Whole Genome Shotgun Metagenomics Research Articles

Insights into the Adolescent Cystic Fibrosis Airway Microbiome Using Shotgun Metagenomics.

Cystic fibrosis (CF) is an inherited genetic disorder which manifests primarily in airway disease. Recent advances in molecular technologies have unearthed the diverse polymicrobial nature of the CF airway. Numerous studies have characterised the genus-level composition of this airway community using targeted 16S rDNA sequencing. Here, we employed whole-genome shotgun metagenomics to provide a more comprehensive understanding of the early CF airway microbiome. We collected 48 sputum samples from 11 adolescents and children with CF over a 12-month period and performed shotgun metagenomics on the Illumina NextSeq platform. We carried out functional and taxonomic analysis of the lung microbiome at the species and strain levels. Correlations between microbial diversity measures and independent demographic and clinical variables were performed. Shotgun metagenomics detected a greater diversity of bacteria than culture-based methods. A large proportion of the top 25 most-dominant species were anaerobes. Samples dominated by Staphylococcus aureus and Prevotella melaninogenica had significantly higher microbiome diversity, while no CF pathogen was associated with reduced microbial diversity. There was a diverse resistome present in all samples in this study, with 57.8% agreement between shotgun metagenomics and culture-based methods for detection of resistance. Pathogenic sequence types (STs) of S. aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Stenotrophomonas maltophilia were observed to persist in young CF patients, while STs of S. aureus were both persistent and shared between patients. This study provides new insight into the temporal changes in strain level composition of the microbiome and the landscape of the resistome in young people with CF. Shotgun metagenomics could provide a very useful one-stop assay for detecting pathogens, emergence of resistance and conversion to persistent colonisation in early CF disease.

Integrated multi-omics reveal gut microbiota-mediated bile acid metabolism alteration regulating immunotherapy responses to anti-α4β7-integrin in Crohn’s disease

ABSTRACT Gut microbiota and related metabolites are both crucial factors that significantly influence how individuals with Crohn’s disease respond to immunotherapy. However, little is known about the interplay among gut microbiota, metabolites, Crohn’s disease, and the response to anti-α4β7-integrin in current studies. Our research utilized 2,4,6-trinitrobenzene sulfonic acid to induce colitis based on the humanized immune system mouse model and employed a combination of whole-genome shotgun metagenomics and non-targeted metabolomics to investigate immunotherapy responses. Additionally, clinical cases with Crohn’s disease initiating anti-α4β7-integrin therapy were evaluated comprehensively. Particularly, 16S-rDNA gene high-throughput sequencing and targeted bile acid metabolomics were conducted at weeks 0, 14, and 54. We found that anti-α4β7-integrin therapy has shown significant potential for mitigating disease phenotypes in remission-achieving colitis mice. Microbial profiles demonstrated that not only microbial composition but also microbially encoded metabolic pathways could predict immunotherapy responses. Metabonomic signatures revealed that bile acid metabolism alteration, especially elevated secondary bile acids, was a determinant of immunotherapy responses. Especially, the remission mice significantly enriched the proportion of the beneficial Lactobacillus and Clostridium genera, which were correlated with increased gastrointestinal levels of BAs involving lithocholic acid and deoxycholic acid. Moreover, most of the omics features observed in colitis mice were replicated in clinical cases. Notably, anti-α4β7 integrin provided sustained therapeutic benefits in clinical remitters during follow-up, and long-lasting remission was linked to persistent changes in the microbial-related bile acids. In conclusion, gut microbiota-mediated bile acid metabolism alteration could play a crucial role in regulating immunotherapy responses to anti-α4β7-integrin in Crohn’s disease. Therefore, the identification of prognostic microbial signals facilitates the advancement of targeted probiotics that activate anti-inflammatory bile acid metabolic pathways, thereby improving immunotherapy responses. The integrated multi-omics established in our research provide valuable insights into potential mechanisms that impact treatment responses in complex diseases.

Airway 'Resistotypes' and Clinical Outcomes in Bronchiectasis.

Application of whole-genome shotgun metagenomics to the airway microbiome in bronchiectasis highlights a diverse pool of antimicrobial resistance genes: the 'resistome', the clinical significance of which remains unclear. Individuals with bronchiectasis were prospectively recruited into cross-sectional and longitudinal cohorts (n=280) including the international multicentre cross-sectional Cohort of Asian and Matched European Bronchiectasis 2 study (CAMEB 2; n=251) and two independent cohorts, one describing patients experiencing acute exacerbation and a further cohort of patients undergoing P. aeruginosa eradication treatment. Sputum was subjected to metagenomic sequencing and the bronchiectasis resistome evaluated in association with clinical outcomes and underlying host microbiomes. The bronchiectasis resistome features a unique resistance gene profile and elevated counts of aminoglycoside, bicyclomycin, phenicol, triclosan and multi-drug resistance genes. Longitudinally, it exhibits within-patient stability over time and during exacerbations despite between-patient heterogeneity. Proportional differences in baseline resistome profiles including increased macrolide and multi-drug resistance genes associate with shorter intervals to next exacerbation, while distinct resistome archetypes associate with frequent exacerbations, poorer lung function, geographic origin, and the host microbiome. Unsupervised analysis of resistome profiles identified two clinically relevant 'resistotypes' RT1 and RT2, the latter characterized by poor clinical outcomes, increased multi-drug resistance and P. aeruginosa. Successful targeted eradication in P. aeruginosa-colonized individuals mediated reversion from RT2 to RT1, a more clinically favourable resistome profile demonstrating reduced resistance gene diversity. The bronchiectasis resistome associates with clinical outcomes, geographic origin, and the underlying host microbiome. Bronchiectasis 'resistotypes' link to clinical disease and are modifiable through targeted antimicrobial therapy.

Association Between Bowel Movement Pattern and Cognitive Function: Prospective Cohort Study and a Metagenomic Analysis of the Gut Microbiome.

Little is known regarding the association between intestinal motility patterns and cognitive function in individuals who are baseline cognitively healthy. The gut microbiome may contribute to the association. We examined the association between bowel movement (BM) pattern and cognitive function and explored the role of the gut microbiome in explaining this association. In this prospective study, we leveraged 3 cohort studies, Nurses' Health Study (NHS), NHSII, and Health Professionals Follow-Up Study (HPFS). Participants reported BM frequency and subjective cognitive function. In a subset of NHSII participants, we assessed cognitive function using an objective neuropsychological battery. We profiled the gut microbiome in a subset of participants using whole-genome shotgun metagenomics. General linear models, Poisson regression, and logistic regression were used to quantify the association of BM frequency with different cognitive measurements. We followed 112,753 men and women (women: 87.6%) with a mean age of 67.2 years at baseline (NHS: 76 years, NHSII: 59 years, HPFS: 75 years) for a median follow-up of 4 years (NHSII and HPFS: 4 years, NHS: 2 years). Compared with those with BM once daily, participants with BM frequency every 3+ days had significantly worse objective cognitive function, equivalent to 3.0 (95% confidence interval [CI],1.2-4.7) years of chronological cognitive aging. We observed similar J-shape dose-response relationships of BM frequency with the odds of subjective cognitive decline and the likelihood of having more subsequent subjective cognitive complaints (both p nonlinearity < 0.001). BM frequencies of every 3+ days and ≥twice/day, compared with once daily, were associated with the odds ratios of subjective cognitive decline of 1.73 (95% CI 1.60-1.86) and 1.37 (95% CI 1.33-1.44), respectively. BM frequency and subjective cognitive decline were significantly associated with the overall gut microbiome configuration (both p < 0.005) and specific microbial species in the 515 participants with microbiome data. Butyrate-producing microbial species were depleted in those with less frequent BM and worse cognition, whereas a higher abundance of proinflammatory species was associated with BM frequency of ≥twice/day and worse cognition. Lower BM frequency was associated with worse cognitive function. The gut microbial dysbiosis may be a mechanistic link underlying the association.

Metagenomic Insight into the Microbiome and Virome Associated with Aedes aegypti Mosquitoes in Manado (North Sulawesi, Indonesia).

The aim of this study was to investigate the microbial diversity encompassing bacteria, fungi, and viruses within the composite microbial community associated with Aedes aegypti mosquitoes in Manado, Indonesia, using a whole-genome shotgun metagenomics approach. Female mosquitoes were collected and grouped into pools of 50 individuals, from which genomic DNA (gDNA) and RNA were extracted separately. Whole-genome shotgun metagenomics were performed on gDNA samples. The bioinformatics analysis encompassed quality assessment, taxonomic classification, and visualization. The evaluation of the microbial community entailed an assessment of taxa abundance and diversity using Kraken version 2.1.2. The study delineated the prevalence of dominant bacterial phyla, including Proteobacteria, with varying abundance of Firmicutes, Bacteroidota, and Actinobacteria, and notable occurrence of Tenericutes. Furthermore, the presence of the fungal phylum Ascomycota was also detected. Among the identified barcodes, Barcode04 emerged as the most abundant and diverse, while Barcode06 exhibited greater evenness. Barcode03, 05, and 07 displayed moderate richness and diversity. Through an analysis of the relative abundance, a spectrum of viruses within Ae. aegypti populations was unveiled, with Negarnaviricota constituting the most prevalent phylum, followed by Nucleocytoviricota, Uroviricota, Artverviricota, Kitrinoviricota, Peploviricota, Phixviricota, and Cossaviricota. The presence of Negarnaviricota viruses raises pertinent public health concerns. The presence of other viral phyla underscores the intricate nature of virus-mosquito interactions. The analysis of viral diversity provides valuable insights into the range of viruses carried by Ae. aegypti. The community exhibits low biodiversity, with a few dominant species significantly influencing its composition. This has implications for healthcare and ecological management, potentially simplifying control measures but also posing risks if the dominant species are harmful. This study enriches our comprehension of the microbiome and virome associated with Ae. aegypti mosquitoes, emphasizing the importance of further research to fully comprehend their ecological significance and impact on public health. The findings shed light on the microbial ecology of Ae. aegypti, offering potential insights into mosquito biology, disease transmission, and strategies for vector control. Future studies should endeavor to establish specific associations with Ae. aegypti, elucidate the functional roles of the identified microbial and viral species, and investigate their ecological implications.

De-correlating immune checkpoint inhibitor toxicity and response in melanoma via the microbiome.

9569 Background: Immune-checkpoint inhibitor (ICI) immunotherapy increases survival in patients with melanoma. Yet only half of the patients respond, and 10-40% of patients experience immune-related adverse events (irAEs). Previous studies have found a correlation between the development of an irAE and treatment response. Modifying the gut microbiome could positively affect response to ICIs and reduce toxicities. We sought to determine if the microbiome at the onset of treatment, during an irAE, and at 12 weeks, can predict the response or toxicity during ICI treatment for metastatic melanoma. Methods: Melanoma patients (pts) &gt; 18 yo treated with ICI enrolled in a prospective observational cohort study at The Ohio State University Comprehensive Cancer Center Skin Cancer Clinic. Excluded were patients on corticosteroids at the start of ICI cycle 1, except for adrenal physiologic replacement. Stools were collected at baseline, within 2 days of an adverse event assessed by physician chart review using CTCAE (Common Terminology Criteria for Adverse Events) v5.0, and at 12 weeks. Response (ORR) to ICIs was assessed by Response Evaluation Criteria in Solid Tumors (RECIST v1.1) q12 weeks and progression-free survival recorded. Metagenomic whole-genome shotgun sequencing was performed on an Illumina NovaSeq 6000 and classified using HUMAnN3. The effect of microbe relative abundances on irAEs was modeled by logistic regression with the R package glmm. Results: Of the 88 patients enrolled, 48 had metastatic disease with 32 assessable for response. ORR at 12 weeks was 28% (1 CR, 8 PR, 17 SD, and 6 PD) and 24 patients showed PFS at 12 months. Grade &gt; 2 irAEs were observed in 11/48 pts. Abundance of Bacteroides dorei (False Discovery Rate Corrected p-value = 9.5E-07) and Blautia species CAG:257 (padj = 0.001) were enriched in responders, Prevotella Stercorea (padj = 1.1E-07) and a phage with a predicted target of Salmonella (padj = 8.3E-09) in non-responders. Responders with an irAE had enrichment of Bacteroides plebeius (padj = 4.2E-13) and Bacteroides coprophilus (padj = 0.0002) whereas responders without irAE had enrichment of Eubacterium siraeum (padj = 7.2E-05). Conclusions: Longitudinal and event-driven biospecimen collection in patients treated with ICIs showed several bacteria and viruses but no fungi associated with response, with and without the development of an irAE. The abundance of the two high-taxonomic rank microbe groups is significantly associated with irAEs. The association with the Bacteroides Genus is consistent with previous studies and is associated with response to ICIs. A microbiome abundant in Bacteroides and lacking Prevotella is associated with response to ICI. An abundance of Bacteroides could be a biomarker for irAEs, and an abundance of Eubacterium, a biomarker for response without irAEs. Future analyses are necessary to assign causal roles for microbe biomarkers with specific irAEs. Clinical trial information: NCT05102773 .

Shotgun metagenomics reveals abnormal short-chain fatty acid-producing bacteria and glucose and lipid metabolism of the gut microbiota in patients with schizophrenia.

Evidence has shown that the gut microbiota is closely related to the pathogenesis of schizophrenia, but temporal changes in the gut microbiota of patients with schizophrenia (SZ) during treatment remain unclear. Here, to evaluate temporal changes in the gut microbiota in schizophrenia, we performed whole-genome shotgun metagenomics on fecal samples from 36 healthy controls (HCs) and 19 baseline-period patients, and followed up with patients upon treatment. Compared to that in HCs, beta diversity in SZ was significantly distinct. The genera Bacteroides, Prevotella and Clostridium were the top 3 altered genera between SZ and HCs, and the Bacteroides-Prevotella ratio was significantly increased in SZ. Thirty-three percent of differentially abundant species were short-chain fatty acid (SCFA)-producing bacteria. Functional analysis showed that glucose and lipid metabolism of the gut microbiota was decreased in SZ compared with those in HCs. The abundances of two rate-limiting enzymes in glucose and lipid metabolism, phosphofructokinase (PFK) and acetyl-CoA carboxylase (ACC), were significantly decreased in SZ, and differentially abundant metabolism-related enzymes were significantly associated with SCFA-producing bacteria. Next, we found that the abundance of SCFA-producing bacteria also changed after treatment and that Clostridium was significantly negatively correlated with the total positive and negative syndrome scale (PANSS) score in patients. Functional analysis showed that glycoside hydrolase family 30 incrementally increased in abundance during treatment and were significantly associated with SCFA-producing bacteria. Our findings help to provide evidence for the role of gut microbiota in the occurrence and development of schizophrenia.

Abstract PR004: Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC

Abstract The gut microbiome changes with age and affects many aspects of human health, including response to cancer treatments. Cancer survival rates have improved with new treatment options, including immune checkpoint blockade (ICB); however, the objective response rate remains low. Manipulation of the microbiome is a promising approach to improving cancer outcomes, but the effect of age is understudied. Here, we sought to understand whether (1) specific microbes are associated with treatment response in older adults with non-small cell lung cancer (NSCLC) and (2) whether these microbes are the same as for younger adults. Next, we explored the causal effects of the microbiome on ICB response in mouse models and the relationship with blood-based markers of T-cell senescence. We conducted a prospective cohort study of adults ≥60 years with a new diagnosis of NSCLC who received any treatment modality. Stool was collected, and metagenomic whole-genome shotgun sequencing was performed. Blood T-cells were isolated, the RNA purified and then assessed for markers of senescence by nanostring. Response to treatment was determined by RECIST v1.1 criteria. Generalized linear regression was used to relate baseline microbiome abundances to treatment response and non-parametric correlations associated with CDKN2A (p16) expression to microbe abundances. To assess the causal role of the gut microbiome in ICB response, we gavaged gut microbiome samples from responders and non-responders into C57BL/6 mice to create human-microbiome avatar models. The mice were then injected with MC38 cancer cells and treated with anti-PD1 or isotype control antibodies, and tumor volume was measured over time. Biospecimens and best response data at three months were captured from 23 patients, of which five had a complete response, eight had a partial response, eight had stable disease, and two had progressive disease. Over 50 microbes were associated with a response after p-value adjustment. Responder stool was enriched for microbes associated with youth and ICB response (Bifidobacterium adolescentis, p = 2.64e-20). However, microbial taxa associated with response differed from those reported in younger populations (Firmicutes sp. CAG 145, p = 1.58e-20, Oscillibacter sp. 57-20, p = 7.96e-24). Stool from non-responders (NRs) was enriched in taxa previously linked to treatment-related toxicities and shorter progression-free survival (Streptococcus lutetiensis, p = 4.55E-24) but also contained microbes previously linked to response in younger adults (e.g., Roseburia sp. CAG 309, p = 5.16e-15). The T cell senescence marker, p16, correlated with the most enriched taxon in non-responders NRs (Streptococcus thermophilus, r = 0.45, p = 0.02), suggesting a connection between immune aging and the microbiome. Preliminary fecal transplant studies in mice showed improved ICB response in mice engrafted with stool from responders versus non-responders. Together, these data identify potential differences in the gut microbiomes of young and older adult NSCLC patients who respond to ICB. Citation Format: Daniel Spakowicz, Rebecca Hoyd, Caroline E. Wheeler, Nyelia Williams, Amna Bibi, Marium Husain, Srichandhana Rajamouli, Shankar Suman, Joseph Amann, Madison Grogan, Pooja Vibhakar, Dwight H. Owen, David P. Carbone, Ashley Rosko, Christin E. Burd, Carolyn J. Presley. Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR004.

Functional Interpretation of Cross-Talking Pathways with Emphasis on Amino Acid Metabolism in Rhizosphere Microbiome of the Wild Plant Moringa oleifera

The functional processes and mutual benefits of the wild plant Moringa oleifera and its rhizosphere microbiome were studied via metagenomic whole-genome shotgun sequencing (mWGS) in comparison with a bulk soil microbiome. The results indicated high gene abundance of the four KEGG categories, “Cellular Processes”, “Environmental Information Processing”, “Genetic Information Processing”, and “Metabolism”, in the rhizosphere microbiome. Most of the enriched enzymes in rhizobacteria are assigned to the pathway “Amino acids metabolism”, where soil-dwelling microbes use amino acids as a defense mechanism against phytopathogens, while promoting growth, colonizing the cohabiting commensal microbes and conferring tolerance against abiotic stresses. In the present study, it was proven that these beneficial microbes include Bacillus subtilis, Pseudomonas fluorescens, and Escherichia coli. Mineral solubilization in these rhizobacteria can make nutrients available for plant utilization. These rhizobacteria extensively synthesize and metabolize amino acids at a high rate, which makes nitrogen available in different forms for plants and microbes. Amino acids in the rhizosphere might stand mainly as an intermediate switcher for the direction of the soil nitrogen cycle. Indole acetic acid (IAA) was proven to be synthesized by these beneficial rhizobacteria via route indole-3-pyruvate (IPyA) of the pathway “Tryptophan metabolism”. This hormone might stand as a shuttle signaling molecule between M. oleifera and its rhizobacteria. Tryptophan is also metabolized to promote other processes with important industrial applications. Rhizobacteria were also proven to breakdown starch and sucrose into glucose, which is the primary metabolic fuel of living organisms. In conclusion, we assume that the metabolic processes in the rhizosphere microbiome of this wild plant can be eventually utilized in boosting the sustainability of agriculture applications and the plant’s ability to benefit from soil nutrients when they are not in the form available for plant root absorption.

Ca:Mg ratio, medium-chain fatty acids, and the gut microbiome

Ketogenic medium-chain fatty acids (MCFAs) with profound health benefits are commonly found in dairy products, palm kernel oil and coconut oil. We hypothesize that magnesium (Mg) supplementation leads to enhanced gut microbial production of MCFAs and, in turn, increased circulating MCFAs levels. We tested this hypothesis in the Personalized Prevention of Colorectal Cancer Trial (PPCCT) (NCT01105169), a double-blind 2×2 factorial randomized controlled trial enrolling 240 participants. Six 24-hdietary recalls were performed for all participants at the baseline and during the intervention period. Based on the baseline 24-hdietary recalls, the Mg treatment used a personalized dose of Mg supplementation that would reduce the calcium (Ca): Mg intake ratio to around 2.3. We measured plasma MCFAs, sugars, ketone bodies and tricarboxylic acid cycle (TCA cycle) metabolites using the Metabolon's global Precision Metabolomics™ LC-MS platform. Whole-genome shotgun metagenomics (WGS) sequencing was performed to assess microbiota in stool samples, rectal swabs, and rectal biopsies. Personalized Mg treatment (mean dose 205.58mg/day with a range from 77.25to 389.55mg/day) significantly increased the plasma levels of C7:0, C8:0, and combined C7:0 and C8:0 by 18.45%, 25.28%, and 24.20%, respectively, compared to 14.15%, 10.12%, and 12.62% decreases in the placebo arm. The effects remain significant after adjusting for age, sex, race and baseline level (P=0.0126, P=0.0162, and P=0.0031, respectively) and FDR correction at 0.05 (q=0.0324 for both C7:0 and C8:0). Mg treatment significantly reduced the plasma level of sucrose compared to the placebo arm (P=0.0036 for multivariable-adjusted and P=0.0216 for additional FDR correction model) whereas alterations in daily intakes of sucrose, fructose, glucose, maltose and C8:0 from baseline to the end of trial did not differ between two arms. Mediation analysis showed that combined C7:0 and C8:0 partially mediated the effects of Mg treatment on total and individual ketone bodies (P for indirect effect=0.0045, 0.0043, and 0.03, respectively). The changes in plasma levels of C7:0 and C8:0 were significantly and positively correlated with the alterations in stool microbiome α diversity (r=0.51, p=0.0023 and r=0.34, p=0.0497, respectively) as well as in stool abundance for the signatures of MCFAs-related microbiota with acyl-ACP thioesterase gene producing C7:0 (r=0.46, p=0.0067) and C8:0 (r=0.49, p=0.003), respectively, following Mg treatment. Optimizing Ca:Mg intake ratios to around 2.3 through 12-week personalized Mg supplementation leads to increased circulating levels of MCFAs (i.e. C7:0 and C8:0), which is attributed to enhanced production from gut microbial fermentation and, maybe, sucrose consumption.

The effect of the microbiome on immune checkpoint inhibitor toxicity in patients with melanoma.

9568 Background: Immune-checkpoint inhibitor (ICI) immunotherapy has increased survival in patients with melanoma. However, only half of the patients respond, and many experience immune-related adverse events (irAEs). Recent evidence suggests that modification of the gut microbiome may increase response to ICIs and decrease toxicity. Here we describe the first results of a clinical trial to determine if the microbiome can predict the response or toxicity during the first 16 weeks of ICI treatment. Methods: We enrolled patients aged 18 or older in a prospective observational cohort study at The Ohio State University Comprehensive Cancer Center Skin Cancer Clinic (OSUCCC-SCC) who were to receive treatment with pembrolizumab or nivolumab alone or in combination with other treatments (e.g. nivolumab and ipilimumab) for melanoma. Patients receiving systemic or oral corticosteroids at the start of ICI cycle 1 were excluded but were eligible if receiving adrenal physiologic replacement. Patients collected stool samples at baseline, within 2 days of an adverse event (if applicable), and at 12 weeks. The response to ICIs was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST v1.1) at a 12-week computed tomography scan. Metagenomic whole-genome shotgun sequencing was performed on an Illumina NovaSeq 6000 and then classified using HUMAnN3. The effect of microbe relative abundances on potential irAEs was modeled by logistic regression with the R package glmm. Results: In total, 88 patients consented to the trial. Pre-treatment microbiome samples were collected from 49 patients. Potential irAEs were observed in 16 out of the 49 patients for whom pre-treatment microbiome samples were collected. There was no significant difference in the ages (p = 0.150, genders (p = 0.2), stages (p = 0.2) or treatments (p = 0.07) of those who developed potential irAEs. Pretreatment abundance of the family Ruminococaceae was most strongly associated with the development of a potential irAE (p = 0.03), followed by a taxon in an unclassified order within the phylum Firmicutes (p = 0.05). The family Bacteroidaceae was most strongly associated with no potential irAE (p = 0.05). Conclusions: Longitudinal and event-driven biospecimen collection in the context of treatment with immunotherapies was feasible in the OSUCCC-SCC. The abundance of the two high-taxonomic rank microbe groups was significantly associated with potential irAEs. The association with Ruminococaceae is consistent with previous studies where it was associated with response to ICIs and, in separate studies, development of an irAE was associated with a better response. The unclassified taxon is potentially a new biomarker for the prediction of toxicity and a therapeutic target to reduce treatment side effects. Future analyses will associate microbes with treatment response and test for consistent microbiome changes at the time of irAE development. Clinical trial information: NCT05102773.

Evidence of pediatric sepsis caused by a drug resistant Lactococcus garvieae contaminated platelet concentrate

ABSTRACT Owing to an increasing number of infections in adults, Lactococcus (L.) garvieae has gained recognition as an emerging human pathogen, causing bacteraemia and septicaemia. In September 2020, four paediatric onco-hematologic patients received a platelet concentrate from the same adult donor at Bambino Gesù Children’s Hospital IRCCS, Rome. Three of four patients experienced L. garvieae sepsis one day after transfusion. The L. garvieae pediatric isolates and the donor’s platelet concentrates were retrospectively collected for whole-genome sequencing and shot-gun metagenomics, respectively (Illumina HiSeq). By de novo assembly of the L. garvieae genomes, we found that all three pediatric isolates shared a 99.9% identity and were characterized by 440 common SNPs. Plasmid pUC11C (conferring virulence properties) and the temperate prophage Plg-Tb25 were detected in all three strains. Core SNP genome-based maximum likelihood and Bayesian trees confirmed their phylogenetic common origin and revealed their relationship with L. garvieae strains affecting cows and humans (bootstrap values >100 and posterior probabilities = 1.00). Bacterial reads obtained by the donor’s platelet concentrate have been profiled with MetaPhlAn2 (v.2.7.5); among these, 29.9% belonged to Firmicutes, and 5.16% to Streptococcaceae (>97% identity with L. garvieae), confirming the presence of L. garvieae in the platelet concentrate transfusion. These data showed three episodes of sepsis for the first time due to a transfusion-associated transmission of L. garvieae in three pediatric hospitalized hematology patients. This highlights the importance to implement the screening of platelet components with new human-defined pathogens for ensuring the safety of blood supply, and more broadly, for the surveillance of emerging pathogens.

Metagenomic insights into Himalayan glacial and kettle lake sediments revealed microbial community structure, function, and stress adaptation strategies.

Glacial and kettle lakes in the high-altitude Himalayas are unique habitats with significant scope for microbial ecology. The present study provides insights into bacterial community structure and function of the sediments of two high-altitude lakes using 16S amplicon and whole-genome shotgun (WGS) metagenomics. Microbial communities in the sediments of Parvati kund (glacial lake) and Bhoot ground (kettle lake) majorly consist of bacteria and a small fraction of archaea and eukaryota. The bacterial population has an abundance of phyla Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Firmicutes, and Verrucomicrobia. Despite the common phyla, the sediments from each lake have a distinct distribution of bacterial and archaeal taxa. The analysis of the WGS metagenomes at the functional level provides a broad picture of microbial community metabolism of key elements and suggested chemotrophs as the major primary producers. In addition, the findings also revealed that polyhydroxyalkanoates (PHA) are a crucial stress adaptation molecule. The abundance of PHA metabolism in Alpha- and Betaproteobacteria and less representation in other bacterial and archaeal classes in both metagenomes was disclosed. The metagenomic insights provided an incisive view of the microbiome from Himalayan lake's sediments. It has also opened the scope for further bioprospection from virgin Himalayan niches.

Amoxicillin duration and dose for community-acquired pneumonia in children: the CAP-IT factorial non-inferiority RCT.

Data are limited regarding the optimal dose and duration of amoxicillin treatment for community-acquired pneumonia in children. To determine the efficacy, safety and impact on antimicrobial resistance of shorter (3-day) and longer (7-day) treatment with amoxicillin at both a lower and a higher dose at hospital discharge in children with uncomplicated community-acquired pneumonia. A multicentre randomised double-blind 2 × 2 factorial non-inferiority trial in secondary care in the UK and Ireland. Paediatric emergency departments, paediatric assessment/observation units and inpatient wards. Children aged > 6 months, weighing 6-24 kg, with a clinical diagnosis of community-acquired pneumonia, in whom treatment with amoxicillin as the sole antibiotic was planned on discharge. Oral amoxicillin syrup at a dose of 35-50 mg/kg/day compared with a dose of 70-90 mg/kg/day, and 3 compared with 7 days' duration. Children were randomised simultaneously to each of the two factorial arms in a 1 : 1 ratio. The primary outcome was clinically indicated systemic antibacterial treatment prescribed for respiratory tract infection (including community-acquired pneumonia), other than trial medication, up to 28 days after randomisation. Secondary outcomes included severity and duration of parent/guardian-reported community-acquired pneumonia symptoms, drug-related adverse events (including thrush, skin rashes and diarrhoea), antimicrobial resistance and adherence to trial medication. A total of 824 children were recruited from 29 hospitals. Ten participants received no trial medication and were excluded. Participants [median age 2.5 (interquartile range 1.6-2.7) years; 52% male] were randomised to either 3 (n = 413) or 7 days (n = 401) of trial medication at either lower (n = 410) or higher (n = 404) doses. There were 51 (12.5%) and 49 (12.5%) primary end points in the 3- and 7-day arms, respectively (difference 0.1%, 90% confidence interval -3.8% to 3.9%) and 51 (12.6%) and 49 (12.4%) primary end points in the low- and high-dose arms, respectively (difference 0.2%, 90% confidence interval -3.7% to 4.0%), both demonstrating non-inferiority. Resolution of cough was faster in the 7-day arm than in the 3-day arm for cough (10 days vs. 12 days) (p = 0.040), with no difference in time to resolution of other symptoms. The type and frequency of adverse events and rate of colonisation by penicillin-non-susceptible pneumococci were comparable between arms. End-of-treatment swabs were not taken, and 28-day swabs were collected in only 53% of children. We focused on phenotypic penicillin resistance testing in pneumococci in the nasopharynx, which does not describe the global impact on the microflora. Although 21% of children did not attend the final 28-day visit, we obtained data from general practitioners for the primary end point on all but 3% of children. Antibiotic retreatment, adverse events and nasopharyngeal colonisation by penicillin-non-susceptible pneumococci were similar with the higher and lower amoxicillin doses and the 3- and 7-day treatments. Time to resolution of cough and sleep disturbance was slightly longer in children taking 3 days' amoxicillin, but time to resolution of all other symptoms was similar in both arms. Antimicrobial resistance genotypic studies are ongoing, including whole-genome sequencing and shotgun metagenomics, to fully characterise the effect of amoxicillin dose and duration on antimicrobial resistance. The analysis of a randomised substudy comparing parental electronic and paper diary entry is also ongoing. Current Controlled Trials ISRCTN76888927, EudraCT 2016-000809-36 and CTA 00316/0246/001-0006. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 60. See the NIHR Journals Library website for further project information.

Comparison of the distribution and diversity of antimicrobial resistance genes between wild and commercially available Atlantic Mackerel

The global uncontrolled rise of antimicrobial resistance (AMR) is a major societal threat and it is well documented that AMR is already negatively affecting healthcare and intensive livestock farming systems. Nonetheless, capture fisheries are still essential to the global food supply providing over 50% of the world’s aquatic organism production. To facilitate improved management, it is therefore imperative that we better understand reservoirs of AMR and how gene transmission occurs in the aquatic environment. In order to discern which AMR bacteria and genes are present in the marine environment, sediment and seawater samples were collected and Atlantic Mackerel were captured at the beginning of summer and autumn in the Solent Strait (Portsmouth, U.K). In addition, commercially available Atlantic mackerel were purchased at a local fish market. Using culture-dependent techniques we obtained more than 700 bacterial and 20 fungal isolates from skin, intestinal lining, and intestinal content. Ten different cefotaxime-resistant Pseudomonas spp. were isolated from the seawater and market fish skin samples, and one cefotaxime-resistant Rhanella sp. was isolated from wild fish digesta. Results from ongoing whole-genome shotgun metagenomics analysis will be discussed, as well as the connection between AMR bacteria and AMR gene presence within the marine coastal environment and the local fish markets, which are the last link between capture fisheries and consumers.

What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen.

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis—the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)—were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

Identification of Novel Putative Bacterial Feruloyl Esterases From Anaerobic Ecosystems by Use of Whole-Genome Shotgun Metagenomics and Genome Binning.

Feruloyl esterases (FAEs) can reduce the recalcitrance of lignocellulosic biomass to enzymatic hydrolysis, thereby enhancing biorefinery potentials or animal feeding values of the biomass. In addition, ferulic acid, a product of FAE activity, has applications in pharmaceutical and food/beverage industries. It is therefore of great interest to identify new FAEs to enhance understanding about this enzyme family. For this purpose, we used whole-genome shotgun metagenomics and genome binning to explore rumens of dairy cows, large intestines of horses, sediments of freshwater and forest topsoils to identify novel prokaryotic FAEs and trace the responsible microorganisms. A number of prokaryotic genomes were recovered of which, genomes of Clostridiales order and Candidatus Rhabdochlamydia genus showed FAE coding capacities. In total, five sequences were deemed as putative FAE. The BLASTP search against non-redundant protein database of NCBI indicated that these putative FAEs represented novel sequences within this enzyme family. The phylogenetic analysis showed that at least three putative sequences shared evolutionary lineage with FAEs of type A and thus could possess specific activities similar to this type of FAEs, something that is not previously found outside fungal kingdom. We nominate Candidatus Rhabdochlamydia genus as a novel FAE producing taxonomic unit.

Four men in a boat: Ultra-endurance exercise alters the gut microbiome

ObjectivesCompositional and functional adaptions occur in the gut microbiome in response to habitual physical activity. The response of the gut microbiome to sustained, intense exercise in previously active individuals, however, is unknown. This study aimed to prospectively explore the gut microbiome response of four well-trained male athletes to prolonged, high intensity trans-oceanic rowing, describing changes in microbial diversity, abundance and metabolic capacity. DesignA prospective, repeated-measures, within-subject report. MethodsSerial stool samples were obtained from four male athletes for metagenomic whole-genome shotgun sequencing to record microbial community structure and relevant functional gene profiles before, during and after a continuous, unsupported 33-day, 5000 km transoceanic rowing race. Calorific intake and macronutrient composition were recorded by validated food frequency questionnaire and anthropometry was determined by body composition analysis and cardiorespiratory testing. ResultsMicrobial diversity increased throughout the ultra-endurance event. Variations in taxonomic composition included increased abundance of butyrate producing species and species associated with improved metabolic health, including improved insulin sensitivity. The functional potential of bacterial species involved in specific amino and fatty acid biosynthesis also increased. Many of the adaptions in microbial community structure and metaproteomics persisted at three months follow up. ConclusionsThese findings demonstrate that prolonged, intense exercise positively influences gut microbial diversity, increases the relative abundance of some bacterial species and up-regulates the metabolic potential of specific pathways expressing microbial gene products. These adaptions may play a compensatory role in controlling the physiological stress associated with sustained exertion as well as negating the deleterious consequences accompanying endurance exercise.

Leveraging Human Microbiome Features to Diagnose and Stratify Children with Irritable Bowel Syndrome

Accurate diagnosis and stratification of children with irritable bowel syndrome (IBS) remain challenging. Given the central role of recurrent abdominal pain in IBS, we evaluated the relationships of pediatric IBS and abdominal pain with intestinal microbes and fecal metabolites using a comprehensive clinical characterization and multiomics strategy. Using rigorous clinical phenotyping, we identified preadolescent children (aged 7 to 12 years) with Rome III IBS (n = 23) and healthy controls (n = 22) and characterized their fecal microbial communities using whole-genome shotgun metagenomics and global unbiased fecal metabolomic profiling. Correlation-based approaches and machine learning algorithms identified associations between microbes, metabolites, and abdominal pain. IBS cases differed from controls with respect to key bacterial taxa (eg, Flavonifractor plautii and Lachnospiraceae bacterium 7_1_58FAA), metagenomic functions (eg, carbohydrate metabolism and amino acid metabolism), and higher-order metabolites (eg, secondary bile acids, sterols, and steroid-like compounds). Significant associations between abdominal pain frequency and severity and intestinal microbial features were identified. A random forest classifier built on metagenomic and metabolic markers successfully distinguished IBS cases from controls (area under the curve, 0.93). Leveraging multiple lines of evidence, intestinal microbes, genes/pathways, and metabolites were associated with IBS, and these features were capable of distinguishing children with IBS from healthy children. These multi-omics features, and their links to childhood IBS coupled with nutritional interventions, may lead to new microbiome-guided diagnostic and therapeutic strategies.

Abstract 3266: Measurement and comparison of the gut microbial communities in fecal, rectal swab, and mucosal samples

Abstract Background: Studies evaluating the gut microbiome frequently use stool. However, stool, which is predominantly composed of luminal bacteria, may not adequately reflect mucosally adherent bacteria. The purpose of this study is to evaluate similarities and differences in gut bacterial measurements and stability in the microbial communities of three different types of samples that could be used to assess different niches of the gut microbiome: rectal swab, stool, and normal rectal mucosa samples. Design: Sixty-eight participants were selected from a personalized chemoprevention trial population of individuals with previous colorectal adenomas who had donated stool, swab, and mucosal samples at baseline and three months later. 16S rRNA amplicon sequencing was conducted for 60 participants at two time points (n=120 stool and 120 swab). Whole-genome shotgun metagenomics DNA sequencing was conducted for 50 participants at two time points (n=100 stool, 28 swab, and 16 mucosal). Results: In swab-stool comparisons, there were substantial taxa differences with some taxa varying largely by sample type (e.g., Thermaceae) while other taxa were predominantly associated with interindividual subject variation (e.g., Desulfovibrionaceae) or by both sample type and participant (e.g., Enterobacteriaceae). At species-level resolution (with WGS sequences) we observed that bacteria associated with colorectal tumors (Escherichia coli and Fusobacterium nucleatum) were of higher relative abundance in swab than stool. There were also statistically significant differences in other bacteria according to the sample type (e.g., Bifidobacterium longum, Bacteroides fragilis). Comparing all three sample types with whole-genome metagenome shotgun sequencing, swab samples were much closer to stool samples than mucosa samples, although all KEGG functional Level 1 and Level 2 pathways were significantly different across all sample types (e.g., transcription and environmental adaptation). However, the individual signature of participants was also observed and was largely stable between two time points. Thus, we found that while the distribution of some taxa was associated with these different sampling techniques, other taxa largely reflected individual differences in the microbial community that were insensitive to sampling technique. Conclusion: There is substantial variability in the assessment of the gut microbial community according to the type of sample. Citation Format: Roshonda B Jones, Xiangzhu Zhu, Emili Moan, Harvey J. Murff, Qi Dai, Martha J. Shrubsole, Anthony A. Fodor, M. Andrea Azcarate-Peril, Reid M. Ness, Douglas L. Seidner. Measurement and comparison of the gut microbial communities in fecal, rectal swab, and mucosal samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3266.