V3 Sequences Research Articles

The Effect of Maternal Postpartum Practices on Infant Gut Microbiota: A Chinese Cohort Study

(1) Background: The human gut microbiota at early life is shaped by numerous factors, especially factors from mothers, which have huge influence on infants’ gut microbiotas. The aim of this study was to investigate the effect of maternal adherence to Chinese traditional postpartum practices of “doing the month” on the development of infant gut microbiota at 6-month postpartum. (2) Methods: A cohort of 62 Chinese women at late pregnancy was recruited from a tertiary general hospital in a central region of China. The participants and their babies were followed up to 6 months postpartum. Finally, 50 mother-infant dyads were enrolled in the study. Women’s adherence to the traditional postpartum practices was measured by adherence to doing the month practices (ADP). Infant fecal samples were collected at six months of age and were analyzed using 16S rRNA V3 and V4 gene region sequences. (3) Results: Ruminococcus gnavus was significantly less abundant in infants whose mothers had a better adherence to the traditional postpartum practices of “doing the month.” Infants receiving Clostridium-butyricum during the first month after delivery had a significant dominance of Escherichia/Shigella. (4) Conclusions: Adherence to the traditional postpartum practices of “doing the month” can impact an infant’s gut microbiota at 6 months of age. Infants receiving probiotics during the first month after delivery had a significant dominance of opportunistic pathogens.

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat.

SummaryWheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.

Skin Microbiota Perturbations Are Distinct and Disease Severity–Dependent in Hidradenitis Suppurativa

Skin Microbiota Perturbations Are Distinct and Disease Severity–Dependent in Hidradenitis Suppurativa

Consumption of a Leuconostoc holzapfelii-enriched synbiotic beverage alters the composition of the microbiota and microbial extracellular vesicles

Synbiotics, the combination of probiotics and prebiotics, are known to confer health benefits via intestinal microbiota modulation. However, significant intestinal microbiota alterations can be difficult to determine in intervention studies based on solely bacterial stool metagenomic analysis. Intestinal microbiota constituents secrete 20–200-nm-sized extracellular vesicles (EVs) containing microbial DNA, proteins, and lipids that are distributed throughout the body, providing an alternative target for microbiota metagenomic analysis. Here, we determined the impact of a synbiotic beverage enriched with the kimchi-derived bacterium Leuconostoc holzapfelii (L. holzapfelii) on the intestinal microbiota and local and circulatory microbiota-derived EV composition of healthy Korean adults. We isolated microbial DNA from stool bacteria, stool EVs, and urinary EVs and conducted next-generation sequencing of the 16S rDNA V3–V4 regions before and after synbiotic consumption. The species diversity of circulating urinary EVs was significantly increased after synbiotic consumption, while stool bacterial and EV diversity remained unchanged. Furthermore, we found that while a single genus was decreased among the stool bacteria constituents, stool EVs and urinary EVs showed significant alterations in four and eight genera, respectively. Blood chemistry assays revealed that synbiotic consumption significantly lowered aspartate aminotransferase (AST) serum levels, particularly in subjects with starting levels above the normal range (>40 UI/L). In conclusion, the L. holzapfelii-enriched synbiotic beverage greatly altered serum AST levels and microbial EV composition in urine and stool, while only minor changes were observed in the gut microbiota composition. Based on these findings, we suggest the potential use of microbiota-derived EVs as surrogate markers in future predictive diagnosis studies.

Structural and functional microbial diversity along a eutrophication gradient of interconnected lakes undergoing anthropopressure

We present the results of an analysis of the 16S rRNA-based taxonomical structure of bacteria together with an analysis of carbon source utilization ability using EcoPlate (Biolog, USA) metabolic fingerprinting assessment against the backdrop of physicochemical parameters in fifteen interconnected lakes. The lakes exhibit a wide spectrum of trophic gradients and undergo different intensities of anthropopressure. Sequences of V3–V4 16S rRNA genes binned by taxonomic assignment to family indicated that bacterial communities in the highly eutrophicated lakes were distinctly different from the bacterial communities in the meso-eutrophic lakes (ANOSIM r = 0.99, p = 0.0002) and were characterized by higher richness and more diverse taxonomical structure. Representatives of the Actinobacteria, Proteobacteria, Cyanobacteria, Planctomycetes, Verrucomicrobia, Bacteroides phyla predominated. In most cases their relative abundance was significantly correlated with lake trophic state. We found no similar clear relationship of community-level physiological profiling with lake trophic state. However, we found some significant links between the taxonomic and metabolic structure of the microbes in the studied lakes (Mantel’s correlation r = 0.22, p = 0.006). The carbon source utilization ability of the studied microorganisms was affected not only by the taxonomic groups present in the lakes but also by various characteristics like a high PO43− concentration inhibiting the utilization of phosphorylated carbon.

Chronic obstructive pulmonary disease upper airway microbiome is associated with select clinical characteristics.

BackgroundChronic obstructive pulmonary disease (COPD) is an inflammatory lung disorder associated with lung microbiome dysbiosis. Although the upper airway microbiome is the source of the lung microbiome, the relationships between the oral, nasal, and sputum microbiota are incompletely understood. Our objective was to determine features that differentiate the oral, nasal, and sputum microbiome among subjects with stable COPD.MethodsWe recruited 15 current or former smokers to provide oral and sputum samples on day 1. On day 2, another oral sample and a nasal sample were obtained. Each sample and control underwent DNA extraction, 16S V4 rRNA amplification, 16S V4 sequencing, and qPCR of 16S rRNA. Data were analyzed using dada2 and R.ResultsMost (14 of 15) subjects were male with a mean age of 65.2. One subject had no pulmonary obstruction, while 5 had mild COPD, 7 had moderate COPD, and 2 had severe COPD. Three subjects (20%) were current tobacco users and 2 subjects (13%) used inhaled corticosteroids (ICS). Subjects had a mean of 49.1 pack-years of tobacco exposure. Bacterial biomass was associated with anatomic site, but no differences in biomass were observed with age, FEV1 percent predicted (FEV1pp), ICS use, smoking status, or edentulous state. Shannon index was associated with site (lower nasal diversity than oral and sputum diversity, p<0.001), but not age, ICS use, FEV1pp, tobacco use, or edentulous state. β-diversity was illustrated by principal coordinate analysis using Bray-Curtis dissimilarity and PERMANOVA analyses, showing sample clustering by anatomic site (p = 0.001) with nasal samples forming a cluster separate from the combined oral wash samples and sputum samples. Clustering was also observed with ICS use (p = 0.029) and edentulous state (p = 0.019), while FEV1pp and current tobacco use were not significant. In an amplicon sequencing variant (ASV)-level analysis of oral samples using a linear regression model with Benjamini-Hochberg correction at an FDR<0.10, 10 ASVs were associated with age while no ASVs were associated with FEV1pp or smoking status. Sputum sample analysis demonstrated that 51 ASVs (25 unique genera) were associated with age, 61 ASVs (32 genera) were associated with FEV1pp, and no ASVs were associated with smoking status. In a combined dataset, the frequent exacerbator phenotype, rather than ICS use, was associated with decreased sputum Shannon diversity.ConclusionsAmong the upper airway microbiota of COPD subjects, anatomic site was associated with bacterial biomass, Shannon diversity, and β-diversity. ICS use and edentulous state were both associated with β-diversity. Age was associated with taxa relative abundance in oral and sputum samples, while FEV1pp was associated with taxa relative abundance in sputum samples only.

Molecular and geographic characterization of hiv-1 bf recombinant viruses

The Human Immunodeficiency Virus Type 1 (HIV-1) presents a wide genetic variability, which is represented by four groups, nine subtypes of group M and several recombinant forms. Among these, the BF recombinants have been distinguished by a high global dispersion and an increase in number and diversity. To date, 15 BF Circulating Recombinant Forms (CRFs) and diverse BF Unique Recombinant Forms (URFs) have been described. In Brazil, nine CRF_BF have been identified. The aim of this work was to perform molecular and geographic characterization of HIV-1 BF recombinant strains. Near full-length genomes of 265 BF recombinant viruses were collected from public databases and molecular analyses were performed. These sequences were originally retrieved between 1993–2006 and isolated from 16 countries (51.3% from Brazil). Diagnostic's year analysis showed that BF recombinants circulate in Brazil since at least 1985. Most sequences displayed recombination in the pol (84.9%), gag (69.3%) and env (51.4%) regions. The subtype B predominated in all accessory and regulatory genes, except in vif, in which the F subtype was predominant (40.4%). Twelve regions with a recombination rate higher than 10% were identified, especially one region inside p24 gene (1359–1397) whose recombination was present in more than 30% of the sequences. Coreceptor usage prediction during viral entry showed that BF recombinants preferentially use CCR5 (67.2%) and the most frequent tetrapeptides found in the V3 loop were GPGR (47.9%) and GPGQ (21.1%). The frequency of X4/dual viruses was lower amongst F subtype (25.8%) V3 sequences, compared with B subtype (43%). In addition, mutations associated with intermediate or high resistance levels to PI (10.6%), NRTI (15.0%), NNRTI (14.0%) and INSTI (2.6%) were identified. The great diversity of the recombination patterns evidences that the recombination between the subtypes B and F is frequent, reflecting a probable high rate of dual infection and the acquisition of advantageous characteristics for viral fitness.

Abstract 1669: Full length transcriptome sequencing of melanoma cell line complements long read sequencing assessment of genomic rearrangements

Abstract Transcriptome sequencing has proven to be an important tool for understanding the biological changes in cancer genomes including the consequences of structural rearrangements. Short read sequencing has been the method of choice, as the high throughput at low cost allows for transcript quantitation and the detection of even rare transcripts. However, the reads are generally too short to reconstruct complete isoforms. Conversely, long-read approaches can provide unambiguous full-length isoforms, but lower throughput has complicated quantitation and high RNA input requirements has made working with cancer samples challenging. Recently, the COLO 829 cell line was sequenced to 50-fold coverage with PacBio SMRT Sequencing. To validate and extend the findings from this effort, we have generated long-read transcriptome data using an updated PacBio Iso-Seq method, the results of which will be shared at the AACR 2019 General Meeting. With this complimentary transcriptome data, we demonstrate how recent innovations in the PacBio Iso-Seq method sample preparation and sequencing chemistry have made long-read sequencing of cancer transcriptomes more practical. In particular, library preparation has been simplified and throughput has increased. The improved protocol has reduced sample prep time from several days to one day while reducing the sample input requirements ten-fold. In addition, the incorporation of unique molecular identifier (UMI) tags into the workflow has improved the bioinformatics analysis. Yield has also increased, with v3 sequencing chemistry typically delivering &amp;gt; 30 Gb per SMRT Cell 1M. By integrating long and short read data, we demonstrate that the Iso-Seq method is a practical tool for annotating cancer genomes with high-quality transcript information. Citation Format: Elizabeth Tseng, Brendan Galvin, Ting Hon, Wigard P. Kloosterman, Meredith Ashby. Full length transcriptome sequencing of melanoma cell line complements long read sequencing assessment of genomic rearrangements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1669.

Taking insight into the gut microbiota of three spider species: No characteristic symbiont was found corresponding to the special feeding style of spiders.

Microorganisms in insect guts have been recognized as having a great impact on their hosts' nutrition, health, and behavior. Spiders are important natural enemies of pests, and the composition of the gut microbiota of spiders remains unclear. Will the bacterial taxa in spiders be same as the bacterial taxa in insects, and what are the potential functions of the gut bacteria in spiders? To gain insight into the composition of the gut bacteria in spiders and their potential function, we collected three spider species, Pardosa laura, Pardosa astrigera, and Nurscia albofasciata, in the field, and high‐throughput sequencing of the 16S rRNA V3 and V4 regions was used to investigate the diversity of gut microbiota across the three spider species. A total of 23 phyla and 150 families were identified in these three spider species. The dominant bacterial phylum across all samples was Proteobacteria. Burkholderia, Ralstonia, Ochrobactrum, Providencia, Acinetobacter, Proteus, and Rhodoplanes were the dominant genera in the guts of the three spider species. The relative abundances of Wolbachia and Rickettsiella detected in N. albofasciata were significantly higher than those in the other two spider species. The relative abundance of Thermus, Amycolatopsis, Lactococcus, Acinetobacter Microbacterium, and Koribacter detected in spider gut was different among the three spider species. Biomolecular interaction networks indicated that the microbiota in the guts had complex interactions. The results of this study also suggested that at the genus level, some of the gut bacteria taxa in the three spider species were the same as the bacteria in insect guts.

Characteristics of Envelope Genes in a Chinese Chronically HIV-1 Infected Patient With Broadly Neutralizing Activity.

Exploring the characteristics of the HIV-1 envelope glycoprotein (env) gene in a natural HIV-1 infected individual, with broadly neutralizing activity, may provide insight into the generation of such broadly neutralizing antibodies and initiate the design of an appropriate immunogen. Recently, a chronically HIV-1 infected patient with broadly neutralization activity was identified and a VRC01-class neutralizing antibody DRVIA7 (A7) was isolated from the patient. In the present study, 155 full length HIV-1 env gene fragments (including 68 functionally Env clones) were amplified longitudinally from the plasma of six time points spanning over 5 years in this donor. Viral features were analyzed by comparing Env clones of different time points, as well as 165 Chinese HIV-1 subtype B env sequences from HIV Sequence Database (Chinese B_database). Shorter V1 length, less potential glycan and a lower ratio of NXT: NXS in gp160 were observed in the first five time points compared to that from the last time points, as well that from the Chinese B_database. A sequence analysis and a neutralization assay of Env-pseudoviruses showed that the increasing diversity of env sequences in the patient was consistent with the appearance and maturation of A7 lineage antibodies. The potent neutralization activity and viruses that escaped from the neutralization of the concurrent autologous plasma, are consistent with higher residue variations at the antibody recognition sites. Almost all viruses from the plasmas were neutralization-resistant to VRC01 and A7 lineage antibodies. For a chronically HIV-1 infected individual over 10 years, we found that greater viral diversity, short V1 sequences and less potential N-linked glycosylation (PNGS) in V1, might be associated with the development of broadly neutralizing antibody responses.

Integrated Analysis of Human Milk Microbiota With Oligosaccharides and Fatty Acids in the CHILD Cohort.

Background: Human milk contains many bioactive components that are typically studied in isolation, including bacteria. We performed an integrated analysis of human milk oligosaccharides and fatty acids to explore their associations with milk microbiota.Methods: We studied a sub-sample of 393 mothers in the CHILD birth cohort. Milk was collected at 3–4 months postpartum. Microbiota was analyzed by 16S rRNA gene V4 sequencing. Oligosaccharides and fatty acids were analyzed by rapid high-throughput high performance and gas liquid chromatography, respectively. Dimension reduction was performed with principal component analysis for oligosaccharides and fatty acids. Center log-ratio transformation was applied to all three components. Associations between components were assessed using Spearman rank correlation, network visualization, multivariable linear regression, redundancy analysis, and structural equation modeling. P-values were adjusted for multiple comparisons. Key covariates were considered, including fucosyltransferase-2 (FUT2) secretor status of mother and infant, method of feeding (direct breastfeeding or pumped breast milk), and maternal fish oil supplement use.Results: Overall, correlations were strongest between milk components of the same type. For example, FUT2-dependent HMOs were positively correlated with each other, and Staphylococcus was negatively correlated with other core taxa. Some associations were also observed between components of different types. Using redundancy analysis and structural equation modeling, the overall milk fatty acid profile was significantly associated with milk microbiota composition. In addition, some individual fatty acids [22:6n3 (docosahexaenoic acid), 22:5n3, 20:5n3, 17:0, 18:0] and oligosaccharides (fucosyl-lacto-N-hexaose, lacto-N-hexaose, lacto-N-fucopentaose I) were associated with microbiota α diversity, while others (C18:0, 3′-sialyllactose, disialyl-lacto-N-tetraose) were associated with overall microbiota composition. Only a few significant associations between individual HMOs and microbiota were observed; notably, among mothers using breast pumps, Bifidobacterium prevalence was associated with lower abundances of disialyl-lacto-N-hexaose. Additionally, among non-secretor mothers, Staphylococcus was positively correlated with sialylated HMOs.Conclusion: Using multiple approaches to integrate and analyse milk microbiota, oligosaccharides, and fatty acids, we observed several associations between different milk components and microbiota, some of which were modified by secretor status and/or breastfeeding practices. Additional research is needed to further validate and mechanistically characterize these associations and determine their relevance to infant gut and respiratory microbiota development and health.

Comparison of PI-RADS v1 and v2 for multiparametric MRI detection of prostate cancer with whole-mount histological workup as reference standard

PurposeThe aim of this study was to compare Prostate Imaging Reporting and Data System (PI-RADS) versions v1 and v2 for the detection of prostate cancer (PCa) in multiparametric MRI (mpMRI) using whole-mount histological workup as reference standard. Material and methodsMRI data of 40 patients with positive transrectal ultrasound-guided biopsy were analyzed retrospectively by two blinded readers (5 and 4 years’ experience) with PI-RADS v1 and v2 for cancer-suspicious lesions. Prior to radical prostatectomy, patients had undergone IRB-approved mpMRI at 3 T according to PI-RADS recommendations: T2-weighted (T2w), diffusion-weighted (DWI) and dynamic contrast-enhanced (DCE) imaging. The reference standard was provided by whole-mount sections of the prostatectomy specimens. Versions v1 and v2 were compared with respect to sensitivity and positive predictive value (PPV) per lesion. Subgroups stratified by tumor location (peripheral vs. transition zone) and aggressiveness (high vs. low grade) were also analyzed. We also evaluated the concordance of the dominant MRI sequence in v2 (DWI or T2w) and the highest individual score under v1. Interobserver agreement for PI-RADS v1 and v2 was assessed by Cohen’s kappa statistics. ResultsReader 1 (R1) described 66 and Reader 2 (R2) 72 MRI lesions. The average Gleason score of 58 PCa lesions was 6.5 (range: 6 = 3 + 3 to 8 = 4 + 4), most of them (65.5%) located in the peripheral zone. PI-RADS v2 showed a trend towards lower sensitivities, but differences were not significant for both readers: R1 72.4% (v1) vs. 63.8% (v2) (P = 0.426) and R2 77.6% (v1) vs. 69.0% (v2) (P = 0.402). The trends were more pronounced in the transition zone and for low-grade cancers but remained insignificant (p-values from 0.313 to 0.691). Likewise, the apparent PPV differences, overall as well as in each zone, were not significant. Agreement between high-score v1 and dominant v2 sequence was 48% for R1 and 53% for R2. Cohen’s κ of PCa detection for two readers was 0.48 for both v1 and v2. ConclusionOur findings indicate that the simplified, zone-specific approach of PI-RADS v2 (2015) for MRI assessment of prostate cancer may not necessarily be better than the original v1 criteria (2012). In specific cases, a strict interpretation of v2 criteria may even lead to false-negative findings. Therefore, the current PI-RADS criteria should be reconsidered, despite the low statistical evidence here.

Humoral Response to the HIV-1 Envelope V2 Region in a Thai Early Acute Infection Cohort.

Reduced risk of HIV-1 infection correlated with antibody responses to the envelope variable 1 and 2 regions in the RV144 vaccine trial. To understand the relationship between antibody responses, V2 sequence, and structure, plasma samples (n = 16) from an early acute HIV-1 infection cohort from Thailand infected with CRF01_AE strain were analyzed for binding to V2 peptides by surface plasmon resonance. Five participants with a range of V2 binding responses at week 24 post-infection were further analyzed against a set of four overlapping V2 peptides that were designed based on envelope single-genome amplification. Antibody responses that were relatively consistent over the four segments of the V2 region or a focused response to the C-strand (residues 165–186) of the V2 region were observed. Viral escape in the V2 region resulted in significantly reduced antibody binding. Structural modeling indicated that the C-strand and the sites of viral variation were highly accessible in the open conformation of the HIV-1 Env trimer. V2 residues, 165–186 are preferentially targeted during acute infection. Residues 169–184 were also preferentially targeted by the protective immune response in the RV144 trial, thus emphasizing the importance of these residues for vaccine design.

Encrustations on ureteral stents from patients without urinary tract infection reveal distinct urotypes and a low bacterial load

BackgroundCurrent knowledge of the urinary tract microbiome is limited to urine analysis and analysis of biofilms formed on Foley catheters. Bacterial biofilms on ureteral stents have rarely been investigated, and no cultivation-independent data are available on the microbiome of the encrustations on the stents.ResultsThe typical encrustations of organic and inorganic urine-derived material, including microbial biofilms formed during 3–6 weeks on ureteral stents in patients treated for kidney and ureteral stones, and without reported urinary tract infection at the time of stent insertion, were analysed. Next-generation sequencing of the 16S rRNA gene V3–V4 region revealed presence of different urotypes, distinct bacterial communities. Analysis of bacterial load was performed by combining quantification of 16S rRNA gene copy numbers by qPCR with microscopy and cultivation-dependent analysis methods, which revealed that ureteral stent biofilms mostly contain low numbers of bacteria. Fluorescence microscopy indicates the presence of extracellular DNA. Bacteria identified in biofilms by microscopy had mostly morphogenic similarities to gram-positive bacteria, in few cases to Lactobacillus and Corynebacterium, while sequencing showed many additional bacterial genera. Weddellite crystals were absent in biofilms of patients with Enterobacterales and Corynebacterium-dominated microbiomes.ConclusionsThis study provides novel insights into the bacterial burden in ureteral stent encrustations and the urinary tract microbiome. Short-term (3–6 weeks) ureteral stenting is associated with a low load of viable and visible bacteria in ureteral stent encrustations, which may be different from long-term stenting. Patients could be classified according to different urotypes, some of which were dominated by potentially pathogenic species. Facultative pathogens however appear to be a common feature in patients without clinically manifested urinary tract infection.Trial registrationClinicalTrials.gov, NCT02845726. Registered on 30 June 2016—retrospectively registered.

Overfeeding a High-Fat Diet Promotes Sex-Specific Alterations on the Gut Microbiota of the Zebrafish (Danio rerio).

Diet modulates the gut microbiota and is one of the main factors promoting obesity and overweight. In the present study, we investigated the effect of a high-fat diet (HFD) on the gut microbiota of the zebrafish (Danio rerio). Fish were separated into three groups and fed in different regimes: low fat, high fat, and high fat overfed; the experiments were performed on males and females separately. We analyzed more than 2.6 million sequences of variable region V3 of the 16S rRNA gene generated by the Illumina Miniseq platform, clustered to 97% similarity with vsearch and classified with the EzBioCloud database. The weight gain, condition factor (K), and body mass index were calculated as indicators of obesity. Multivariate analysis (PERMANOVA and ANOSIM) and diversity indices (Shannon and Dominance) revealed that overfeeding a HFD disturbs the gut microbiota differently in males and females suggesting that sex is a significant factor (p < 0.05) for the composition of the gut microbiota of zebrafish. The results also indicate that a HFD provided in a basal caloric regime does not promote obesity or alterations in the gut microbiota.

Fecal Microbiome, Metabolites, and Stem Cell Transplant Outcomes: A Single-Center Pilot Study.

BackgroundAccumulating evidence suggests that the intestinal microbiome may dramatically affect the outcomes of hematopoietic stem cell transplant (HSCT) recipients. Providing 16S ribosomal RNA based microbiome characterization in a clinically actionable time frame is currently problematic. Thus, determination of microbial metabolites as surrogates for microbiome composition could offer practical biomarkers.MethodsLongitudinal fecal specimens (n = 451) were collected from 44 patients before HSCT through 100 days after transplantation, as well as 1-time samples from healthy volunteers (n = 18) as controls. Microbiota composition was determined using 16S ribosomal RNA V4 sequencing. Fecal indole and butyrate levels were determined using liquid chromatography tandem mass spectrometry.ResultsAmong HSCT recipients, both fecal indole and butyrate levels correlated with the Shannon diversity index at baseline (P = .02 and P = .002, respectively) and directly after transplantation (P = .006 and P < .001, respectively). Samples with high butyrate levels were enriched for Clostridiales, whereas samples containing high indole were also enriched for Bacteroidales. A lower Shannon diversity index at the time of engraftment was associated with increased incidence of acute intestinal graft-vs-host disease (iGVHD) (P = .02) and transplant-related deaths (P = .03). Although fecal metabolites were not associated with acute iGVHD or overall survival, patients contracting bloodstream infections within 30 days after transplantation had significantly lower levels of fecal butyrate (P = .03).ConclusionsLongitudinal analysis of fecal microbiome and metabolites after HSCT identified butyrate and indole as potential surrogate markers for microbial diversity and specific taxa. Further studies are needed to ascertain whether fecal metabolites can be used as biomarkers of acute iGVHD or bacteremia after HSCT.

Feeding practice influences gut microbiome composition in very low birth weight preterm infants and the association with oxidative stress: A prospective cohort study

Knowledge about the development of the preterm infant gut microbiota is emerging and is critical to their health. Very-low-birth-weight (VLBW; birth weight, <1500 g) infants usually have special dietary needs while showing increased oxidative stress related to intensive care. This prospective cohort study assessed the effect of feeding practice on gut microbiome development and oxidative stress in preterm infants.Fecal samples were collected from each infant in the early (1–2 weeks of enteral feeding) and late (2–4 weeks of enteral feeding) feeding stages. We performed high-throughput sequencing of V3–V4 regions of the 16S rRNA gene to analyze the fecal microbiome composition of 20 VLBW preterm infants and to determine the association of gut bacterial composition with feeding practice using an oxidative stress marker (urinary F2-isoprostane). Our results showed that feeding practices in the late stage significantly influenced the gut microbiome composition and oxidative stress in preterm infants. Preterm infants fed human milk + human milk fortifier and only formula diets showed a significant increase in F2-isoprostane levels (P < 0.05) compared with those fed human milk + formula diet. The gut microbiome of the infants fed the human milk + Human milk fortifier diet showed the lower relative abundance of Veillonella (P < 0.05) compared with that of the infants fed the human milk + formula diet. The gut microbiome of the infants fed the only formula diet showed the lowest microbial diversity and the highest relative abundance of Terrisporobacter (P < 0.05) compared with the gut microbiome of the infants fed the other diets. Correlation network analysis showed that urinary F2-isoprostane level was positively correlated with Terrisporobacter and Enterobacteriaceae abundance (P < 0.05) in the preterm infants.In conclusion, these data suggest that feeding practice affects the bacterial diversity and composition in the gut microbiome and is associated with oxidative stress in VLBW preterm infants.

Integrase strand transfer inhibitor-based regimen is related with a limited HIV-1 V3 loop evolution in clinical practice.

Integrase-strand-transfer inhibitors (INSTIs) are known to rapidly reduce HIV-1 plasma viral load, replication cycles, and new viral integrations, thus potentially limiting viral evolution. Here, we assessed the role of INSTIs on HIV-1 V3 evolution in a cohort of 89 HIV-1-infected individuals starting an INSTI- (N = 41, [dolutegravir: N = 1; elvitegravir: N = 3; raltegravir: N = 37]) or a non-INSTI-based (N = 48) combined antiretroviral therapy (cART), with two plasma RNA V3 genotypic tests available (one before [baseline] and one during cART). V3 sequences were analysed for genetic distance (Tajima-Nei model) and positive selection (dN/dS ratio). Individuals were mainly infected by B subtype (71.9%). Median (interquartile-range, IQR) plasma viral load and CD4 + T cell count at baseline were 4.8 (3.5-5.5) log10 copies/mL and 207 (67-441) cells/mm3, respectively. Genetic distance (median, IQR) between the V3 sequences obtained during cART and those obtained at baseline was 0.04 (0.01-0.07). By considering treatment, genetic distance was significantly lower in INSTI-treated than in non-INSTI-treated individuals (median [IQR]: 0.03[0.01-0.04] vs. 0.05[0.02-0.08], p = 0.026). In line with this, a positive selection (defined as dN/dS ≥ 1) was observed in 36.6% of V3 sequences belonging to the INSTI-treated group and in 56.3% of non-INSTI group (p = 0.05). Multivariable logistic regression confirmed the independent correlation of INSTI-based regimens with a lower probability of both V3 evolution (adjusted odds-ratio: 0.35 [confidence interval (CI) 0.13-0.88], p = 0.027) and positive selection (even if with a trend) (adjusted odds-ratio: 0.46 [CI 0.19-1.11], p = 0.083). Overall, this study suggests a role of INSTI-based regimen in limiting HIV-1 V3 evolution over time. Further studies are required to confirm these findings.

Guanidine thiocyanate solution facilitates sample collection for plant rhizosphere microbiome analysis.

The interactions between rhizosphere microorganisms and plants are important for the health and development of crops. Analysis of plant rhizosphere bacterial compositions, particularly of those with resistance to biotic/abiotic stresses, may improve their applications in sustainable agriculture. Large-scale rhizosphere samplings in the field are usually required; however, such samples, cannot be immediately frozen. We found that the storage of samples at room temperature for 2 days leads to a considerable reduction in the operational taxonomic unit (OTU) number and the indices of bacterial alpha-diversity of rhizosphere communities. In this study, in order to overcome these problems, we established a method using guanidine thiocyanate (GTC) solution for the preservation of rhizosphere samples after their collection. This method allowed the maintenance of the samples for at least 1 day at room temperature prior to their cryopreservation and was shown to be compatible with conventional DNA isolation protocols. Illumina sequencing of V3 and V4 hypervariable regions of the 16S rRNA gene was used to assess the feasibility and reliability of this method, and no significant differences were observed in the number of OTUs and in the Chao and Shannon indices between samples stored at −70 °C and those stored in GTC solution. Moreover, the representation of Pseudomonas spp. in samples stored in GTC solution was not significantly different from that in samples stored at −70 °C, as determined by real-time quantitative polymerase chain reaction (p > 0.05). Both types of samples were shown to cluster together according to principal coordinate analysis. Furthermore, GTC solution did not affect the bacterial taxon profiles at different storage periods compared with those observed when storing the samples below −70 °C. Even incubation of thawed samples (frozen at −70 °C) for 15 min at room temperature induced minor changes in the bacterial composition. Taken together, our results demonstrated that GTC solution may provide a reliable alternative for the preservation of rhizosphere samples in the field.

Age-related changes in the marmoset gut microbiome.

The gut microbiome is known to play a significant role in human health but its role in aging remains unclear. The objective of this study was to compare the gut microbiome composition between young adult and geriatric non-human primates (marmosets) as a model of human health and disease. Stool samples were collected from geriatric (8+ years) and young adult males (2-5 years). Stool 16S ribosomal RNA V4 sequences were amplified and sequenced on the Illumina MiSeq platform. Sequences were clustered into operational taxonomic units and classified via Mothur's Bayesian classifier referenced against the Greengenes database. A total of 10 young adult and 10 geriatric marmosets were included. Geriatric marmosets had a lower mean Shannon diversity compared with young marmosets (3.15 vs. 3.46; p = 0.0191). Geriatric marmosets had a significantly higher mean abundance of Proteobacteria (0.22 vs. 0.09; p = 0.0233) and lower abundance of Firmicutes (0.15 vs. 0.19; p = 0.0032) compared with young marmosets. Geriatric marmosets had a significantly higher abundance of Succinivibrionaceae (0.16 vs. 0.01; p = 0.0191) and lower abundance of Porphyromonadaceae (0.07 vs. 0.11; p = 0.0494). In summary, geriatric marmosets had significantly altered microbiome diversity and composition compared with young adult marmosets. Further studies are needed to test microbiome-targeted therapies to improve healthspan and lifespan.