The human microbiome has emerged as a major player in regulating human health and disease. Translation studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that shotgun whole genome sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

Rationale: T-regulatory cells (Tregs) are potent immunomodulators in allergic asthma. Objectives: We evaluated the functional effects of Tregs by adoptively transferring naturally occurring CD4 1 CD25 1 Tregs (NTregs) and CD4 1 CD25 2 inducible Tregs (iTregs) from lung and spleens of green fluorescent protein (GFP)-transgenic Balb/c mice into cockroachsensitized and -challenged mice. Methods: GFP-labeled NTregs and iTregs were adoptively transferred into cockroach-sensitized and -challenged mice. Airway hyperresponsiveness (AHR) to methacholine was examined using a single-chamber, whole-body plethysmograph and invasive tracheostomy. Measurements and Main Results: Adoptive transfer of either NTregs or iTregs from lung or spleen reversed airway inflammation and AHR to methacholine, and the effect lasted for at least 4 weeks. GFP-labeled iTregs up-regulated CD25 and forkhead-winged transcriptional factor box protein 3 and migrated to lymph node and lung. Lung CD4 1 CD25 1 T cells isolated from each group of recipient mice were inducible costimulatory molecule-high and programmed death (PD)-1-positive; however, higher expression of PD-1 was found in the spleen iTregs (S25 2 ) and lung iTregs (L25 2 ) groups. Higher levels of transforming growth factor-b and IL-10 mRNA transcripts and bronchoalveolar lavage fluid IL-10 and INF-g levels were observed in lung CD4 1 CD25 1 cells from the L25 2 and S25 2 cell-recipient mice than from lung NTregs (L25 1 ) and spleen NTregs (S25 1 ) cell-recipient mice. Adoptive transfer of either cell type significantly reduced bronchoalveolar lavage fluid IL-4, IL-5, and IL-13 levels. Conclusions: Tregs reverse AHR and airway inflammation; however iTregs that differentiated into IL-10-producing CD4 1 type 1 cells in the lung exert their suppressive activity likely by higher levels of transforming growth factor-b, IL-10, IFN-g, and elevated levels of PD-1 compared with NTregs. Hence, PD-1 may be a conduit for reversing AHR by Tregs and a plausible target for treating asthma.

Recent studies have established that the human urine contains a complex microbiome, including a virome about which little is known. Following immunosuppression in kidney transplant patients, BK polyomavirus (BKV) has been shown to induce nephropathy (BKVN), decreasing graft survival. In this study we investigated the urine virome profile of BKV+ and BKV− kidney transplant recipients. Virus-like particles were stained to confirm the presence of VLP in the urine samples. Metagenomic DNA was purified, and the virome profile was analyzed using metagenomic shotgun sequencing. While the BK virus was predominant in the BKV+ group, it was also found in the BKV− group patients. Additional viruses were also detected in all patients, notably including JC virus (JCV) and Torque teno virus (TTV) and interestingly, we detected multiple subtypes of the BKV, JCV and TTV. Analysis of the BKV subtypes showed that nucleotide polymorphisms were detected in the VP1, VP2 and Large T Antigen proteins, suggesting potential functional effects for enhanced pathogenicity. Our results demonstrate a complex urinary virome in kidney transplant patients with multiple viruses with several distinct subtypes warranting further analysis of virus subtypes in immunosuppressed hosts.

Recent studies have established that a complex community of microbes colonize the human urinary tract; however their role in kidney transplant patients treated with prophylactic antibiotics remains poorly investigated. Our aim was to investigate the urinary microbiome of kidney transplant recipients. Urine samples from 21 patients following kidney transplantation and 8 healthy controls, were collected. All patients received prophylactic treatment with the antibiotic trimethoprim/sulfamethoxazole. Metagenomic DNA was isolated from urine samples, sequenced using metagenomics shotgun sequencing approach on Illumina HiSeq2000 platform, and analyzed for microbial taxonomic and functional annotations. Our results demonstrate that the urine microbiome of kidney transplants was markedly different at all taxonomic levels from phyla to species, had decreased microbial diversity and increased abundance of potentially pathogenic species compared to healthy controls. Specifically, at the phylum level we detected a significant decrease in Actinobacteria and increase in Firmicutes due to increases in Enterococcus faecalis. In addition, there was an increase in the Proteobacteria due to increases in E. coli. Analysis of predicted functions of the urinary metagenome revealed increased abundance of enzymes in the folate pathway including dihydrofolate synthase that are not inhibited by trimethoprim/ sulfamethoxazole, but can augment folate metabolism. This report characterizes the urinary microbiome of kidney transplants using shotgun metagenomics approach. Our results indicate that the urinary microbiota may be modified in the context of prophylactic antibiotics, indicating that a therapeutic intervention may shift the urinary microbiota to select bacterial species with increased

BackgroundDifferences in asthma severity may be related to inflammation in the airways. The lower airway microbiota has been associated with clinical features such as airway obstruction, symptom control, and response to corticosteroids.ObjectiveTo assess the relationship between local airway inflammation, severity of disease, and the lower airway microbiota in atopic asthmatics.MethodsA cohort of young adult, atopic asthmatics with intermittent or mild/moderate persistent symptoms (n = 13) were assessed via bronchoscopy, lavage, and spirometry. These individuals were compared to age matched non-asthmatic controls (n = 6) and to themselves after six weeks of treatment with fluticasone propionate (FP). Inflammation of the airways was assessed via a cytokine and chemokine panel. Lower airway microbiota composition was determined by metagenomic shotgun sequencing.ResultsUnsupervised clustering of cytokines and chemokines prior to treatment with FP identified two asthmatic phenotypes (AP), termed AP1 and AP2, with distinct bronchoalveolar lavage inflammatory profiles. AP2 was associated with more obstruction, compared to AP1. After treatment with FP reduced MIP-1β and TNF-α and increased IL-2 was observed. A module of highly correlated cytokines that include MIP-1β and TNF-α was identified that negatively correlated with pulmonary function. Independently, IL-2 was positively correlated with pulmonary function. The airway microbiome composition correlated with asthmatic phenotypes. AP2, prior to FP treatment, was enriched with Streptococcus pneumoniae. Unique associations between IL-2 or the cytokine module and the microbiota composition of the airways were observed in asthmatics subjects prior to treatment but not after or in controls.ConclusionThe underlying inflammation in atopic asthma is related to the composition of microbiota and is associated with severity of airway obstruction. Treatment with inhaled corticosteroids was associated with changes in the airway inflammatory response to microbiota.