Publications by authors named "Barbara Methe"

92 Publications

Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis.

Cell Rep 2021 Oct;37(3):109829

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA. Electronic address:

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution.
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http://dx.doi.org/10.1016/j.celrep.2021.109829DOI Listing
October 2021

"Longitudinal Fecal Microbiome Study of Total Body Irradiated Mice Treated With Radiation Mitigators Identifies Bacterial Associations With Survival".

Front Cell Infect Microbiol 2021 21;11:715396. Epub 2021 Sep 21.

Center for Medicine and the Microbiome, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States.

Total body irradiation (TBI) has been demonstrated to alter the intestinal microbiome, but the effects of successful small molecule ionizing radiation mitigators on the intestinal microbiome are not well-known. Our survival experiments examined the effects of anti-cell death radiation mitigators on and in conjunction with the host's microbiota. Mice received 9.25 Gy TBI and then were administered radiation mitigators 24 hours later. Passed stool were collected pre-irradiation, then on days 1, 3, 5, 7, 10, 14, 21, and 30 post-irradiation for 16S rRNA gene (V4 region) sequencing. The Cox proportional hazards (CPH) model was fit with taxonomic composition (time varying covariates) and treatment as predictors. In the first experiment, mice were administered drugs for "granulocyte stimulation and anti-apoptosis" in four protocol combinations: JP4-039 (anti-apoptosis), granulocyte colony-stimulating factor (G-CSF, granulopoietic precursor cell stimulator), both mitigators, and control. Survival improved relative to control (30.0%) for G-CSF (80%, p-value = 0.025), G-CSF/JP4-039 (70%, p-value = 0.084), but not for JP4-039 (50.0%). In the second experiment, mice were administered mitigation drugs "inhibiting programmed cell death" pathways: JP4-039 (anti-apoptosis), necrostatin-1 (anti-necroptosis), and baicalein (anti-ferroptosis), in eight combinations. The survival of JP4-039/baicalein (60.0%, p-value = 0.010) and JP4-039/baicalein/necrostatin-1 (60.0%, p-value = 0.06) treatment combinations were significantly different from the control (26.7%). The JP4-039/necrostatin-1 (46.7%) and baicalein/necrostatin-1 (40.0%) and singlet treatment combinations (26.7%) were not significantly different from the control. Despite differences between the baseline microbiota compositions of the two experiments, consistent changes in composition after irradiation were found: decreased post-irradiation, relative to baseline. By day 7, microbiota perturbations had incompletely reversed, and no drug-specific differences were identifiable. The CPH model identified and members of , including , as protective and as deleterious. By day 30, the microbiota of surviving mice had not returned to baseline, but the differences between experiments suggest the resultant microbiota composition of the survivors are stochastic or batch specific in nature, rather than a requirement for survival. In conclusion, the study determined that key taxa identified in fecal samples, when applied towards the prediction of TBI survival, improves the survival model relative to treatment information alone.
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http://dx.doi.org/10.3389/fcimb.2021.715396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490782PMC
October 2021

SARS-CoV-2 Viremia is Associated with COVID-19 Severity and Predicts Clinical Outcomes.

Clin Infect Dis 2021 Aug 10. Epub 2021 Aug 10.

University of Pittsburgh School of Medicine, Department of Medicine, Division of Infectious Diseases, Pittsburgh, PA, USA.

Background: SARS-CoV-2 viral RNA (vRNA) is detected in the bloodstream of some patients with COVID-19 ("RNAemia") but it is not clear whether this RNAemia reflects viremia (i.e., virus particles) and how RNAemia/viremia is related to host immune responses and outcomes.

Methods: SARS-CoV-2 vRNA was quantified by ultra-sensitive RT-PCR in plasma samples (0.5-1.0 ml) from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-ICU), and 23 ICU patients, and vRNA levels compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in pelleted plasma.

Results: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6% and 11.1% of ICU, non-ICU, and outpatients respectively. Virions were detected in plasma pellets by electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (p<0.0001); and for inpatient, plasma vRNA levels were strongly associated with higher WHO score at admission (p=0.01), maximum WHO score (p=0.002) and discharge disposition (p=0.004). A plasma vRNA level >6,000 copies/ml was strongly associated with mortality (HR: 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (p<0.01) but not with plasma neutralizing antibody titers (p=0.8).

Conclusions: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia quantified by ultrasensitive RT-PCR correlate strongly with disease severity, patient outcome and specific inflammatory biomarkers but not neutralizing antibody titers.
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http://dx.doi.org/10.1093/cid/ciab686DOI Listing
August 2021

Plasma 1,3-β-d-glucan levels predict adverse clinical outcomes in critical illness.

JCI Insight 2021 07 22;6(14). Epub 2021 Jul 22.

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.

BACKGROUNDThe fungal cell wall constituent 1,3-β-d-glucan (BDG) is a pathogen-associated molecular pattern that can stimulate innate immunity. We hypothesized that BDG from colonizing fungi in critically ill patients may translocate into the systemic circulation and be associated with host inflammation and outcomes.METHODSWe enrolled 453 mechanically ventilated patients with acute respiratory failure (ARF) without invasive fungal infection and measured BDG, innate immunity, and epithelial permeability biomarkers in serially collected plasma samples.RESULTSCompared with healthy controls, patients with ARF had significantly higher BDG levels (median [IQR], 26 pg/mL [15-49 pg/mL], P < 0.001), whereas patients with ARF with high BDG levels (≥40 pg/mL, 31%) had higher odds for assignment to the prognostically adverse hyperinflammatory subphenotype (OR [CI], 2.88 [1.83-4.54], P < 0.001). Baseline BDG levels were predictive of fewer ventilator-free days and worse 30-day survival (adjusted P < 0.05). Integrative analyses of fungal colonization and epithelial barrier disruption suggested that BDG may translocate from either the lung or gut compartment. We validated the associations between plasma BDG and host inflammatory responses in 97 hospitalized patients with COVID-19.CONCLUSIONBDG measurements offered prognostic information in critically ill patients without fungal infections. Further research in the mechanisms of translocation and innate immunity recognition and stimulation may offer new therapeutic opportunities in critical illness.FUNDINGUniversity of Pittsburgh Clinical and Translational Science Institute, COVID-19 Pilot Award and NIH grants (K23 HL139987, U01 HL098962, P01 HL114453, R01 HL097376, K24 HL123342, U01 HL137159, R01 LM012087, K08HK144820, F32 HL142172, K23 GM122069).
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http://dx.doi.org/10.1172/jci.insight.141277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410081PMC
July 2021

Dynamics of hepatic steatosis resolution and changes in gut microbiome with weight loss in nonalcoholic fatty liver disease.

Obes Sci Pract 2021 Apr 4;7(2):217-225. Epub 2021 Jan 4.

Cancer Epidemiology and Prevention Program University of Pittsburgh Medical Center Hillman Cancer Center Pittsburgh Pennsylvania USA.

Background: Weight loss is recommended as the primary treatment for nonalcoholic fatty liver disease (NAFLD). However, the magnitude and velocity of hepatic steatosis resolution with weight loss is unclear, making it difficult to counsel patients seeking weight loss for treatment of NAFLD. The aim of this study was to determine the rate of hepatic steatosis improvement and stool microbiome changes associated with rapid diet-induced weight loss in NAFLD.

Methods: Fourteen NAFLD patients (mean ± standard deviation, body mass index [BMI] 36.4 ± 4 kg/m) enrolled in a 12-week meal replacement program underwent frequent measurement of Fibroscan-controlled attenuation parameter (CAP). Magnetic resonance imaging (MRI-Dixon method) for hepatic fat quantitation and stool microbiome analysis (16S rRNA gene sequencing) were completed in 11 subjects at baseline and Week 12.

Results: At Week 12, mean (95% confidence interval) weight loss was -13.4 (-15.2, -11.5)% and CAP score -26.6 (-35.6, -17.6)% (both  < 0.001). CAP scores changed at a rate of -4.9 dB/m/kg (-30.1 dB/m per unit BMI) in Weeks 1-4 and -0.6 dB/m/kg (-2.4 dB/m per unit BMI) in Weeks 8-12. MRI-determined hepatic fat fraction decreased by -74.1% ( < 0.001) at a rate of -0.51%/kg (-3.19% per unit BMI), with complete steatosis resolution in 90% patients. BMI change was associated with decreased stool microbial diversity (coefficient = 0.17; Shannon Index), increased abundance of (Bacteroidetes; coefficient = 0.96) and decreased abundance of (Firmicutes; coefficient = -0.42) (both  < 0.05).

Conclusions: Diet-induced intensive weight loss is associated with rapid improvement and complete resolution of hepatic steatosis and decreased stool microbial diversity. These findings highlight the dynamic nature of hepatic fat and may help clinicians to develop evidence-based treatment goals for patients with NAFLD and obesity who undertake weight loss interventions. Further research is warranted to understand the effects of intensive weight loss and gut microbiome changes on long-term NAFLD resolution.
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http://dx.doi.org/10.1002/osp4.476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019274PMC
April 2021

Bifurcated monocyte states are predictive of mortality in severe COVID-19.

bioRxiv 2021 Feb 10. Epub 2021 Feb 10.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations , ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality . Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by (MIP-1β) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1β and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.
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http://dx.doi.org/10.1101/2021.02.10.430499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885916PMC
February 2021

COVID-19 versus Non-COVID-19 Acute Respiratory Distress Syndrome: Comparison of Demographics, Physiologic Parameters, Inflammatory Biomarkers, and Clinical Outcomes.

Ann Am Thorac Soc 2021 07;18(7):1202-1210

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Acute Lung Injury Center of Excellence, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.

There is an urgent need for improved understanding of the mechanisms and clinical characteristics of acute respiratory distress syndrome (ARDS) due to coronavirus disease (COVID-19). To compare key demographic and physiologic parameters, biomarkers, and clinical outcomes of COVID-19 ARDS and ARDS secondary to direct lung injury from other etiologies of pneumonia. We enrolled 27 patients with COVID-19 ARDS in a prospective, observational cohort study and compared them with a historical, pre-COVID-19 cohort of patients with viral ARDS ( = 14), bacterial ARDS ( = 21), and ARDS due to culture-negative pneumonia ( = 30). We recorded clinical demographics; measured respiratory mechanical parameters; collected serial peripheral blood specimens for measurement of plasma interleukin (IL)-6, IL-8, and IL-10; and followed patients prospectively for patient-centered outcomes. We conducted between-group comparisons with nonparametric tests and analyzed time-to-event outcomes with Kaplan-Meier and Cox proportional hazards models. Patients with COVID-19 ARDS had higher body mass index and were more likely to be Black, or residents of skilled nursing facilities, compared with those with non-COVID-19 ARDS ( < 0.05). Patients with COVID-19 had lower delivered minute ventilation compared with bacterial and culture-negative ARDS ( < 0.01) but not compared with viral ARDS. We found no differences in static compliance, hypoxemic indices, or carbon dioxide clearance between groups. Patients with COVID-19 had lower IL-6 levels compared with bacterial and culture-negative ARDS at early time points after intubation but no differences in IL-6 levels compared with viral ARDS. Patients with COVID-19 had longer duration of mechanical ventilation but similar 60-day mortality in both unadjusted and adjusted analyses. COVID-19 ARDS bears several similarities to viral ARDS but demonstrates lower minute ventilation and lower systemic levels of IL-6 compared with bacterial and culture-negative ARDS. COVID-19 ARDS was associated with longer dependence on mechanical ventilation compared with non-COVID-19 ARDS. Such detectable differences of COVID-19 do not merit deviation from evidence-based management of ARDS but suggest priorities for clinical research to better characterize and treat this new clinical entity.
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http://dx.doi.org/10.1513/AnnalsATS.202008-1026OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328355PMC
July 2021

Intractable Coronavirus Disease 2019 (COVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication in a Chimeric Antigen Receptor-Modified T-Cell Therapy Recipient: A Case Study.

Clin Infect Dis 2021 08;73(3):e815-e821

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

A chimeric antigen receptor-modified T-cell therapy recipient developed severe coronavirus disease 2019, intractable RNAemia, and viral replication lasting >2 months. Premortem endotracheal aspirate contained >2 × 1010 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intrahost virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.
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http://dx.doi.org/10.1093/cid/ciab072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929077PMC
August 2021

Uremia Coupled with Mucosal Damage Predisposes Mice with Kidney Disease to Systemic Infection by Commensal .

Immunohorizons 2021 01 15;5(1):16-24. Epub 2021 Jan 15.

Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261; and

Infections are the second major cause of mortality in patients with kidney disease and accompanying uremia. Both vascular access and non-access-related infections contribute equally to the infection-related deaths in patients with kidney disease. Dialysis is the most common cause of systemic infection by in these patients. also reside in the gastrointestinal tract as a commensal fungus. However, the contribution of gut-derived in non-access-related infections in kidney disease is unknown. Using a mouse model of kidney disease, we demonstrate that uremic animals showed increased gut barrier permeability, impaired mucosal defense, and dysbiosis. The disturbance in gut homeostasis is sufficient to drive the translocation of microbiota and intestinal pathogen to extraintestinal sites but not Interestingly, a majority of uremic animals showed fungal translocation only when the gut barrier integrity is disrupted. Our data demonstrate that uremia coupled with gut mucosal damage may aid in the translocation of and cause systemic infection in kidney disease. Because most of the individuals with kidney disease suffer from some form of gut mucosal damage, these results have important implications in the risk stratification and control of non-access-related opportunistic fungal infections in these patients.
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http://dx.doi.org/10.4049/immunohorizons.2000114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913778PMC
January 2021

Topographic heterogeneity of lung microbiota in end-stage idiopathic pulmonary fibrosis: the Microbiome in Lung Explants-2 (MiLEs-2) study.

Thorax 2021 03 2;76(3):239-247. Epub 2020 Dec 2.

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

Background: Lung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown.

Methods: We performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene.

Findings: Explants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death.

Interpretation: IPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions.
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http://dx.doi.org/10.1136/thoraxjnl-2020-214770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203183PMC
March 2021

Distinctive gene and protein characteristics of extremely piezophilic Colwellia.

BMC Genomics 2020 Oct 6;21(1):692. Epub 2020 Oct 6.

Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, 92093-0202, USA.

Background: The deep ocean is characterized by low temperatures, high hydrostatic pressures, and low concentrations of organic matter. While these conditions likely select for distinct genomic characteristics within prokaryotes, the attributes facilitating adaptation to the deep ocean are relatively unexplored. In this study, we compared the genomes of seven strains within the genus Colwellia, including some of the most piezophilic microbes known, to identify genomic features that enable life in the deep sea.

Results: Significant differences were found to exist between piezophilic and non-piezophilic strains of Colwellia. Piezophilic Colwellia have a more basic and hydrophobic proteome. The piezophilic abyssal and hadal isolates have more genes involved in replication/recombination/repair, cell wall/membrane biogenesis, and cell motility. The characteristics of respiration, pilus generation, and membrane fluidity adjustment vary between the strains, with operons for a nuo dehydrogenase and a tad pilus only present in the piezophiles. In contrast, the piezosensitive members are unique in having the capacity for dissimilatory nitrite and TMAO reduction. A number of genes exist only within deep-sea adapted species, such as those encoding d-alanine-d-alanine ligase for peptidoglycan formation, alanine dehydrogenase for NADH/NAD homeostasis, and a SAM methyltransferase for tRNA modification. Many of these piezophile-specific genes are in variable regions of the genome near genomic islands, transposases, and toxin-antitoxin systems.

Conclusions: We identified a number of adaptations that may facilitate deep-sea radiation in members of the genus Colwellia, as well as in other piezophilic bacteria. An enrichment in more basic and hydrophobic amino acids could help piezophiles stabilize and limit water intrusion into proteins as a result of high pressure. Variations in genes associated with the membrane, including those involved in unsaturated fatty acid production and respiration, indicate that membrane-based adaptations are critical for coping with high pressure. The presence of many piezophile-specific genes near genomic islands highlights that adaptation to the deep ocean may be facilitated by horizontal gene transfer through transposases or other mobile elements. Some of these genes are amenable to further study in genetically tractable piezophilic and piezotolerant deep-sea microorganisms.
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http://dx.doi.org/10.1186/s12864-020-07102-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542103PMC
October 2020

Strategies to Promote ResiliencY (SPRY): a randomised embedded multifactorial adaptative platform (REMAP) clinical trial protocol to study interventions to improve recovery after surgery in high-risk patients.

BMJ Open 2020 09 29;10(9):e037690. Epub 2020 Sep 29.

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Introduction: As the population ages, there is interest in strategies to promote resiliency, especially for frail patients at risk of its complications. The physiological stress of surgery in high-risk individuals has been proposed both as an important cause of accelerated age-related decline in health and as a model testing the effectiveness of strategies to improve resiliency to age-related health decline. We describe a randomised, embedded, multifactorial, adaptative platform (REMAP) trial to investigate multiple perioperative interventions, the first of which is metformin and selected for its anti-inflammatory and anti-ageing properties beyond its traditional blood glucose control features.

Methods And Analysis: Within a multihospital, single healthcare system, the Core Protocol for Strategies to Promote ResiliencY (SPRY) will be embedded within both the electronic health record (EHR) and the healthcare culture generating a continuously self-learning healthcare system. Embedding reduces the administrative burden of a traditional trial while accessing and rapidly analysing routine patient care EHR data. SPRY-Metformin is a placebo-controlled trial and is the first SPRY domain evaluating the effectiveness of three metformin dosages across three preoperative durations within a heterogeneous set of major surgical procedures. The primary outcome is 90-day hospital-free days. Bayesian posterior probabilities guide interim decision-making with predefined rules to determine stopping for futility or superior dosing selection. Using response adaptative randomisation, a maximum of 2500 patients allows 77%-92% power, detecting >15% primary outcome improvement. Secondary outcomes include mortality, readmission and postoperative complications. A subset of patients will be selected for substudies evaluating the microbiome, cognition, postoperative delirium and strength.

Ethics And Dissemination: The Core Protocol of SPRY REMAP and associated SPRY-Metformin Domain-Specific Appendix have been ethically approved by the Institutional Review Board and are publicly registered. Results will be publicly available to healthcare providers, patients and trial participants following achieving predetermined platform conclusions.

Trial Registration Number: NCT03861767.
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http://dx.doi.org/10.1136/bmjopen-2020-037690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526307PMC
September 2020

Functional gene categories differentiate maize leaf drought-related microbial epiphytic communities.

PLoS One 2020 18;15(9):e0237493. Epub 2020 Sep 18.

Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, NC, United States of America.

The phyllosphere epiphytic microbiome is composed of microorganisms that colonize the external aerial portions of plants. Relationships of plant responses to specific microorganisms-both pathogenic and beneficial-have been examined, but the phyllosphere microbiome functional and metabolic profile responses are not well described. Changing crop growth conditions, such as increased drought, can have profound impacts on crop productivity. Also, epiphytic microbial communities provide a new target for crop yield optimization. We compared Zea mays leaf microbiomes collected under drought and well-watered conditions by examining functional gene annotation patterns across three physically disparate locations each with and without drought treatment, through the application of short read metagenomic sequencing. Drought samples exhibited different functional sequence compositions at each of the three field sites. Maize phyllosphere functional profiles revealed a wide variety of metabolic and regulatory processes that differed in drought and normal water conditions and provide key baseline information for future selective breeding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237493PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500591PMC
October 2020

Lower respiratory tract myeloid cells harbor SARS-CoV-2 and display an inflammatory phenotype.

medRxiv 2020 Aug 14. Epub 2020 Aug 14.

SARS-CoV-2 pneumonia may induce an aberrant immune response with brisk recruitment of myeloid cells into the lower respiratory tract, which may contribute to morbidity and mortality. We describe endotracheal aspirate samples from seven patients with SARS-CoV-2 pneumonia requiring mechanical ventilation. We note SARS-CoV-2 virions within lower respiratory tract myeloid cells shown by electron tomography, immunofluorescence confocal imaging, and immuno-electron microscopy. Endotracheal aspirates are primarily composed of mononuclear and polymorphonuclear leukocytes. These myeloid cells that harbor virus are frequently positive for CD14 and/or CD16 and most display an inflammatory phenotype marked by expression of IL-6 and tissue factor mRNA transcript and protein expression.
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http://dx.doi.org/10.1101/2020.08.11.20171967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430612PMC
August 2020

Respiratory Tract Dysbiosis Is Associated with Worse Outcomes in Mechanically Ventilated Patients.

Am J Respir Crit Care Med 2020 12;202(12):1666-1677

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine and University of Pittsburgh Medical Center.

Host inflammatory responses have been strongly associated with adverse outcomes in critically ill patients, but the biologic underpinnings of such heterogeneous responses have not been defined. We examined whether respiratory tract microbiome profiles are associated with host inflammation and clinical outcomes of acute respiratory failure. We collected oral swabs, endotracheal aspirates (ETAs), and plasma samples from mechanically ventilated patients. We performed 16S ribosomal RNA gene sequencing to characterize upper and lower respiratory tract microbiota and classified patients into host-response subphenotypes on the basis of clinical variables and plasma biomarkers of innate immunity and inflammation. We derived diversity metrics and composition clusters with Dirichlet multinomial models and examined our data for associations with subphenotypes and clinical outcomes. Oral and ETA microbial communities from 301 mechanically ventilated subjects had substantial heterogeneity in α and β diversity. Dirichlet multinomial models revealed a cluster with low α diversity and enrichment for pathogens (e.g., high or relative abundance) in 35% of ETA samples, associated with a hyperinflammatory subphenotype, worse 30-day survival, and longer time to liberation from mechanical ventilation (adjusted  < 0.05), compared with patients with higher α diversity and relative abundance of typical oral microbiota. Patients with evidence of dysbiosis (low α diversity and low relative abundance of "protective" oral-origin commensal bacteria) in both oral and ETA samples (17%, ) had significantly worse 30-day survival and longer time to liberation from mechanical ventilation than patients without dysbiosis (55%; adjusted  < 0.05). Respiratory tract dysbiosis may represent an important, modifiable contributor to patient-level heterogeneity in systemic inflammatory responses and clinical outcomes.
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http://dx.doi.org/10.1164/rccm.201912-2441OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737572PMC
December 2020

Author Correction: Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection.

Nat Commun 2020 06 16;11(1):3132. Epub 2020 Jun 16.

Departmento de Enfermedades Infecciosas e Inmunología Pediátrica, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17020-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298031PMC
June 2020

Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection.

Nat Commun 2020 05 21;11(1):2537. Epub 2020 May 21.

Departmento de Enfermedades Infecciosas e Inmunología Pediátrica, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.

Infection with influenza can be aggravated by bacterial co-infections, which often results in disease exacerbation. The effects of influenza infection on the upper respiratory tract (URT) microbiome are largely unknown. Here, we report a longitudinal study to assess the temporal dynamics of the URT microbiomes of uninfected and influenza virus-infected humans and ferrets. Uninfected human patients and ferret URT microbiomes have stable healthy ecostate communities both within and between individuals. In contrast, infected patients and ferrets exhibit large changes in bacterial community composition over time and between individuals. The unhealthy ecostates of infected individuals progress towards the healthy ecostate, coinciding with viral clearance and recovery. Pseudomonadales associate statistically with the disturbed microbiomes of infected individuals. The dynamic and resilient microbiome during influenza virus infection in multiple hosts provides a compelling rationale for the maintenance of the microbiome homeostasis as a potential therapeutic target to prevent IAV associated bacterial co-infections.
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http://dx.doi.org/10.1038/s41467-020-16429-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242466PMC
May 2020

The microbiome of pediatric patients with chronic rhinosinusitis.

Int Forum Allergy Rhinol 2021 01 22;11(1):31-39. Epub 2020 Jun 22.

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA.

Background: This study aimed to compare the microbiota of pediatric patients with chronic rhinosinusitis (CRS) who are undergoing adenoidectomy to treat their disease with that of healthy control patients.

Methods: Patients undergoing adenoidectomy-only for obstructive sleep apnea (n = 50) and CRS (n = 37) were recruited. Preoperative 22-item Sino-Nasal Outcome Test (SNOT-22) or Sinus and Nasal Quality of Life Survey (SN-5) were collected. Each patient had samples collected from their nasopharynx (adenoid bed) and nasal cavity (sinus) at the onset of surgery. 16S ribosomal ribonucleic acid (rRNA) gene sequencing was subsequently performed to obtain per sample taxonomic abundances. Statistical analyses included permutational multivariate analysis of variance (PERMANOVA), alpha (within sample) diversity measures, and changes in taxonomic abundance.

Results: Moraxella was the most abundant organism. Nasopharyngeal swabs demonstrated higher alpha diversity compared to the nasal cavity. The diversity was not different based on CRS vs obstructive history. There was an increase in diversity with increasing age, and eczema contributed to a greater difference in diversity between the nasopharynx and nasal cavity. Diversity was not affected by adenoid size; however, use of nasal steroids, inhaled steroids, and antihistamines influenced diversity in both the nasopharynx and nasal cavity. Nasopharyngeal samples were higher in relative abundance for Fusobacterium, Prevotella, Porphyromonas, and Campylobacter compared to the nasal cavity.

Conclusion: The nasopharynx and nasal cavity differed in both microbiota composition and diversity. In contrast, no significant difference in composition or diversity were found in CRS vs control patients. Ecological changes in the nasopharyngeal and sinus site may contribute to the etiology for adenoid hypertrophy in both healthy controls and CRS patients.
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http://dx.doi.org/10.1002/alr.22597DOI Listing
January 2021

Rifaximin on intestinally-related pathologic changes in sickle cell disease.

Am J Hematol 2020 04 3;95(4):E83-E86. Epub 2020 Feb 3.

Division of Hematology and Hemostasis, Department of Medicine, New York Medical College, Valhalla, New York.

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http://dx.doi.org/10.1002/ajh.25722DOI Listing
April 2020

A prospective cohort analysis of gut microbial co-metabolism in Alaska Native and rural African people at high and low risk of colorectal cancer.

Am J Clin Nutr 2020 02;111(2):406-419

Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

Background: Alaska Native (AN) people have the world's highest recorded incidence of sporadic colorectal cancer (CRC) (∼91:100,000), whereas rural African (RA) people have the lowest risk (<5:100,000). Previous data supported the hypothesis that diet affected CRC risk through its effects on the colonic microbiota that produce tumor-suppressive or -promoting metabolites.

Objectives: We investigated whether differences in these metabolites may contribute to the high risk of CRC in AN people.

Methods: A cross-sectional observational study assessed dietary intake from 32 AN and 21 RA healthy middle-aged volunteers before screening colonoscopy. Analysis of fecal microbiota composition by 16S ribosomal RNA gene sequencing and fecal/urinary metabolites by 1H-NMR spectroscopy was complemented with targeted quantification of fecal SCFAs, bile acids, and functional microbial genes.

Results: Adenomatous polyps were detected in 16 of 32 AN participants, but not found in RA participants. The AN diet contained higher proportions of fat and animal protein and less fiber. AN fecal microbiota showed a compositional predominance of Blautia and Lachnoclostridium, higher microbial capacity for bile acid conversion, and low abundance of some species involved in saccharolytic fermentation (e.g., Prevotellaceae, Ruminococcaceae), but no significant lack of butyrogenic bacteria. Significantly lower concentrations of tumor-suppressive butyrate (22.5 ± 3.1 compared with 47.2 ± 7.3 SEM µmol/g) coincided with significantly higher concentrations of tumor-promoting deoxycholic acid (26.7 ± 4.2 compared with 11 ± 1.9 µmol/g) in AN fecal samples. AN participants had lower quantities of fecal/urinary metabolites than RA participants and metabolite profiles correlated with the abundance of distinct microbial genera in feces. The main microbial and metabolic CRC-associated markers were not significantly altered in AN participants with adenomatous polyps.

Conclusions: The low-fiber, high-fat diet of AN people and exposure to carcinogens derived from diet or environment are associated with a tumor-promoting colonic milieu as reflected by the high rates of adenomatous polyps in AN participants.
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http://dx.doi.org/10.1093/ajcn/nqz301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997097PMC
February 2020

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study.

Respir Res 2019 Nov 27;20(1):265. Epub 2019 Nov 27.

Center for Medicine and the Microbiome, University of Pittsburgh, Pittsburgh, PA, USA.

Background: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens.

Methods: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing.

Results: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa.

Conclusions: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.
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http://dx.doi.org/10.1186/s12931-019-1218-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882222PMC
November 2019

Intestinal IL-17R Signaling Constrains IL-18-Driven Liver Inflammation by the Regulation of Microbiome-Derived Products.

Cell Rep 2019 Nov;29(8):2270-2283.e7

Richard King Mellon Foundation Institute for Pediatric Research, Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA; Departments of Medicine and Pediatrics, Center for Translational Research in Infection and Inflammation, Tulane University School of Medicine, New Orleans, LA 70112, USA. Electronic address:

Interleukin (IL)-17 signaling to the intestinal epithelium regulates the intestinal microbiome. Given the reported links between intestinal dysbiosis, bacterial translocation, and liver disease, we hypothesize that intestinal IL-17R signaling plays a critical role in mitigating hepatic inflammation. To test this, we study intestinal epithelium-specific IL-17RA-deficient mice in an immune-driven hepatitis model. At the naive state, these mice exhibit microbiome dysbiosis and increased translocation of bacterial products (CpG DNA), which drives liver IL-18 production. Upon disease induction, absence of enteric IL-17RA signaling exacerbates hepatitis and hepatocyte cell death. IL-18 is necessary for disease exacerbation and is associated with increased activated hepatic lymphocytes based on Ifng and Fasl expression. Thus, intestinal IL-17R regulates translocation of TLR9 ligands and constrains susceptibility to hepatitis. These data connect enteric Th17 signaling and the microbiome in hepatitis, with broader implications on the effects of impaired intestinal immunity and subsequent release of microbial products observed in other extra-intestinal pathologies.
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http://dx.doi.org/10.1016/j.celrep.2019.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886715PMC
November 2019

Alterations in Oral Microbiota in HIV Are Related to Decreased Pulmonary Function.

Am J Respir Crit Care Med 2020 02;201(4):445-457

Division of Pulmonary, Allergy and Critical Care Medicine and.

Mechanisms of HIV-associated chronic obstructive pulmonary disease (COPD) are poorly understood. The oral microbiome shapes the lung microbiome, and gut dysbiosis can affect lung diseases; however, relationships of the oral and gut microbiome to COPD in HIV have not been explored. To examine alterations in the oral and gut microbiome associated with pulmonary disease in persons with HIV (PWH). Seventy-five PWH and 93 HIV-uninfected men from the MACS (Multicenter AIDS Cohort Study) performed pulmonary function testing. Sequencing of bacterial 16S ribosomal RNA in saliva and stool was performed. We used nonmetric multidimensional scaling, permutational multivariate ANOVA, and linear discriminant analysis to analyze communities by HIV and lung function. Oral microbiome composition differed by HIV and smoking status. Alterations of oral microbial communities were observed in PWH with abnormal lung function with increases in relative abundance of , , and . There were no significant associations between the oral microbiome and lung function in HIV-uninfected individuals. No associations with HIV status or lung function were seen with the gut microbiome. Alterations of oral microbiota in PWH were related to impaired pulmonary function and to systemic inflammation. These results suggest that the oral microbiome may serve as a biomarker of lung function in HIV and that its disruption may contribute to COPD pathogenesis.
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http://dx.doi.org/10.1164/rccm.201905-1016OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049920PMC
February 2020

Rectal Swabs from Critically Ill Patients Provide Discordant Representations of the Gut Microbiome Compared to Stool Samples.

mSphere 2019 07 24;4(4). Epub 2019 Jul 24.

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

The role of the gut microbiome in critical illness is being actively investigated, but the optimal sampling methods for sequencing studies of gut microbiota remain unknown. Stool samples are generally considered the reference standard but are not practical to obtain in the intensive care unit (ICU), and thus, rectal swabs are often used. However, the reliability of rectal swabs for gut microbiome profiling has not been established in the ICU setting. In this study, we compared 16S rRNA gene sequencing results between rectal swab and stool samples collected at three time points from mechanically ventilated critically ill adults. Rectal swabs comprised 89% of the samples collected at the baseline time point, but stool samples became more extensively available at later time points. Significant differences in alpha-diversity and beta-diversity between rectal swabs and stool samples were observed, but these differences were primarily due to baseline samples. Higher relative abundances of members of the phylum (typically skin microbes) were present in rectal swabs than in stool samples ( = 0.05), a difference that was attenuated over time. The progressively increasing similarity of rectal swabs and stool samples likely resulted from increasing levels of stool coating of the rectal vault and direct soiling of the rectal swabs taken at later time points. Therefore, inferences about the role of the gut microbiome in critical illness should be drawn cautiously and should take into account the type and timing of samples analyzed. Rectal swabs have been proposed as potential alternatives to stool samples for gut microbiome profiling in outpatients or healthy adults, but their reliability in assessment of critically ill patients has not been defined. Because stool sampling is not practical and often not feasible in the intensive care unit, we performed a detailed comparison of gut microbial sequencing profiles between rectal swabs and stool samples in a longitudinal cohort of critically ill patients. We identified systematic differences in gut microbial profiles between rectal swabs and stool samples and demonstrated that the timing of the rectal swab sampling had a significant impact on sequencing results. Our methodological findings should provide valuable information for the design and interpretation of future investigations of the role of the gut microbiome in critical illness.
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http://dx.doi.org/10.1128/mSphere.00358-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656869PMC
July 2019

Intestinal injury and gut permeability in sickle cell disease.

J Transl Med 2019 05 30;17(1):183. Epub 2019 May 30.

Division of Hematology and Hemostasis, New York Medical College, 19 Bradhurst Avenue, Suite 2575S, Hawthorne, Valhalla, NY, 10595, USA.

Background: Due to recurrent hypoxia-reperfusion injury induced by vaso-occlusive crises (VOC), patients with sickle cell disease (SCD) may have intestinal injury and increased permeability. These may explain the qualitative and quantitative neutrophil abnormalities observed in these patients.

Methods: Serum intestinal fatty-acid binding protein (iFABP), lipopolysaccharides (LPS), and CD62L were measured by ELISA. Multicolor flow cytometry was used to measure circulating aged neutrophils.

Results: Compared to controls, SCD individuals had higher iFABP (median: 1.38 ng/ml vs 0.81 ng/ml; p = 0.04) and LPS (median: 2.15 μg/ml vs 0.69 μg/ml; p = 0.03), indicating intestinal injury and increased intestinal bacterial translocation into the systemic circulation. They also had higher soluble CD62L (median: 1.38 μg/ml vs 1.11 μg/ml; p = 0.04). Among SCD individuals, soluble CD62L correlated positively with circulating aged neutrophils (R = 0.7, p = 0.03) and LPS (R = 0.66, p = 0.027). Surprisingly, serum iFABP in SCD correlated negatively with both LPS (R = - 0.7, p = 0.02) and soluble CD62L (R = - 0.56, p = 0.08).

Conclusions: Since LPS translocation across the intestinal barrier may be due to increases in the intestinal bacterial density, gut permeability, or both, the negative correlations between iFABP and LPS, and CD62L raise the possibility that any damage-associated molecular patterns induced by intestinal injury may modulate the degree of bacterial translocation. Our results provide the first evidence of the presence of intestinal injury and increased gut permeability in SCD.
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http://dx.doi.org/10.1186/s12967-019-1938-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543649PMC
May 2019

Effects of rifaximin on circulating aged neutrophils in sickle cell disease.

Am J Hematol 2019 06 3;94(6):E175-E176. Epub 2019 Apr 3.

Division of Hematology and Hemostasis, New York Medical College, Valhalla, New York.

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http://dx.doi.org/10.1002/ajh.25467DOI Listing
June 2019

Elevated urinary 3-indoxyl sulfate in sickle cell disease.

Am J Hematol 2019 06 20;94(6):E162-E164. Epub 2019 Mar 20.

Division of Hematology and Hemostasis, New York Medical College, Valhalla, New York.

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http://dx.doi.org/10.1002/ajh.25456DOI Listing
June 2019

Invasive non-typhoidal Salmonella in sickle cell disease in Africa: is increased gut permeability the missing link?

J Transl Med 2018 08 30;16(1):239. Epub 2018 Aug 30.

Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA.

Non-typhoidal Salmonella usually induces self-limiting gastroenteritis. However, in many parts of Africa, especially in individuals who are malnourished, infected with malaria, or have sickle cell disease, the organism causes serious and potentially fatal systemic infections. Since the portal of entry of non-typhoidal Salmonella into the systemic circulation is by way of the intestine, we argue that an increased gut permeability plays a vital role in the initiation of invasive non-typhoidal Salmonella in these patients. Here, we will appraise the evidence supporting a breach in the intestinal barrier and propose the mechanisms for the increased risks for invasive non-typhoidal Salmonella infections in these individuals.
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http://dx.doi.org/10.1186/s12967-018-1622-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116559PMC
August 2018
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