Abstract
While metagenomic sequencing has become the tool of preference to study host-associated microbial communities, downstream analyses and clinical interpretation of microbiome data remains ...challenging due to the sparsity and compositionality of sequence matrices. Here, we evaluate both computational and experimental approaches proposed to mitigate the impact of these outstanding issues. Generating fecal metagenomes drawn from simulated microbial communities, we benchmark the performance of thirteen commonly used analytical approaches in terms of diversity estimation, identification of taxon-taxon associations, and assessment of taxon-metadata correlations under the challenge of varying microbial ecosystem loads. We find quantitative approaches including experimental procedures to incorporate microbial load variation in downstream analyses to perform significantly better than computational strategies designed to mitigate data compositionality and sparsity, not only improving the identification of true positive associations, but also reducing false positive detection. When analyzing simulated scenarios of low microbial load dysbiosis as observed in inflammatory pathologies, quantitative methods correcting for sampling depth show higher precision compared to uncorrected scaling. Overall, our findings advocate for a wider adoption of experimental quantitative approaches in microbiome research, yet also suggest preferred transformations for specific cases where determination of microbial load of samples is not feasible.
The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 ...and 2), differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, non-synonymous mutations, indels and genome rearrangements of these 23 strains and 4 previously sequenced ones, has revealed new subclasses in the two main groups, some of them being associated with the country of isolation. Integrative analysis of in vitro gene essentiality and mutation rates enabled the identification of several putative virulence factors and antigenic proteins; revealing recombination machinery, glycerol metabolism and peroxide production as possible factors in the genetics and physiology of these pathogenic strains. Additionally, the transcriptomes and proteomes of two representative strains, one from each of the two main groups, have been characterized to evaluate the impact of mutations on RNA and proteins levels. This study has revealed that type 2 strains show higher expression levels of CARDS toxin, a protein recently shown to be one of the major factors of inflammation. Thus, we propose that type 2 strains could be more toxigenic than type 1 strains of M. pneumoniae.
Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome‐reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 ...residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non‐coding RNAs (ncRNAs) non‐overlapping with essential genes is 5% higher than of non‐transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non‐coding regions, thus changing the focus when aiming to define the minimal essential genome.
Synopsis
A genome essentiality analysis in the genome‐reduced bacterium Mycoplasma pneumoniae, reveals that protein essentiality should be considered at the domain level and that small proteins (< 100 aa) and ncRNAs are frequently essential genomic elements.
A genome essentiality analysis is performed using two mini‐transposon mutant libraries of M. pneumoniae.
The results indicate that ORF essentiality should be considered at the protein domain level.
Small ORFs are as essential as conventional ORFs and they can interact with DNA.
Some essential antisense ncRNAs are involved in the regulation of essential ORF expression.
A genome essentiality analysis in the genome‐reduced bacterium Mycoplasma pneumoniae, reveals that protein essentiality should be considered at the domain level and that small proteins (< 100 aa) and ncRNAs are frequently essential genomic elements.
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•DPFs have been loaded with 0.6wt.% of either Pt or an optimized CePr active phase.•Pt–DPF and CePr–DPF are regenerated at equal temperature in most conditions.•Both Pt and CePr ...active phases are stable after several DPF regeneration cycles.•The presence of H2O and CO2 affects equally to Pt–DPF and CePr–DPF.•CePr is a promising candidate to replace Pt in real soot removal DPFs.
Diesel Particulate Filters (DPFs) have been loaded with the same amount (0.6wt.%) of either Pt or an optimized CePr active phase, and an experimental set-up has been designated and used to investigate the catalytic combustion of soot under realistic reaction conditions. Both active phases were stable under reaction conditions, with no evidences of deactivation in consecutive combustion experiments. The presence of H2O and CO2 together with NOx and O2 in the gas mixture slightly delays soot combustion to higher temperatures, but the effect is equal for the Pt and CePr active phases. The mass of soot loaded into the filters had no effect in the catalytic regeneration of the DPFs for soot:catalyst weigh ratios below 0.4, while it was hindered above this ratio. The Pt and CePr active phases behave equal until 0.6 soot:catalyst weigh ratio and Pt performance was slightly better for higher soot loading. This difference is explaining according to the main soot combustion mechanisms occurring during Pt–DPF (NO2-assited) and CePr–DPF regeneration (active oxygen). The CO2 selectivity is near 100% for both catalysts in all the experimental reaction conditions evaluated. According to this study, the CePr active phase seems to be a promising candidate to replace Pt in real applications.
The composition of the human gut microbiome is well resolved, but predictive understanding of its dynamics is still lacking. Here, we followed a bottom-up strategy to explore human gut community ...dynamics: we established a synthetic community composed of three representative human gut isolates (
L1-82,
A2-165 and
S5a33) and explored their interactions under well-controlled conditions in vitro. Systematic mono- and pair-wise fermentation experiments confirmed competition for fructose and cross-feeding of formate. We quantified with a mechanistic model how well tri-culture dynamics was predicted from mono-culture data. With the model as reference, we demonstrated that strains grown in co-culture behaved differently than those in mono-culture and confirmed their altered behavior at the transcriptional level. In addition, we showed with replicate tri-cultures and simulations that dominance in tri-culture sensitively depends on the initial conditions. Our work has important implications for gut microbial community modeling as well as for ecological interaction detection from batch cultures.
Microbial culturing and meta-omic profiling technologies have significantly advanced our understanding of the taxonomic and functional variation of the human microbiome and its impact on host ...processes. The next increase in resolution will come by understanding the role of low-abundant and less-prevalent bacteria and the study of individual cell behaviors that underlie the complexity of microbial ecosystems. To this aim, single-cell techniques are being rapidly developed to isolate, culture, and characterize the genomes and transcriptomes of individual microbes in complex communities. Here, we discuss how these single-cell technologies are providing unique insights into the biology and behavior of human microbiomes.
This review describes a variety of recently developed single-cell approaches for studying the human microbiome and remaining challenges for the field.
Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report ...contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper (n = 58) and lower (n = 35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We find that time in the intensive care unit and type of oxygen support, as well as associated treatments such as antibiotic usage, explain the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load has a reduced impact. Specifically, mechanical ventilation is linked to altered community structure and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomics of the lower respiratory tract of COVID-19 patients identifies specific oral bacteria in physical association with proinflammatory immune cells, which show higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients.
•Over 9% of patients hospitalized for COVID-19 will present a co-infection.•Independent risk factors for co-infection were identified.•When procalcitonin values are <0.2 ng/mL, co-infection is very ...rare.•High ferritin values and oxygen saturation >94% are also uncommon in co-infection.
We described the current incidence and risk factors of bacterial co-infection in hospitalized patients with COVID-19.
Observational cohort study was performed at the Hospital Clinic of Barcelona (February 2020–February 2021). All patients with COVID-19 who were admitted for >48 hours with microbiological sample collection and procalcitonin (PCT) determination within the first 48 hours were included.
A total of 1125 consecutive adults met inclusion criteria. Co-infections were microbiologically documented in 102 (9.1%) patients. Most frequent microorganisms were Streptococcus pneumoniae (79%), Staphylococcus aureus (6.8%), and Haemophilus influenzae (6.8%). Test positivity was 1% (8/803) for blood cultures, 10.1% (79/780) for pneumococcal urinary antigen test, and 11.4% (15/132) for sputum culture. Patients with PCT higher than 0.2, 0.5, 1, and 2 ng/mL had significantly more co-infections than those with lower levels (p=0.017, p=0.031, p<0.001, and p<0.001, respectively). In multivariate analysis, oxygen saturation ≤94% (OR 2.47, CI 1.57–3.86), ferritin levels <338 ng/mL (OR 2.63, CI 1.69–4.07), and PCT higher than 0.2 ng/mL (OR 1.74, CI 1.11–2.72) were independent risk factors for co-infection at hospital admission owing to COVID-19.
Bacterial co-infection in patients hospitalized for COVID-19 is relatively common. However, clinicians could spare antibiotics in patients with PCT values <0.2, especially with high ferritin values and oxygen saturation >94%.
•Genome-reduced bacteria have lost regulatory proteins acting at most regulatory levels.•Minimal bacteria have retained sequence features to regulate transcription.•Non-transcription factor ...regulation can occur at genome-wide, operon and transcript level.
Transcription is a core process of bacterial physiology, and as such it must be tightly controlled, so that bacterial cells maintain steady levels of each RNA molecule in homeostasis and modify them in response to perturbations. The major regulators of transcription in bacteria (and in eukaryotes) are transcription factors. However, in genome-reduced bacteria, the limited number of these proteins is insufficient to explain the variety of responses shown upon changes in their environment. Thus, alternative regulators may play a central role in orchestrating RNA levels in these microorganisms. These alternative mechanisms rely on intrinsic features within DNA and RNA molecules, suggesting they are ancestral mechanisms shared among bacteria that could have an increased relevance on transcriptional regulation in minimal cells. In this review, we summarize the alternative elements that can regulate transcript abundance in genome-reduced bacteria and how they contribute to the RNA homeostasis at different levels.
The purpose of this study is to evaluate the in-hospital mortality of community-acquired pneumonia (CAP) treated with ceftaroline in comparison with standard therapy. This was a retrospective ...observational study in two centers. Hospitalized patients with CAP were grouped according to the empiric regimen (ceftaroline versus standard therapy) and analyzed using a propensity score matching (PSM) method to reduce confounding factors. Out of the 6981 patients enrolled, 5640 met the inclusion criteria, and 89 of these received ceftaroline. After PSM, 78 patients were considered in the ceftaroline group (cases) and 78 in the standard group (controls). Ceftaroline was mainly prescribed in cases with severe pneumonia (67% vs. 56%,
p
= 0.215) with high suspicion of
Staphylococcus aureus
infection (9% vs. 0%,
p
= 0.026). Cases had a longer length of hospital stay (13 days vs. 10 days,
p
= 0.007), while an increased risk of in-hospital mortality was observed in the control group compared to the case group (13% vs. 21%, HR 0.41; 95% CI 0.18 to 0.62,
p
= 0.003). The empiric use of ceftaroline in hospitalized patients with severe CAP was associated with a decreased risk of in-hospital mortality.
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