no major differences in microbial diversity were observed between both preservation methods for most samples, saliva samples stored in DNA/RNA Shield exhibited significantly higher diversity. Both preservation methods performed in a highly comparable manner in their ability to retrieve consistent biological readouts, such as site-specificity and health status. CONCLUSION:Storing oral samples at room temperature in DNA/RNA Shield buffer provides a reliable and efficient alternative to freezing for preserving microbiome samples. Furthermore, it may enhance DNA yield while preserving microbial diversity in the samples.

Periodontitis (PD) is an age-associated infectious periodontal disease driven by dysbiosis and hyperinflammatory host response, resulting in bone and tissue loss. Often linked to systemic inflammatory comorbidities, modulating host response emerged as promising clinical target. This study investigates whether nutritional intervention mitigates age-associated systemic impact of oral infection with PD key pathogen Porphyromonas gingivalis. Young (5 weeks) and aged (≥73 weeks) mice received palmitic acid-enriched Western-diet (PA-ED) or Mediterranean-diet enriched with oleic acid (OA-ED), both known for immunomodulatory properties. PA-ED exacerbated oral bone loss and systemic effects, particularly in aged mice, correlated with gut microbiota destabilization and impaired stress responses. Further, PA-ED enhanced osteoclast differentiation and primed bone marrow cells towards inflammation. Contrarily, OA-ED mitigated these effects. Osteoblasts showed baseline inflammation and reduced responsiveness in aged mice, thereby promoting a pro-inflammatory micro-environment. Findings suggest OA-ED as protective, especially for the elderly, by limiting local and systemic tissue damage associated with PD.

Bathing strategies with antiseptic agents, such as Chlorhexidine and Octenidine, have been widely adopted to mitigate infection risks in intensive care units (ICU). However, concerns exist regarding their long-term effects on skin microbiome structures and potential unintended consequences, including antibiotic cross-resistance. This longitudinal study characterized the compositional changes of the skin microbiome of ICU patients upon these two antiseptic bathing strategies when compared to standard water and soap bathing. Samples were collected in a three-armed cluster randomized decolonization trial (registration number DRKS00010475). Skin swabs from 5 different sites and three time points were analyzed by culture-based methods, 16S rRNA-gene amplicon sequencing and multiplex Taq-Man assays for detection of antimicrobial resistance genes (ARG). Our results show that Chlorhexidine bathing led to a sustained reduction of the bacterial biomass on different skin sites, as measured by both molecular and culture-based methods. Thereby, the microbial structures remained largely unaltered both in their diversity and their taxonomic composition. However, the loss of microbiome site-specificity observed on the skin of ICU patients remained unchanged independently from the bathing strategy applied and persisted even after discharge. None of the antiseptic bathing strategies led to an increase or accumulation of antibiotic-resistance determinants on any of the skin sites investigated in this study. Thus, this study suggests that daily patient bathing with 2% Chlorhexidine impregnated cloths or 0.08% Octenidine wash mitts does not impact skin microbiome structures and antibiotic resistance gene accumulation in ICU patients when compared to non-antiseptic water and soap bathing routine.

Background: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is known as a major receptor for oxidized low-density lipoproteins (oxLDL) and plays a significant role in the genesis of atherosclerosis. Recent research has shown its involvement in cancer, ischemic stroke, and diabetes. LOX-1 is a C-type lectin receptor and is involved in the activation of immune cells and inflammatory processes. It may further interact with pathogens, suggesting a role in infections or the host's response. Summary: This review compiles the current knowledge of potential implications of LOX-1 in inflammatory processes and in host-pathogen interactions with a particular emphasis on its regulatory role in immune responses. Also discussed are genomic and structural variations found in LOX-1 homologs across different species as well as potential involvements of LOX-1 in inflammatory processes from the angle of different cell types and organ-specific interactions. Key Messages: The results presented reveal both similar and different structures in human and murine LOX-1 and provide clues as to the possible origins of different modes of interaction. These descriptions raise concerns about the suitability, particularly of mouse models, that are often used in the analysis of its functionality in humans. Further research should also aim to better understand the mostly unknown binding and interaction mechanisms between LOX-1 and different pathogens. This pursuit will not only enhance our understanding of LOX-1 involvement in inflammatory processes but also identify potential targets for immunomodulatory approaches.

Background: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is known as a major receptor for oxidized low-density lipoproteins (oxLDL) and plays a significant role in the genesis of atherosclerosis. Recent research has shown its involvement in cancer, ischemic stroke, and diabetes. LOX-1 is a C-type lectin receptor and involved in the activation of immune cells and inflammatory processes. It may further interact with pathogens, suggesting a role in infections or the host's response. Summary: This review compiles the current knowledge of potential implications of LOX-1 in inflammatory processes and in host-pathogen interactions with a particular emphasis on its regulatory role in immune responses. Also discussed are genomic and structural variations found in LOX-1 homologues across different species as well as potential involvements of LOX-1 in inflammatory processes from the angle of different cell types and organ-specific interactions. Key messages: The results presented reveal both similar and different structures in human and murine LOX-1 and provide clues as to the possible origins of different modes of interaction. These descriptions raise concerns about the suitability, particularly of mouse models, that are often used in the analysis of its functionality in humans. Further research should also aim to better understand the mostly unknown binding and interaction mechanisms between LOX-1 and different pathogens. This pursuit will not only enhance our understanding of LOX-1 involvement in inflammatory processes, but also identify potential targets for immunomodulatory approaches.

An increasing amount of evidence has linked critical illness with dysbiotic microbiome signatures in different body sites. The disturbance of the indigenous microbiota structures has been further associated with disease severity and outcome and has been suggested to pose an additional risk for complications in intensive care units (ICUs), including hospital-acquired infections. A better understanding of the microbial dysbiosis in critical illness might thus help to develop strategies for the prevention of such complications. While most of the studies addressing microbiome changes in ICU patients have focused on the gut, the lung, or the oral cavity, little is known about the microbial communities on the skin of ICU patients. Since the skin is the outermost organ and the first immune barrier against pathogens, its microbiome might play an important role in the risk management for critically ill patients. This observational study characterizes the skin microbiome in ICU patients covering five different body sites at the time of admission. Our results show a profound dysbiosis on the skin of critically ill patients, which is characterized by a loss of site specificity and an overrepresentation of gut bacteria on all skin sites when compared to a healthy group. This study opens a new avenue for further investigations on the effect of skin dysbiosis in the ICU setting and points out the need of strategies for the management of dysbiosis in critically ill patients.IMPORTANCEUnbalanced gut microbiota in critically ill patients has been associated with poor outcome and complications during the intensive care unit (ICU) stay. Whether the disturbance of the microbial communities in these patients is extensive for other body sites, such as the skin, is largely unknown. The skin not only is the largest organ of the body but also serves as the first immune barrier against potential pathogens. This study characterized the skin microbiota on five different body sites in ICU patients at the time of admission. The observed disturbance of the bacterial communities might help to develop new strategies in the risk management of critically ill patients.

Staphylococcus aureus (Sa) and Acinetobacter baumannii (Ab) are frequently co-isolated from polymicrobial infections that are severe and refractory to therapy. Here, we apply a combination of wet-lab experiments and in silico modeling to unveil the intricate nature of the Ab/Sa interaction using both, representative laboratory strains and strains co-isolated from clinical samples. This comprehensive methodology allowed uncovering Sa's capability to exert a partial interference on Ab by the expression of phenol-soluble modulins. In addition, we observed a cross-feeding mechanism by which Sa supports the growth of Ab by providing acetoin as an alternative carbon source. This study is the first to dissect the Ab/Sa interaction dynamics wherein competitive and cooperative strategies can intertwine. Through our findings, we illuminate the ecological mechanisms supporting their coexistence in the context of polymicrobial infections. Our research not only enriches our understanding but also opens doors to potential therapeutic avenues in managing these challenging infections.

Staphylococcus aureus evades antibiotic therapy and antimicrobial defenses by entering human host cells. Bacterial transcriptomic analysis represents an invaluable tool to unravel the complex interplay between host and pathogen. Therefore, the extraction of high-quality RNA from intracellular S. aureus lays the foundation to acquire meaningful gene expression data. In this study, we present a novel and straightforward strategy to isolate RNA from internalized S. aureus after 90 min, 24 h, and 48 h postinfection. Real-time PCR data were obtained for the target genes agrA and fnba, which play major roles during infection. The commonly used reference genes gyrB, aroE, tmRNA, gmk, and hu were analyzed under different conditions: bacteria from culture (condition I), intracellular bacteria (condition II), and across both conditions I and II. The most stable reference genes were used for the normalization of agrA and fnbA. Delta Cq (quantification cycle) values had a relatively low variability and thus demonstrated the high quality of the extracted RNA from intracellular S. aureus during the early phase of infection. The established protocol allows the extraction and purification of intracellular staphylococcal RNA while minimizing the amount of host RNA in the sample. This approach can leverage reproducible gene expression data to study host–pathogen interactions.

Exposure of human monocytes to lipopolysaccharide (LPS) or other pathogen-associated molecular pattern (PAMPs) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance (ET), associated with the pathogenesis of sepsis. In this study, we aimed to characterize the cellular state of human monocytes from healthy donors stimulated with Staphylococcus aureus in comparison to TLR2-specific ligands. We analyzed S. aureus induced gene expression changes after 2 and 24 hours by amplicon sequencing (RNA-AmpliSeq) and compared the pro-inflammatory response after 2 hours with the response in re-stimulation experiments. In parallel, glycoprotein expression changes in human monocytes after 24 hours of S. aureus stimulation were analyzed by proteomics and compared to stimulation experiments with TLR2 ligands Malp-2 and Pam3Cys and TLR4 ligand LPS. Finally, we analyzed peripheral blood monocytes of patients with S. aureus bloodstream infection for their ex vivo inflammatory responses towards S. aureus stimulation and their glycoprotein expression profiles. Our results demonstrate that monocytes from healthy donors stimulated with S. aureus and TLR ligands of Gram-positive bacteria entered the tolerant cell state after activation similar to LPS treatment. In particular reduced gene expression of pro-inflammatory cytokines (TNF, IL1β) and chemokines (CCL20, CCL3, CCL4, CXCL2, CXCL3 and CXCL8) could be demonstrated. Glycoprotein expression changes in monocytes tolerized by the different TLR agonists were highly similar while S. aureus -stimulated monocytes shared some of the PAMP-induced changes but also exhibited a distinct expression profile. 11 glycoproteins (CD44, CD274, DSC2, ICAM1, LAMP3, LILRB1, PTGS2, SLC1A3, CR1, FGL2, and HP) were similarly up- or downregulated in all four comparisons in the tolerant cell state. Monocytes from patients with S. aureus bacteremia revealed preserved pro-inflammatory responsiveness to S. aureus stimulation ex vivo, expressed increased CD44 mRNA but no other glycoprotein of the tolerance signature was differentially expressed.

Neutrophils are critical phagocytic cells in innate immunity, playing a significant role in defending against invasive fungal pathogens. This study aimed to explore the transcriptional activation of human neutrophils in response to different fungal pathogens, including Candida albicans and Aspergillus fumigatus, compared to the bacterial pathogen Escherichia coli. We identified distinct transcriptional profiles and stress-related pathways in neutrophils during fungal infections, highlighting their functional diversity and adaptability. The transcriptional response was largely redundant across all pathogens in immune-relevant categories and cytokine pathway activation. However, differences in the magnitude of differentially expressed genes (DEGs) were observed, with A. fumigatus inducing a lower transcriptional effect compared to C. albicans and E. coli. Notably, specific gene signatures associated with cell death were differentially regulated by fungal pathogens, potentially increasing neutrophil susceptibility to autophagy, pyroptosis, and neutrophil extracellular trap (NET) formation. These findings provide valuable insights into the complex immunological responses of neutrophils during fungal infections, offering new avenues for diagnostic and therapeutic strategies, particularly in the management of invasive fungal diseases.

Cystic fibrosis (CF) is an autosomal recessive disease which affects over 100,000 people worldwide. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), leading to malfunction of exocrine tissues and severe damage to the respiratory and the digestive tracts. In the last decades, improved therapeutic approaches for most of the CFTR mutations have significantly increased the life expectancy of CF patients. The frequent antibiotic interventions of these patients, however, represent an important manipulation of the microbial communities in the already biased microbiome structures in different organs. The particular effects of long-term and short-term antibiotic treatments on the compositional microbiome changes and the potential accumulation of antibiotic resistance genes (ARGs) are yet largely unknown.

Depression is highly prevalent (6% 1-year prevalence) and is the second leading cause of disability worldwide. Available treatment options for depression are far from optimal, with response rates only around 50%. This is most likely related to a heterogeneous clinical presentation of major depression disorder (MDD), suggesting different manifestations of underlying pathophysiological mechanisms.Poorer treatment outcomes to first-line antidepressants were reported in MDD patients endorsing an "atypical" symptom profile that is characterized by preserved reactivity in mood, increased appetite, hypersomnia, a heavy sensation in the limbs, and interpersonal rejection sensitivity. In recent years, evidence has emerged that immunometabolic biological dysregulation is an important underlying pathophysiological mechanism in depression, which maps more consistently to atypical features.In the last few years human microbial residents have emerged as a key influencing variable associated with immunometabolic dysregulations in depression. The microbiome plays a critical role in the training and development of key components of the host's innate and adaptive immune systems, while the immune system orchestrates the maintenance of key features of the host-microbe symbiosis. Moreover, by being a metabolically active ecosystem commensal microbes may have a huge impact on signaling pathways, involved in underlying mechanisms leading to atypical depressive symptoms. In this review, we discuss the interplay between the microbiome and immunometabolic imbalance in the context of atypical depressive symptoms. Although research in this field is in its infancy, targeting biological determinants in more homogeneous clinical presentations of MDD may offer new avenues for the development of novel therapeutic strategies for treatment-resistant depression.This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

Hintergrund Pulmonale Beteiligungen bei Covid-19-Erkrankungen umfassen das Spektrum von asymptomatischen Verläufen bis hin zu schwersten akuten Lungenschädigungen im Sinne eines ARDS, aber auch in ihrer Ausprägung variable Long-Covid-Syndrom-Verläufe. Neben einer Hyperinflammation treten auch fibrosierende Lungenveränderungen auf, deren Pathogenese weiterhin nicht hinreichend verstanden wird. Das Ziel unserer Pilotstudie war die Charakterisierung von Fibrose-assoziierten Pathways bei Pat. mit Covid-19-ARDS und Long-Covid mit interstitieller Lungenbeteiligung (ILD) im Vergleich zu ILD anderer Genese.

Introduction: Pulmonary manifestations of COVID-19 range from asymptomatic cases over severe acute lung injuries such as ARDS to pulmonary long-COVID manifestations. In addition to hyperinflammation, fibrotic lung changes may occur, whose pathogenesis is not yet fully understood. Aims and objectives: The aim of our pilot study was to characterize differences of fibrosis-associated pathways in patients with acute COVID-19 ARDS and long-COVID-ILD compared to non COVID-19 ARDS and other ILD, respectively. Methods: We performed comprehensive transcriptome analyses for RNA sequencing (TruSeq-RNA Kit, Fa. Illumina) on 18 formalin-fixed, paraffin-embedded (FFPE) lung samples (autopsy/intraoperative tissue samples, lung biopsies) from patients with COVID-19 ARDS +/- invasive ventilation and long-COVID-ILD compared to 30 control samples from non COVID-ARDS, NSIP, COP, IPF and healthy lung tissue. Results: In general, transcriptome analyses on FFPE material were challenging due to RNA denaturation. Both homologous and significantly differentially regulated gene expression patterns were identifiable relating to specific fibrosis pathways in COVID-19, non COVID-ARDS, COP and NSIP (see Figure 1). Conclusions: Our preliminary transcriptome analyses suggest distinct gene expression patterns in the various subgroups and may set the basis for future molecular biological studies to understand specific lung fibrosis pathways.