Genomic characterization of the C. tuberculostearicum species complex, a prominent member of the human skin microbiome.

IMPORTANCE: Amplicon sequencing data combined with isolate whole genome sequencing have expanded our understanding of Corynebacterium on the skin. Healthy human skin is colonized by a diverse collection of Corynebacterium species, but Corynebacterium tuberculostearicum predominates on many skin sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex, including a potentially new species we term Corynebacterium hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomic analysis, and growth in skin-like media. This isolate collection and high-quality genome resource set the stage for developing engineered strains for both basic and translational clinical studies.

Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. OBJECTIVE: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. METHODS: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. RESULTS: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. CONCLUSION: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.

Genomic characterization of the C. tuberculostearicum species complex, a ubiquitous member of the human skin microbiome.

Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A & B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates are largely syntenic and carry a diversity of methylation patterns, plasmids and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species.

Author Correction: Interaction between the microbiome and TP53 in human lung cancer.

Following publication of the original paper [1], the authors submitted a new Additional file 5 to replace the one containing formatting issues. The updated Additional file 5 is published in this correction.

Interaction between the microbiome and TP53 in human lung cancer.

BACKGROUND: Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort. RESULTS: Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas. CONCLUSIONS: The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization.

UNLABELLED: Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists' actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms. IMPORTANCE: In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing.

UNLABELLED: Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant "genomes" are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. IMPORTANCE: The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community.

Commensal-dendritic-cell interaction specifies a unique protective skin immune signature.

The skin represents the primary interface between the host and the environment. This organ is also home to trillions of microorganisms that play an important role in tissue homeostasis and local immunity. Skin microbial communities are highly diverse and can be remodelled over time or in response to environmental challenges. How, in the context of this complexity, individual commensal microorganisms may differentially modulate skin immunity and the consequences of these responses for tissue physiology remains unclear. Here we show that defined commensals dominantly affect skin immunity and identify the cellular mediators involved in this specification. In particular, colonization with Staphylococcus epidermidis induces IL-17A(+) CD8(+) T cells that home to the epidermis, enhance innate barrier immunity and limit pathogen invasion. Commensal-specific T-cell responses result from the coordinated action of skin-resident dendritic cell subsets and are not associated with inflammation, revealing that tissue-resident cells are poised to sense and respond to alterations in microbial communities. This interaction may represent an evolutionary means by which the skin immune system uses fluctuating commensal signals to calibrate barrier immunity and provide heterologous protection against invasive pathogens. These findings reveal that the skin immune landscape is a highly dynamic environment that can be rapidly and specifically remodelled by encounters with defined commensals, findings that have profound implications for our understanding of tissue-specific immunity and pathologies.

Complete Genome Sequence of a Klebsiella pneumoniae Isolate with Chromosomally Encoded Carbapenem Resistance and Colibactin Synthesis Loci.

Klebsiella pneumoniae is an important nosocomial pathogen, and multidrug-resistant strains have become a worldwide concern. Here, we report the complete genome of a K. pneumoniae isolate with chromosomally integrated blaKPC genes and a colibactin synthesis locus.

Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae.

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care-associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.

Topographic diversity of fungal and bacterial communities in human skin.

Traditional culture-based methods have incompletely defined the microbial landscape of common recalcitrant human fungal skin diseases, including athlete's foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms and provides a home for diverse commensal microbiota. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also have major roles in microbial community stability, human health and disease. Genomic methodologies to identify fungal species and communities have been limited compared with those that are available for bacteria. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes. Here we sequenced and analysed fungal communities of 14 skin sites in 10 healthy adults. Eleven core-body and arm sites were dominated by fungi of the genus Malassezia, with only species-level classifications revealing fungal-community composition differences between sites. By contrast, three foot sites--plantar heel, toenail and toe web--showed high fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that physiologic attributes and topography of skin differentially shape these two microbial communities. These results provide a framework for future investigation of the contribution of interactions between pathogenic and commensal fungal and bacterial communities to the maintainenace of human health and to disease pathogenesis.

Probiotic/prebiotic supplementation of antiretrovirals improves gastrointestinal immunity in SIV-infected macaques.

HIV infection results in gastrointestinal (GI) tract damage, microbial translocation, and immune activation, which are not completely ameliorated with suppression of viremia by antiretroviral (ARV) therapy. Furthermore, increased morbidity and mortality of ARV-treated HIV-infected individuals is associated with these dysfunctions. Thus, to enhance GI tract physiology, we treated SIV-infected pigtail macaques with ARVs, probiotics, and prebiotics or with ARVs alone. This synbiotic treatment resulted in increased frequency and functionality of GI tract APCs, enhanced reconstitution and functionality of CD4+ T cells, and reduced fibrosis of lymphoid follicles in the colon. Thus, ARV synbiotic supplementation in HIV-infected individuals may improve GI tract immunity and thereby mitigate inflammatory sequelae, ultimately improving prognosis.

Compartmentalized control of skin immunity by resident commensals.

Intestinal commensal bacteria induce protective and regulatory responses that maintain host-microbial mutualism. However, the contribution of tissue-resident commensals to immunity and inflammation at other barrier sites has not been addressed. We found that in mice, the skin microbiota have an autonomous role in controlling the local inflammatory milieu and tuning resident T lymphocyte function. Protective immunity to a cutaneous pathogen was found to be critically dependent on the skin microbiota but not the gut microbiota. Furthermore, skin commensals tuned the function of local T cells in a manner dependent on signaling downstream of the interleukin-1 receptor. These findings underscore the importance of the microbiota as a distinctive feature of tissue compartmentalization, and provide insight into mechanisms of immune system regulation by resident commensal niches in health and disease.

Inhibition of transforming growth factor-ß restores endothelial thromboresistance in vein grafts.

BACKGROUND: Thrombosis is a major cause of the early failure of vein grafts (VGs) implanted during peripheral and coronary arterial bypass surgeries. Endothelial expression of thrombomodulin (TM), a key constituent of the protein C anticoagulant pathway, is markedly suppressed in VGs after implantation and contributes to local thrombus formation. While stretch-induced paracrine release of transforming growth factor-ß (TGF-ß) is known to negatively regulate TM expression in heart tissue, its role in regulating TM expression in VGs remains unknown. METHODS: Changes in relative mRNA expression of major TGF-ß isoforms were measured by quantitative polymerase chain reaction (qPCR) in cultured human saphenous vein smooth muscle cells (HSVSMCs) subjected to cyclic stretch. To determine the effects of paracrine release of TGF-ß on endothelial TM mRNA expression, human saphenous vein endothelial cells (HSVECs) were co-cultured with stretched HSVSMCs in the presence of 1D11, a pan-neutralizing TGF-ß antibody, or 13C4, an isotype-control antibody. Groups of rabbits were then administered 1D11 or 13C4 and underwent interpositional grafting of jugular vein segments into the carotid circulation. The effect of TGF-ß inhibition on TM gene expression was measured by qPCR; protein C activating capacity and local thrombus formation were measured by in situ chromogenic substrate assays; and VG remodeling was assessed by digital morphometry. RESULTS: Cyclic stretch induced TGF-ß(1) expression in HSVSMCs by 1.9 ± 0.2-fold (P < .001) without significant change in the expressions of TGF-ß(2) and TGF-ß(3). Paracrine release of TGF-ß(1) by stretched HSVSMCs inhibited TM expression in stationary HSVECs placed in co-culture by 57 ± 12% (P = .03), an effect that was abolished in the presence of 1D11. Similarly, TGF-ß(1) was the predominant isoform induced in rabbit VGs 7 days after implantation (3.5 ± 0.4-fold induction; P < .001). TGF-ß(1) protein expression localized predominantly to the developing neointima and coincided with marked suppression of endothelial TM expression (16% ± 2% of vein controls; P < .03), a reduction in situ activated protein C (APC)-generating capacity (53% ± 9% of vein controls; P = .001) and increased local thrombus formation (3.7 ± 0.8-fold increase over vein controls; P < .01). External stenting of VGs to limit vessel distension significantly reduced TGF-ß(1) induction and TM downregulation. Systemic administration of 1D11 also effectively prevented TM downregulation, preserved APC-generating capacity, and reduced local thrombus in rabbit VGs without observable effect on neointima formation and other morphometric parameters 6 weeks after implantation. CONCLUSION: TM downregulation in VGs is mediated by paracrine release of TGF-ß(1) caused by pressure-induced vessel stretch. Systemic administration of an anti-TGF-ß antibody effectively prevented TM downregulation and preserved local thromboresistance without negative effect on VG remodeling.

A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis.

Two common inflammatory skin disorders with impaired barrier, atopic dermatitis (AD) and psoriasis, share distinct genetic linkage to the Epidermal Differentiation Complex (EDC) locus on 1q21. The EDC is comprised of tandemly arrayed gene families encoding proteins involved in skin cell differentiation. Discovery of semi-dominant mutations in filaggrin (FLG) associated with AD and a copy number variation within the LCE genes associated with psoriasis provide compelling evidence for the role of EDC genes in the pathogenesis of these diseases. To date, little is known about the potentially complex regulatory landscape within the EDC. Here, we report a computational approach to identify conserved non-coding elements (CNEs) in the EDC queried for regulatory function. Coordinate expression of EDC genes during mouse embryonic skin development and a striking degree of synteny and linearity in the EDC locus across a wide range of mammalian (placental and marsupial) genomes suggests an evolutionary conserved regulatory milieu in the EDC. CNEs identified by comparative genomics exhibit dynamic regulatory activity (enhancer or repressor) in differentiating or proliferating conditions. We further demonstrate epidermal-specific, developmental in vivo enhancer activities (DNaseI and transgenic mouse assays) in CNEs, including one within the psoriasis-associated deletion, LCE3C_LCE3B-del. Together, our multidisciplinary study features a network of regulatory elements coordinating developmental EDC gene expression as an unexplored resource for genetic variants in skin diseases.

Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors.

OBJECTIVE: Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathological thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We investigated the effects of proteasome inhibitors on endothelial thrombomodulin expression and function. METHODS AND RESULTS: Proteasome inhibitors as a class markedly induced the expression of thrombomodulin and enhanced the protein C activating capacity of endothelial cells. Thrombomodulin upregulation was independent of NF-kappaB signaling, a principal target of proteasome inhibitors, but was instead a direct consequence of increased expression of the Krüppel-like transcription factors, KLF2 and KLF4. These effects were confirmed in vivo, where systemic administration of a proteasome inhibitor enhanced thrombomodulin expression that was paralleled by changes in the expression of KLF2 and KLF4. CONCLUSIONS: These findings identify a novel mechanism of action of proteasome inhibitors that may help to explain their clinically observed thromboprotective effects.

Topographical and temporal diversity of the human skin microbiome.

Human skin is a large, heterogeneous organ that protects the body from pathogens while sustaining microorganisms that influence human health and disease. Our analysis of 16S ribosomal RNA gene sequences obtained from 20 distinct skin sites of healthy humans revealed that physiologically comparable sites harbor similar bacterial communities. The complexity and stability of the microbial community are dependent on the specific characteristics of the skin site. This topographical and temporal survey provides a baseline for studies that examine the role of bacterial communities in disease states and the microbial interdependencies required to maintain healthy skin.

Hemodynamic modulation of endocardial thromboresistance.

BACKGROUND: Patients with heart failure are at increased risk for thromboembolic events, including stroke. Historically attributed to blood stasis, little is known about the adverse effects of elevated chamber filling pressure on endocardial function, which could predispose to intracardiac thrombus formation. METHODS AND RESULTS: We investigated changes in the expression of thrombomodulin, a key component of the anticoagulant protein C pathway, in rats subjected to acute atrial pressure overload caused by aortic banding. Acute elevation of left atrial filling pressure, without an associated decline in ventricular systolic function, caused a 70% inhibition of atrial endocardial thrombomodulin expression and resulted in increased local thrombin generation. Targeted restoration of atrial thrombomodulin expression with adenovirus-mediated gene transfer successfully reduced thrombin generation to baseline levels. In vitro co-culture studies revealed that thrombomodulin downregulation is caused by the paracrine release of transforming growth factor-beta from cardiac connective tissue in response to mechanical stretch. This was confirmed in vivo by administration of a neutralizing transforming growth factor-beta antibody, which effectively prevented thrombomodulin downregulation during acute pressure overload. CONCLUSIONS: These findings suggest that increased hemodynamic load adversely affects endocardial function and is a potentially important contributor to thromboembolus formation in heart failure.

Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B.

Inflammation and thrombosis are increasingly recognized as interrelated biologic processes. Endothelial cell expression of thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is potently inhibited by inflammatory cytokines. Because the mechanism underlying this effect is largely unknown, we investigated a potential role for the inflammatory transcription factor nuclear factor-kappa B (NF-kappaB). Blocking NF-kappaB activation effectively prevented cytokine-induced down-regulation of TM, both in vitro and in a mouse model of tumor necrosis factor-alpha (TNF-alpha)-mediated lung injury. Although the TM promoter lacks a classic NF-kappaB consensus site, it does contain tandem Ets transcription factor binding sites previously shown to be important for both constitutive TM gene expression and cytokine-induced repression. Using electrophoretic mobility shift assay and chromatin immunoprecipitation, we found that multiple Ets species bind to the TNF-alpha response element within the TM promoter. Although cytokine exposure did not alter Ets factor binding, it did reduce binding of p300, a coactivator required by Ets for full transcriptional activity. Overexpression of p300 also prevented TM repression by cytokines. We conclude that NF-kappaB is a critical mediator of TM repression by cytokines. Further evidence suggests a mechanism involving competition by NF-kappaB for limited pools of the transcriptional coactivator p300 necessary for TM gene expression.