Single-cell approaches in human microbiome research.

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.

Effectiveness and Safety of Postoperative Hospital at Home for Surgical Patients: A Cohort Study.

OBJECTIVE: To determine the feasibility and effectiveness of a Hospital at Home (HaH) enabled early transfer pathways for surgical patients. BACKGROUND: HaH serves as a safe alternative to traditional hospitalization by providing acute care to patients in their homes through a comprehensive range of hospital-level interventions. To our knowledge, no studies have been published to date reporting a large cohort of early home-transferred patients after surgery through a HaH unit. METHODS: Cohort study enrolling every patient admitted to the HaH unit of a tertiary hospital who underwent any of 6 surgeries with a predefined early transfer pathway and fitting both general and surgery inclusion criteria (clinical and hemodynamic stability, uncomplicated surgery, presence of a caregiver, among others) from November 2021 to May 2023. Protocols were developed for each pathway between surgical services and HaH to deliver the usual postoperative care in the home setting. Discharge was decided according to protocol. An urgent escalation pathway was also established. RESULTS: During the study period, 325 patients were included: 141 were bariatric surgeries, 85 kidney transplants, 45 thoracic surgeries, 37 cystectomies, 10 appendicectomies, and 7 ventral hernia repairs. The overall escalation of care during HaH occurred in 7.3% of patients and 30-day readmissions in 7%. Most adverse events were managed at home and the overall mortality was zero. The total mean length of stay was 8 days (interquartile range 2-14), and patients with HaH were transferred home 3 days (interquartile range 1-6) earlier than the usual pathway; a total of 1551 bed-days were saved. CONCLUSIONS: The implementation of early home transfer pathways for surgical patients through HaH is feasible and effective, with favorable safety outcomes.

Qualitative Subgenomic RNA to Monitor the Response to Remdesivir in Hospitalized Patients With Coronavirus Disease 2019: Impact on the Length of Hospital Stay and Mortality.

BACKGROUND: There is no reliable microbiological marker to guide the indication and the response to antiviral treatment in patients with coronavirus disease 2019 (COVID-19). We aimed to evaluate the dynamics of subgenomic RNA (sgRNA) in patients with COVID-19 before and after receiving treatment with remdesivir. METHODS: We included consecutive patients admitted for COVID-19 who received remdesivir according to our institutional protocol and accepted to participate in the study. A nasopharyngeal swab for quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was collected at baseline and after 3 and 5 days of treatment with remdesivir. Genomic and sgRNA were analyzed in those samples and main comorbidities and evolution were collected for the analyses. The main outcomes were early discharge (≤10 days) and 30-day mortality. RESULTS: A total of 117 patients were included in the study, of whom 24 had a negative sgRNA at baseline, with 62.5% (15/24) receiving early discharge (≤10 days) and no deaths in this group. From the 93 remaining patients, 62 had a negative sgRNA at day 5 with 37/62 (59.6%) with early discharge and a mortality rate of 4.8% (3/62). In the subgroup of 31 patients with positive sgRNA after 5 days of remdesivir, the early discharge rate was 29% (9/31) and the mortality rate was 16.1% (5/31). In multivariable analyses, the variables associated with early discharge were negative sgRNA at day 3 and not needing treatment with corticosteroids or intensive care unit admission. CONCLUSIONS: Qualitative sgRNA could help in monitoring the virological response in patients who receive remdesivir. Further studies are needed to confirm these findings.

Time from symptoms onset to remdesivir is associated with the risk of ICU admission: a multicentric analyses.

BACKGROUND: Shorter duration of symptoms before remdesivir has been associated with better outcomes. Our goal was to evaluate variables associated with the need of ICU admission in a cohort of hospitalized patients for COVID-19 under remdesivir including the period from symptoms onset to remdesivir. METHODS: We conducted a retrospective multicentric study analysing all patients admitted with COVID-19 in 9 Spanish hospitals who received treatment with remdesivir in October 2020. The main outcome was the need of ICU admission after 24 h of the first dose of remdesivir. RESULTS: In our cohort of 497 patients, the median of days from symptom onset to remdesivir was 5 days, and 70 of them (14.1%) were later admitted into ICU. The clinical outcomes associated with ICU admission were days from symptoms onset (5 vs. 6; p = 0.023), clinical signs of severe disease (respiratory rate, neutrophil count, ferritin levels and very-high mortality rate in SEIMC-Score) and the use of corticosteroids and anti-inflammatory drugs before ICU. The only variable significatively associated with risk reduction in the Cox-regression analyses was ≤ 5 days from symptoms onset to RDV (HR: 0.54, CI95%: 0.31-0.92; p = 0.024). CONCLUSION: For patients admitted to the hospital with COVID-19, the prescription of remdesivir within 5 days from symptoms onset diminishes the need of ICU admission.

Personalized Therapy Approach for Hospitalized Patients with Coronavirus Disease 2019.

Hospitalized patients with coronavirus disease 2019 (COVID-19) experiencing respiratory symptoms have different complications (inflammatory, co-infection, and thrombotic) that are identifiable by analytics patterns. Personalized treatment decisions decreased early mortality (odds ratio [OR] .144; 95% confidence interval [CI] .03-.686; P = .015). Increasing age (OR 1.06; P = .038) and therapeutic effort limitation (OR 9.684; P < .001) were associated with higher mortality.

Efficacy of early transfusion of convalescent plasma with high-titer SARS-CoV-2 neutralizing antibodies in hospitalized patients with COVID-19.

BACKGROUND: Despite most controlled trials have shown no measurable benefit of COVID-19 convalescent plasma (CCP) in patients with COVID-19, some studies suggest that early administration of CCP with high-titer anti-SARS-CoV-2 can be beneficial in selected patients. We investigated the efficacy of early administration of high-titer CCP to patients with COVID-19 who required hospitalization, STUDY DESIGN AND METHODS: Observational, propensity score (PS) matched case-control study of COVID-19 patients treated with CCP within 72 h of hospital admission and untreated controls from August 2020 to February 2021. All CCP donations had a Euroimmun anti-SARS-CoV-2 sample-to-cutoff ratio ≥3. PS matching was based on prognostic factors and presented features with high-standardized differences between the treated and control groups. The primary endpoint was mortality within 30 days of diagnosis. RESULTS: A total of 1604 patients were analyzed, 261 of whom received CCP, most (82%) within 24 h after admission. Median age was 67 years (interquartile range: 56-79), and 953 (60%) were men. Presenting factors independently associated with higher 30-day mortality were increased age, cardiac disease, hypoxemic respiratory failure, renal failure, and plasma d-dimer >700 ng/ml. After PS matching, transfusion of CCP was associated with a significant reduction in the 30-day mortality rate (odds ratio [OR]; 0.94, 95% confidence interval [CI]: 0.91-0.98; p = .001) that extended to the 60th day after COVID-19 diagnosis (OR: 0.95; 95% CI: 0.92-0.99; p = .01). CONCLUSION: Our results suggest that CCP can still be helpful in selected patients with COVID-19 and call for further studies before withdrawing CCP from the COVID-19 therapeutic armamentarium.

Impact on in-hospital mortality of ceftaroline versus standard of care in community-acquired pneumonia: a propensity-matched analysis.

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.

Antibiotic-resistant microorganisms in patients with bloodstream infection of intraabdominal origin: risk factors and impact on mortality.

BACKGROUND: Knowledge of resistance patterns is essential to choose empirical treatment. We aimed to determine the risk factors for antibiotic-resistant microorganisms (ARM) in intraabdominal infections (IAI) and their impact on mortality. METHODS: Retrospective cohort study of patients with bacteremia from IAI origin in a single hospital between January 2006 and July 2017. RESULTS: A total of 1485 episodes were recorded, including 381 (25.6%) due to ARM. Independent predictors of ARM were cirrhosis (OR 2; [95% CI 1.15-3.48]), immunosuppression (OR 1.49; 1.12-1.97), prior ceftazidime exposure (OR 3.7; 1.14-11.9), number of prior antibiotics (OR 2.33; 1.61-3.35 for 1 antibiotic), biliary manipulation (OR 1.53; 1.02-2.96), hospital-acquisition (OR 2.77; 1.89-4) and shock (OR 1.48; 1.07-2). Mortality rate of the whole cohort was 11.1%. Age (OR 1.03; 1.01-1.04), cirrhosis (OR 2.32; 1.07-4.38), urinary catheter (OR 1.99; 1.17-3.38), ultimately (OR 2.28; 1.47-3.51) or rapidly (OR 13.3; 7.12-24.9) fatal underlying disease, nosocomial infection (OR 2.76; 1.6-4.75), peritonitis (OR 1.95, 1.1-3.45), absence of fever (OR 2.17; 1.25-3.77), shock (OR 5.96; 3.89-9.13), and an ARM in non-biliary infections (OR 2.14; 1.19-3.83) were independent predictors of 30-day mortality. Source control (OR 0.24; 0.13-0.44) and 2015-2017 period (OR 0.29; 0.14-0.6) were protective. CONCLUSION: Biliary manipulation and septic shock are predictors of ARM. The presence of an ARM from a non-biliary focus is a poor-prognosis indicator. Source control continues to be of paramount importance.

Distinguishing between productive and abortive promoters using a random forest classifier in Mycoplasma pneumoniae.

Distinguishing between promoter-like sequences in bacteria that belong to true or abortive promoters, or to those that do not initiate transcription at all, is one of the important challenges in transcriptomics. To address this problem, we have studied the genome-reduced bacterium Mycoplasma pneumoniae, for which the RNAs associated with transcriptional start sites have been recently experimentally identified. We determined the contribution to transcription events of different genomic features: the -10, extended -10 and -35 boxes, the UP element, the bases surrounding the -10 box and the nearest-neighbor free energy of the promoter region. Using a random forest classifier and the aforementioned features transformed into scores, we could distinguish between true, abortive promoters and non-promoters with good -10 box sequences. The methods used in this characterization of promoters can be extended to other bacteria and have important applications for promoter design in bacterial genome engineering.

Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium.

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.

Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution.

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5'-CTAT-3'), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5'-GAN(7)TAY-3'/3'-CTN(7)ATR-5'). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.

Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?

BACKGROUND: RNA sequencing methods have already altered our view of the extent and complexity of bacterial and eukaryotic transcriptomes, revealing rare transcript isoforms (circular RNAs, RNA chimeras) that could play an important role in their biology. RESULTS: We performed an analysis of chimera formation by four different computational approaches, including a custom designed pipeline, to study the transcriptomes of M. pneumoniae and P. aeruginosa, as well as mixtures of both. We found that rare transcript isoforms detected by conventional pipelines of analysis could be artifacts of the experimental procedure used in the library preparation, and that they are protocol-dependent. CONCLUSION: By using a customized pipeline we show that optimal library preparation protocol and the pipeline to analyze the results are crucial to identify real chimeric RNAs.

Bacteria in metastatic sites: Unveiling hidden players in cancer progression.

Bacteria exhibit key features of cancer metastasis, such as motility, invasion, and modulation of the tumor microenvironment. They migrate through lymphatic and blood systems, invade metastatic tissues, and alter local microenvironments to support metastatic growth. Bacteria also shape the tumor microenvironment, affecting immune responses and inflammation, which influence tumor progression and therapy response. While they hold therapeutic potential, challenges like contamination and complex characterization persist, necessitating advanced sequencing and research for clinical application.

Microbiome confounders and quantitative profiling challenge predicted microbial targets in colorectal cancer development.

Despite substantial progress in cancer microbiome research, recognized confounders and advances in absolute microbiome quantification remain underused; this raises concerns regarding potential spurious associations. Here we study the fecal microbiota of 589 patients at different colorectal cancer (CRC) stages and compare observations with up to 15 published studies (4,439 patients and controls total). Using quantitative microbiome profiling based on 16S ribosomal RNA amplicon sequencing, combined with rigorous confounder control, we identified transit time, fecal calprotectin (intestinal inflammation) and body mass index as primary microbial covariates, superseding variance explained by CRC diagnostic groups. Well-established microbiome CRC targets, such as Fusobacterium nucleatum, did not significantly associate with CRC diagnostic groups (healthy, adenoma and carcinoma) when controlling for these covariates. In contrast, the associations of Anaerococcus vaginalis, Dialister pneumosintes, Parvimonas micra, Peptostreptococcus anaerobius, Porphyromonas asaccharolytica and Prevotella intermedia remained robust, highlighting their future target potential. Finally, control individuals (age 22-80 years, mean 57.7 years, standard deviation 11.3) meeting criteria for colonoscopy (for example, through a positive fecal immunochemical test) but without colonic lesions are enriched for the dysbiotic Bacteroides2 enterotype, emphasizing uncertainties in defining healthy controls in cancer microbiome research. Together, these results indicate the importance of quantitative microbiome profiling and covariate control for biomarker identification in CRC microbiome studies.

Doravirine/Lamivudine/Tenofovir Disoproxil Fumarate for Nonoccupational HIV-1 Postexposure Prophylaxis: A Prospective Open-Label Trial (DORAVIPEP).

Background: New regimens may provide better tolerability, convenience, and safety for nonoccupational human immunodeficiency virus (HIV) postexposure prophylaxis (PEP). For this reason, we evaluated the single-tablet regimen of doravirine/lamivudine/tenofovir disoproxil fumarate (DOR/3TC/TDF) for 28 days. Methods: This was a prospective, open-label, single-arm trial including individuals with potential HIV-1 exposure within 72 hours. The primary endpoint was noncompletion of PEP at day 28. Secondary endpoints were adverse effects, adherence, and rate of seroconversion. We performed follow-up at day 7, week 4, and week 12. Results: Between September 2019 and March 2022, the study enrolled 399 individuals. Median age was 30 (interquartile range [IQR], 27-36) years, and 91% (n = 364) were male. The mode of exposure was sex between men in 84% (n = 331) of cases; risk assessment for HIV-1 transmission was considered as "high" in 97% (n = 385) of the participants. Median time from exposure to consultation was 24 (IQR, 13-40) hours. Noncompletion of PEP was 29% (n = 114) (95% confidence interval [CI], 24%-33%) and 20% (n = 72) (95% CI, 16%-25%) per modified intention-to-treat. Main reasons for noncompletion were loss to follow-up (n = 104 [91%]) and intolerance (n = 8 [7%]). Older age was associated with a lower risk of premature discontinuation (OR, 0.94; P < .001). One hundred twenty-three (31%) participants reported adverse events, mostly mild and self-limited (82%); discontinuation occurred in 8 cases (2%). Adherence to PEP in the assessed users was 96%. There were no HIV seroconversions. Conclusions: DOR/3TC/TDF is a well-tolerated option for nonoccupational PEP. Clinical Trials Registration. NCT04233372.

Hospital at home treatment with remdesivir for patients with COVID-19: real-life experience.

OBJECTIVES: Access and appropriateness of therapeutics for COVID-19 vary because of access or regulatory barriers, the severity of the disease, and for some therapies, the stage of the pandemic and circulating variants. Remdesivir has shown benefits in clinical recovery and is the treatment of choice for selected patients, both hospitalized and nonhospitalized, in main international guidelines. The use of remdesivir in alternatives to conventional hospitalization such as hospital at home (HaH) units remains incompletely explored. In this study, we aim to describe the real-life experience of outpatient remdesivir infusion for COVID-19 in a HaH unit. METHODS: We selected all the consecutive patients receiving remdesivir from a prospective cohort of 507 COVID-19 patients admitted at a HaH unit. Admission criteria included COVID-19 with a fraction of inspired oxygen requirement under 0.35 and respiratory rate under 22 rpm. Patients were daily assessed in person by a nurse and a physician. RESULTS: A total of 236 patients admitted at the HaH unit received remdesivir, 172 of whom were treated at home. Only 2% presented any adverse event related to the infusion, all of them mild. HaH saved 1416 day-beds, with only 5% of the patients requiring transfer back to the hospital. CONCLUSION: Remdesivir infusion in HaH units seems to be a safe and efficient alternative to conventional hospitalization for treating patients with nonsevere COVID-19.

Implementation of a New Protocol for Direct Identification from Urine in the Routine Microbiological Diagnosis.

Background: The direct identification of uropathogens from urine samples, in combination with the rapid detection of resistance, would allow early adjustment of empirical antimicrobial treatment. Methods: Two hundred and ninety-eight urine samples processed between 1 June and 31 December 2020, selected with flow cytometry, with direct identification by MALDI-TOF mass spectrometry, and rapid detection of extended-spectrum beta-lactamase (ESBL) and carbapenemases-producing strains by lateral flow were analyzed. Results: The positive predictive value of the direct identification of the 86 samples that met the flow cytometry criterion (>5000 bacteria/µL) was 96.4%. Reliable direct identification was obtained in 14 of the 27 (51.8%) urinary source bacteraemias. There was 100% agreement between the lateral flow and antibiogram in the detection of ESBL and carbapenemases. Conclusion: the protocol for the direct identification and rapid detection of ESBL and carbapenemases-producing strains from urine samples is a reliable and useful tool.

New Procedure to Maintain Fecal Microbiota in a Dry Matrix Ready to Encapsulate.

Fecal microbiota transplantation (FMT) is one of the recommended treatments for recurrent Clostridioides difficile infection, but endoscopy and available oral formulations still have several limitations in their preparation, storage, and administration. The need for a viable oral formulation that facilitates the implementation of this highly effective therapy in different settings has led us to test the microcrystalline cellulose particles as an adsorbent of concentrated filtered fresh feces in comparison to lyophilized feces. This free-flowing material can provide protection to bacteria and results in a dried product able to maintain the viability of the microbiota for a long time. Adsorbate formulation showed a stabilizing effect in gut microbiota, maintaining bacteria viability and preserving its diversity, and is a competitive option for lyophilized capsules.