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The gut microbiota produce hundreds of small molecules, many of which modulate host physiology. Although efforts have been made to identify biosynthetic genes for secondary metabolites, the chemical output of the gut microbiome consists predominantly of primary metabolites. Here we introduce the gutSMASH algorithm for identification of primary metabolic gene clusters, and we used it to systematically profile gut microbiome metabolism, identifying 19,890 gene clusters in 4,240 high-quality microbial genomes. We found marked differences in pathway distribution among phyla, reflecting distinct strategies for energy capture. These data explain taxonomic differences in short-chain fatty acid production and suggest a characteristic metabolic niche for each taxon. Analysis of 1,135 individuals from a Dutch population-based cohort shows that the level of microbiome-derived metabolites in plasma and feces is almost completely uncorrelated with the metagenomic abundance of corresponding metabolic genes, indicating a crucial role for pathway-specific gene regulation and metabolite flux. This work is a starting point for understanding differences in how bacterial taxa contribute to the chemistry of the microbiome. Taxon-specific primary metabolic pathways are identified using profile hidden Markov models.

Microbial communities might include distinct lineages of closely related organisms that complicate metagenomic assembly and prevent the generation of complete metagenome-assembled genomes (MAGs). Here we show that deep sequencing using long (HiFi) reads combined with Hi-C binning can address this challenge even for complex microbial communities. Using existing methods, we sequenced the sheep fecal metagenome and identified 428 MAGs with more than 90% completeness, including 44 MAGs in single circular contigs. To resolve closely related strains (lineages), we developed MAGPhase, which separates lineages of related organisms by discriminating variant haplotypes across hundreds of kilobases of genomic sequence. MAGPhase identified 220 lineage-resolved MAGs in our dataset. The ability to resolve closely related microbes in complex microbial communities improves the identification of biosynthetic gene clusters and the precision of assigning mobile genetic elements to host genomes. We identified 1,400 complete and 350 partial biosynthetic gene clusters, most of which are novel, as well as 424 (298) potential host–viral (host–plasmid) associations using Hi-C data. Metagenome sequencing can now distinguish closely related microbes using long reads and haplotype phasing.

Microbes play an increasingly recognized role in determining host-associated phenotypes by producing small molecules that interact with other microorganisms or host cells. The production of these molecules is often encoded in syntenic genomic regions, also known as gene clusters. Microbial gene clusters encoding the biosynthesis of primary and secondary metabolites play key roles in shaping microbial ecosystems and driving microbiome-associated phenotypes. Although effective approaches exist to evaluate the metabolic potential of such bacteria through identification of these metabolic gene clusters in their genomes, no automated pipelines exist to profile the abundance and expression levels of such gene clusters in microbiome samples to generate hypotheses about their functional roles, and to find associations with phenotypes of interest. Here, we describe BiG-MAP, a bioinformatic tool to profile abundance and expression levels of gene clusters across metagenomic and metatranscriptomic data and evaluate their differential abundance and expression under different conditions. To illustrate its usefulness, we analyzed 96 metagenomic samples from healthy and caries-associated human oral microbiome samples and identified 252 gene clusters, including unreported ones, that were significantly more abundant in either phenotype. Among them, we found the muc operon, a gene cluster known to be associated with tooth decay. Additionally, we found a putative reuterin biosynthetic gene cluster from a Streptococcus strain to be enriched but not exclusively found in healthy samples; metabolomic data from the same samples showed masses with fragmentation patterns consistent with (poly)acrolein, which is known to spontaneously form from the products of the reuterin pathway and has been previously shown to inhibit pathogenic Streptococcus mutans strains. Thus, we show how BiG-MAP can be used to generate new hypotheses on potential drivers of microbiome-associated phenotypes and prioritize the experimental characterization of relevant gene clusters that may mediate them. IMPORTANCE Microbes play an increasingly recognized role in determining host-associated phenotypes by producing small molecules that interact with other microorganisms or host cells. The production of these molecules is often encoded in syntenic genomic regions, also known as gene clusters. With the increasing numbers of (multi)omics data sets that can help in understanding complex ecosystems at a much deeper level, there is a need to create tools that can automate the process of analyzing these gene clusters across omics data sets. This report presents a new software tool called BiG-MAP, which allows assessing gene cluster abundance and expression in microbiome samples using metagenomic and metatranscriptomic data. Here, we describe the tool and its functionalities, as well as its validation using a mock community. Finally, using an oral microbiome data set, we show how it can be used to generate hypotheses regarding the functional roles of gene clusters in mediating host phenotypes.

Background COVID-19-related ARDS has unique features when compared with ARDS from other origins, suggesting a distinctive inflammatory pathogenesis. Data regarding the host response within the lung are sparse. The objective is to compare alveolar and systemic inflammation response patterns, mitochondrial alarmin release, and outcomes according to ARDS etiology (i.e., COVID-19 vs. non-COVID-19). Methods Bronchoalveolar lavage fluid and plasma were obtained from 7 control, 7 non-COVID-19 ARDS, and 14 COVID-19 ARDS patients. Clinical data, plasma, and epithelial lining fluid (ELF) concentrations of 45 inflammatory mediators and cell-free mitochondrial DNA were measured and compared. Results COVID-19 ARDS patients required mechanical ventilation (MV) for significantly longer, even after adjustment for potential confounders. There was a trend toward higher concentrations of plasma CCL5, CXCL2, CXCL10, CD40 ligand, IL-10, and GM-CSF, and ELF concentrations of CXCL1, CXCL10, granzyme B, TRAIL, and EGF in the COVID-19 ARDS group compared with the non-COVID-19 ARDS group. Plasma and ELF CXCL10 concentrations were independently associated with the number of ventilator-free days, without correlation between ELF CXCL-10 and viral load. Mitochondrial DNA plasma and ELF concentrations were elevated in all ARDS patients, with no differences between the two groups. ELF concentrations of mitochondrial DNA were correlated with alveolar cell counts, as well as IL-8 and IL-1β concentrations. Conclusion CXCL10 could be one key mediator involved in the dysregulated immune response. It should be evaluated as a candidate biomarker that may predict the duration of MV in COVID-19 ARDS patients. Targeting the CXCL10-CXCR3 axis could also be considered as a new therapeutic approach. Trial registration ClinicalTrials.gov, NCT03955887

Non-beneficial stays in the intensive care unit (ICU) may have repercussions for patients and their families, but can also cause suffering among the nursing staff. We aimed explore the perceptions of nursing staff in the ICU about patient stays that are deemed to be \"non-beneficial\" for the patient, to identify areas amenable to intervention, with a view to improving how the nursing staff perceive the patient pathway before, during and after intensive care. Multicentre, qualitative study using individual, semi-structured interviews. All qualified nurses and nurses' aides who were full-time employees in the ICU of three participating centres were invited to participate. Interviews were recorded, transcribed and analyzed using textual content analysis. A total of 21 interviews were performed from February 2020 to October 2021, at which point saturation was reached in the data. Average age of participants was 38.5±7.5 years, and they had an average of 10.7±7.4 years of experience working in the ICU. Four major themes emerged from the interviews, namely: (1) the work is oriented towards life-threatening emergencies, technical procedures and burdensome care; (2) a range of specific criteria and circumstances influence the decisions to admit patients to ICU; (3) there are significant organisational, physical and psychological repercussions associated with a non-beneficial stay in the ICU; (4) respondents made some proposals for improvements to the patient care pathway. Nursing staff have a similar perception to physicians regarding admission decisions and non-beneficial ICU stays. The possibility of future ICU admission needs to be anticipated, discussed systematically with patients and integrated into healthcare goals that are consistent with the patient's wishes and preferences, in multi-professional collaboration including nursing and medical staff.

We investigated the reflections and perceptions of non-ICU physicians about anticipating the need for ICU admission in case of acute decompensation in patients with chronic disease. We performed a qualitative multicentre study using semi-structured interviews among non-ICU specialist physicians. The interview guide, developed in advance, focused on 3 questions: (1) What is your perception of ICU care? (2) How do you think advance directives can be integrated into the patient's healthcare goals? and (3) How can the possibility of a need for ICU admission be integrated into the patient's healthcare goals? Interviews were recorded, transcribed and analysed by thematic analysis. Interviews were performed until theoretical saturation was reached. In total, 16 physicians (8 women, 8 men) were interviewed. The main themes related to intensive care being viewed as a distinct specialty, dispensing very technical care, and with major human and ethical challenges, especially regarding end-of-life issues. The participants also mentioned the difficulty in anticipating an acute decompensation, and the choices that might have to be made in such situations. The timing of discussions about potential decompensation of the patient, the medical culture and the presence of advance directives are issues that arise when attempting to anticipate the question of ICU admission in the patient's healthcare goals or wishes. This study describes the perceptions that physicians treating patients with chronic disease have of intensive care, notably that it is a distinct and technical specialty that presents challenging medical and ethical situations. Our study also opens perspectives for actions that could promote a pluridisciplinary approach to anticipating acute decompensation and ICU requirements in patients with chronic disease.

IntroductionWe investigated the reflections and perceptions of non-ICU physicians about anticipating the need for ICU admission in case of acute decompensation in patients with chronic disease.MethodsWe performed a qualitative multicentre study using semi-structured interviews among non-ICU specialist physicians. The interview guide, developed in advance, focused on 3 questions: (1) What is your perception of ICU care? (2) How do you think advance directives can be integrated into the patient's healthcare goals? and (3) How can the possibility of a need for ICU admission be integrated into the patient's healthcare goals? Interviews were recorded, transcribed and analysed by thematic analysis. Interviews were performed until theoretical saturation was reached.ResultsIn total, 16 physicians (8 women, 8 men) were interviewed. The main themes related to intensive care being viewed as a distinct specialty, dispensing very technical care, and with major human and ethical challenges, especially regarding end-of-life issues. The participants also mentioned the difficulty in anticipating an acute decompensation, and the choices that might have to be made in such situations. The timing of discussions about potential decompensation of the patient, the medical culture and the presence of advance directives are issues that arise when attempting to anticipate the question of ICU admission in the patient's healthcare goals or wishes.ConclusionThis study describes the perceptions that physicians treating patients with chronic disease have of intensive care, notably that it is a distinct and technical specialty that presents challenging medical and ethical situations. Our study also opens perspectives for actions that could promote a pluridisciplinary approach to anticipating acute decompensation and ICU requirements in patients with chronic disease.

Deciding not to re-admit a patient to the intensive care unit (ICU) poses an ethical dilemma for ICU physicians. We aimed to describe and understand the attitudes and perceptions of ICU physicians regarding non-readmission of patients to the ICU. Multicenter, qualitative study using semi-directed interviews between January and May 2019. All medical staff working full-time in the ICU of five participating centres (two academic and three general, non-academic hospitals) were invited to participate. Participants were asked to describe how they experienced non-readmission decisions in the ICU, and to expand on the manner in which the decision was made, but also on the traceability and timing of the decision. Interviews were recorded, transcribed and analyzed using textual content analysis. In total, 22 physicians participated. Interviews lasted on average 26±7 minutes. There were 14 men and 8 women, average age was 35±9 years, and average length of ICU experience was 7±5 years. The majority of respondents said that they regretted that the question of non-readmission was not addressed before the initial ICU admission. They acknowledged that the ICU stay did lead to more thorough contemplation of the overall goals of care. Multidisciplinary team meetings could help to anticipate the question of readmission within the patient's care pathway. Participants reported that there is a culture of collegial decision-making in the ICU, although the involvement of patients, families and other healthcare professionals in this process is not systematic. The timing and traceability of non-readmission decisions are heterogeneous. Non-readmission decisions are a major issue that raises ethical questions surrounding the fact that there is no discussion of the patient's goals of care in advance. Better anticipation, and better communication with the patients, families and other healthcare providers are suggested as areas that could be targeted for improvement.

We sought to describe the characteristics that lead physicians to perceive a stay in the intensive care unit (ICU) as being non-beneficial for the patient. In the first step, we used a multidisciplinary focus group to define the characteristics that lead physicians to consider a stay in the ICU as non-beneficial for the patient. In the second step, we assessed the proportion of admissions that would be perceived by the ICU physicians as non-beneficial for the patient according to our focus group's definition, in a large population of ICU admissions in 4 French ICUs over a period of 4 months. Among 1075 patients admitted to participating ICUs during the study period, 155 stays were considered non-beneficial for the patient, yielding a frequency of 14.4% [95% confidence interval (CI) 8.9, 19.9]. Average age of these patients was 72 ±12.8 years. Mortality was 43.2% in-ICU [95%CI 35.4, 51.0], 55% [95%CI 47.2, 62.8] in-hospital. The criteria retained by the focus group to define a non-beneficial ICU stay were: patient refusal of ICU care (23.2% [95%CI 16.5, 29.8]), and referring physician's desire not to have the patient admitted (11.6% [95%CI 6.6, 16.6]). The characteristics that led physicians to perceive the stay as non-beneficial were: patient's age (36.8% [95%CI 29.2, 44.4]), unlikelihood of recovering autonomy (61.9% [95%CI 54.3, 69.6]), prior poor quality of life (60% [95%CI 52.3, 67.7]), terminal status of chronic disease (56.1% [95%CI 48.3, 63.9]), and all therapeutic options have been exhausted (35.5% [95%CI 27.9, 43.0]). Factors that explained admission to the ICU of patients whose stay was subsequently judged to be non-beneficial included: lack of knowledge of patient's wishes (52% [95%CI 44.1, 59.9]); decisional incapacity (sedation) (69.7% [95%CI 62.5, 76.9]); inability to contact family (34% [95%CI 26.5, 41.5]); pressure to admit (from family or other physicians) (50.3% [95%CI 42.4, 58.2]). Non-beneficial ICU stays are frequent. ICU admissions need to be anticipated, so that patients who would yield greater benefit from other care pathways can be correctly oriented in a timely manner.

Biofilm (BF) growth is believed to play a major role in the development of ventilator-associated pneumonia (VAP) in the intensive care unit. Despite concerted efforts to understand the potential implication of endotracheal tube (ETT)-BF dispersal, clinically relevant data are lacking to better characterize the impact of its mesostructure and microbiological singularity on the occurrence of VAP. We conducted a multicenter, retrospective observational study during the third wave of the COVID-19 pandemic, between March and May 2021. In total, 64 ETTs collected from 61 patients were included in the present BIOPAVIR study. Confocal microscopy acquisitions revealed two main morphological aspects of ETT-deposited BF: (1) a thin, continuous ribbon-shaped aspect, less likely monobacterial and predominantly associated with Enterobacter spp ., Streptococcus pneumoniae or Viridans streptococci, and (2) a thicker, discontinuous, mushroom-shaped appearance, more likely characterized by the association of bacterial and fungal species in respiratory samples. The microbiological characterization of ETT-deposited BF found higher acquired resistance in more than 80% of analyzed BF phenotypes, compared to other colonization sites from the patient’s environment. These findings reveal BF as a singular microbiological compartment, and are of added clinical value, with a view to future ETT-deposited BF-based antimicrobial stewardship in critically ill patients. Trial registration NCT04926493. Retrospectively registered 15 June 2021.