OBJECTIVES To investigate the probiotic Escherichia coli Nissle 1917 (EcN) in canine idiopathic diarrhea and urinary tract infections. ANIMALS/SAMPLES The utility of EcN was explored in a 3-phase study from March 2017 to June 2020. Eighty-nine dogs with idiopathic diarrhea were included in phase 1, 3 healthy dogs were included in phase 2, and uropathogenic E coli (UPEC) isolates from 38 dogs with urinary tract infections were included in phase 3. PROCEDURES In phase 1, dogs with diarrhea were prospectively enrolled in a randomized study to receive EcN (108 EcN bacteria/mL; < 10 kg received 5 mL/dose, 10 to 25 kg received 10 mL/dose, or > 25 kg received 15 mL/dose) or placebo for 3 days, followed by a 15-day observation phase. In phase 2, healthy dogs received EcN as described in phase 1, with feces analyzed for E coli populations and microbiome composition at days 0, 3, and 7. In phase 3, EcN efficacy was tested by in vitro plate assay against UPEC isolates. RESULTS Median duration of abnormal stool consistency, time to response, and duration of diarrhea were shorter for dogs that received EcN (5.0, 3.0, and 2.0 days, respectively) versus the placebo (7.0, 5.0, and 4.0 days, respectively) (P = .21, P = .05, and P = .039, respectively). EcN induced shifts in E coli diversity in healthy dogs while having minimal impact on overall microbiome structure. Furthermore, 68% of the canine UPEC isolates were susceptible to EcN in vitro. CLINICAL RELEVANCE EcN improved the treatment of idiopathic diarrhea, colonized the gastrointestinal tract during the trial, and displayed in vitro competition with UPEC.

IntroductionOrthodontic treatment interferes with oral hygiene and promotes plaque retention, which leads to gingival inflammation and enamel demineralization. Although removable clear aligners (CAs) are designed to improve oral hygiene compared with fixed appliances (FAs), comprehensive studies comparing their respective effects on the oral microbiome are limited. This longitudinal study investigated the microbial changes during orthodontic treatment with FA and CA in correlation with clinical parameters.MethodsClinical parameters and supragingival plaque were collected from 12 study participants for the FA or CA treatment groups at baseline and at least twice at the 1, 3, 6, and 12-month follow-up appointments. The plaque was also harvested from the aligner tray for the CA group. Microbiome composition was determined via 16S rRNA gene sequencing, compared between groups, and correlated with clinical parameters.ResultsPlaque (PI) and gingival indexes (GI) increased significantly in the FA but not the CA group. Beta but not alpha diversities of the microbial communities were distinct between the 2 treatment groups, even though genus-level differences were not significant except for Leptotrichia. The CA tray harbors a unique plaque community. Elevated PI and GI in the FA group correlated with a higher abundance of disease-related genera.ConclusionsOrthodontic treatments trigger appliance-related plaque community shifts from baseline, and the CA tray environment attracts distinct microbial communities. In comparison with FA, the use of CA resulted in better oral health index outcomes, which is reflected by the corresponding PI and GI-associated oral microbial communities.

Steinernema hermaphrodtium is a hermaphroditic species of entomopathogenic (insect-parasitic) nematode that associates with symbiotic bacterium Xenorhabdus griffiniae . During transmission stage, the developmentally-arrested and non-feeding infective juveniles (IJs) carry their symbiotic bacteria in the intestinal pockets, disperse from the insect cadaver and invade another insect host. Once entering an insect, the nematodes go through a process termed IJ recovery: they start feeding, release their symbiotic bacteria which help to kill the insect, and resume reproductive development. Our preliminary data show that the native symbiotic bacteria are better at reducing S. hermaphroditum IJs dispersal behavior in comparison to other bacterial food sources, and they specifically induce IJ recovery. These phenomena suggest that S. hermaphroditum discerns its native symbiont in a species (or strain)-specific manner among environmental microbes during infective (transmission) stage. We hypothesize that host nematode senses particular signaling molecules from the bacterial partner possibly through a neuroendocrine pathway. To better characterize the molecular mechanisms underlying the symbiont discerning behaviors in S. hermaphroditum, we developed a protocol for EMS-based forward genetic screen and CRISPR-Cas9 genome-editing as reverse genetic tools in the nematode host. These host genetic tools will be paired with genetics in the symbiotic bacteria to comprehensively investigate nematode-bacteria recognition. Our research show that the Steinernema-Xenorhabdus symbiotic pair has great potential to reveal molecular pathways underlying naturally occurring mutualistic symbiosis. The bacterial cell wall is comprised of membrane layers and a unique macromolecule called peptidoglycan (PG). PG can be found in nearly all bacterial species and is essential for cell viability. Bacterial growth requires PG biosynthesis. As a critical cell-growth regulator, PG has become a research target for understanding bacterial growth and inhibition. For instance, PG is a valuable target of antibiotics, as these antibiotics have relatively low toxicity to the host because PG doesn't exist in eukaryotic cells. Understanding PG biosynthesis, therefore, is essential for understanding both pathogenesis and mutualism, and for developing technologies to prevent bacterial infection and aid beneficial microbiota. However, much is still unknown about PG synthesis. PG is composed of glycan strands crosslinked by stem-peptide chains. The glycan strands are made of alternating N -acetylmuramic acid (MurNAc) and N acetylglucosamine (GlcNAc) residues connected by beta-1,4-glycosidic linkages. The MurNAc residues have a pentapeptide sidechain, or stem peptide, that forms crosslinks between glycan strands. Although the chemical component of PG is highly conserved, the stem peptides and crosslinking between glycan strands vary among and even within species. In the symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes , the bacteria exist within a specialized light organ of the squid, producing bioluminescence to assist the squid in its nocturnal behaviors. We studied legume nodule microbiomes from both alfalfa ( Medicago sativa ) and a closely related, naturalized California legume ( M. polymorpha ) using both metagenomic and culture-based techniques to identify and isolate plant growth-promoting bacteria (PGPB) that are distinct from the dominant nitrogen-fixing endosymbiont, Ensifer (Rhizobium) meliloti , towards obtaining a deeper understanding of the complexity of soil-grown legume root nodule microbiomes. We investigated potential functions of these non-rhizobial nodule-associated bacteria (NAB), asking, for example, do they serve a neutral or negative role (function as “cheaters”) or do they positively influence nodule development and function? Although this question was not completely answered, our studies found that besides Nutritional interactions in the soil microbiome drive critical environmental and ecological processes including biogeochemical cycling and plant growth. Identifying key nutrients that could predictably modulate soil communities is an attractive way to impact these processes. Nevertheless, the complexity of the soil milieu and diverse microbial community composition make it impossible to experimentally analyze all microbe-microbe interactions simultaneously using current analytical methods. We aim to identify key microbe-microbe interactions by unraveling the nutrient network produced by corrinoids, a group of structurally diverse metabolites used by a majority of the community. Exemplified by its flagship member, Vitamin B12, corrinoids are essential cofactors produced only by a fraction of the bacteria that use them, and thus are shared metabolites. Unlike other shared nutrients, corrinoids are structurally diverse: at least 16 distinct corrinoids have been described. Our group and others have shown that, bacteria that play central roles in bioremediation, plant-bacteria symbiosis, and elemental cycling possess preferences for distinct corrinoids. Thus, corrinoids appear to be an ideal nutrient by which we can pinpoint specific microbial interactions. Here, we test the hypothesis that corrinoids are a key nutrient family that shape soil microbial ecosystems. We optimized a chemical extraction process for corrinoids from an annual grassland soil that circumvents the ability of soil to adsorb corrinoids. Using this method, we show that, despite the ability of many soil-derived organisms to produce alternate corrinoids, Vitamin B12 is the predominant corrinoid in bulk soil. We also demonstrate that Vitamin B12 addition to soil enrichment cultures alters their species composition. Unexpectedly, despite the overwhelming presence of Vitamin B12 in this soil, addition of alternate corrinoids elicited marked responses in enrichment culture assembly. This study identifies key corrinoid-limited taxa that are desirable targets to understand corrinoid sharing in natural soil. Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. We developed CRISPRi knockdown libraries of essential genes in a biofilm-forming Bacillus subtilis strain. To characterize how these knockdown strains compete with a wild-type strain in biofilm colonies, we designed a competitive fitness assay that utilizes fluorescent tags to monitor 2 strains co-cultured in colonies on agar plates. We used this assay to measure the competitive fitness over time of each essential gene knockdown strain during biofilm colony co-culture with wild-type cells. We found that a partial knockdown of several translation-related genes were quickly outcompeted by wild-type cells due to reduced growth rates of the knockdown strains. Additionally, media composition led some knockdowns to compete differentially when grown on a biofilm-promoting medium versus a medium that does not promote biofilms. Intriguingly, we found that cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm coculture via nutrient sharing of D-alanine. This rescue was enhanced and stable over time in co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. This mutualism required growth in 3-dimensional colonies as wild-type cells outcompeted the alrA mutant when growth was confined to 2-dimensions. We next utilized our screening platform to identify other strains that demonstrate mutualism with the matrix-deficient strain. We identified several cases where the mutant cells benefited from growth with the matrix-deficient parent strain, many of which were likely mutualistic interactions involving nutrient and matrix sharing. As in the alrA -depletion mutants, this mutualism was not observed when growth was confined to two dimensions. Thus, we show that growth in a three-dimensional biofilm can promote genetic diversity through the sharing of secreted factors. This work demonstrates that biofilm lifestyles where secreted molecules can be shared across the population may drive evolution of mutualistic behavior. Methyl-coenzyme M reductase (MCR) is the enzyme responsible for methane production in methanogenic archaea. MCR has an α 2 β 2 γ 2 protein subunit stoichiometry and is encoded by the mcrA , mcrB , and mcrG genes, respectively. These genes form an operon with two other genes of unknown function mcrC and mcrD . This is one of the most highly transcribed operons in methanogens, and MCR can account for 10-20% of total cellular protein. Here we present the physiological and global transcriptional response to the reduction of MCR levels in the model methanogen Methanosarcina acetivorans . Suppression of MCR transcription results in transition from exponential to linear growth, consistent with dilution without replacement of a growth-limiting enzyme. Imposing a bottleneck on the flow of C1 units to methane results in a dramatic shift in transcriptional profile of M. characterized methyl-transferase/transporter utilization of methylmercaptopropionate as a potential novel for this methyltransferase system a buildup of methyl groups and reducing in central M. acetivorans . Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is performed by multi-cellular consortia of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). The syntrophic partnership between ANME and SRB is hypothesized to be driven by direct interspecies electron transfer (DIET) from ANME to SRB. This transfer of electrons is likely facilitated by a donor multi-heme cytochrome c complex in the archaea and an acceptor multi-heme containing conduit in the sulfate reducing partner. Within the four different taxonomic clades of SRB (HotSeep1, Seep-SRB2, Seep-SRB1g and Seep-SRB1a) identified as partners of ANME, a similar gene cluster containing a putat

Purpose Studying the legume nodule microbiome is important for understanding the development and nutrition of the plants inhabited by the various microbes within and upon them. We analyzed the microbiomes of these underground organs from both an important crop plant ( Medicago sativa ) and a related legume ( M. polymorpha ) using metagenomic and culture-based techniques to identify the main cultivatable contributors to plant growth enhancement. Methods Using high-throughput sequencing, culturing, and in planta techniques, we identified and analyzed a broad population of the bacterial taxa within Medicago nodules and the surrounding soil. Results Fifty-one distinct bacterial strains were isolated and characterized from nodules of both Medicago species and their growth-promoting activities were studied. Sequencing of 16S rRNA gene amplicons showed that in addition to Ensifer , the dominant genus, a large number of Gram-positive bacteria belonging to the Firmicutes and Actinobacteria were also present. After performing ecological and plant growth-promoting trait analyses, selecting the most promising strains, and then performing in planta assays, we found that strains of Bacillus and Micromonospora among others could play important roles in supporting the growth, health, and productivity of the host plant. Conclusion To our knowledge, the comparison of the biodiversity of the microbiota of undomesticated vs. cultivated Medicago roots and nodules is novel and shows the range of potential Plant Growth-Promoting Bacteria that could be used for plants of agricultural interest. These and other nodule-isolated microbes could also serve as inoculants with rhizobia with the goal of replacing synthetic fertilizers and pesticides for sustainable agriculture.

The mammalian heart switches its main metabolic substrate from glucose to fatty acids shortly after birth. This metabolic switch coincides with the loss of regenerative capacity in the heart. However, it is unknown whether glucose metabolism regulates heart regeneration. Here, we report that glucose metabolism is a determinant of regenerative capacity in the neonatal mammalian heart. Cardiac-specific overexpression of Glut1, the embryonic form of constitutively active glucose transporter, resulted in an increase in glucose uptake and concomitant accumulation of glycogen storage in postnatal heart. Upon cryoinjury, Glut1 transgenic hearts showed higher regenerative capacity with less fibrosis than non-transgenic control hearts. Interestingly, flow cytometry analysis revealed two distinct populations of ventricular cardiomyocytes: Tnnt2-high and Tnnt2-low cardiomyocytes, the latter of which showed significantly higher mitotic activity in response to high intracellular glucose in Glut1 transgenic hearts. Metabolic profiling shows that Glut1-transgenic hearts have a significant increase in the glucose metabolites including nucleotides upon injury. Inhibition of the nucleotide biosynthesis abrogated the regenerative advantage of high intra-cardiomyocyte glucose level, suggesting that the glucose enhances the cardiomyocyte regeneration through the supply of nucleotides. Our data suggest that the increase in glucose metabolism promotes cardiac regeneration in neonatal mouse heart.

Persistent human papillomavirus (HPV) infections is necessary for the development of cervical cancers. Consequently, understanding the biologic mechanisms resulting in clearance is key in cancer prevention. Similar to other mucosal sites, it is expected that the local microbiome plays a significant role in shaping the immune response responsible for HPV clearance. Using cervical wash repository samples from a prospective study of HPV in women, this study investigates the microbiome and its associated inflammatory milieu during HPV 16 pre-acquisition, persistence and clearance states. For comparison, samples from women with no history of HPV ever during the study period were selected. We showed that 9 of 13 inflammatory cytokines were found to be significantly increased in the immediate post-clearance visit compared to the pre-acquisition or infection visits. Gardnerella vaginalis was associated with higher levels of inflammatory cytokines. Women with no history of HPV infection had similar cytokine profiles as those with HPV 16 post-clearance. This in vivo study documented an immune response shortly after HPV 16 clearance. G. vaginalis appeared to be involved in shaping this immune response. The appearance of G. vaginalis may have resulted from a shift from anti-microbial to anti-viral immune response with loss of bacterial control. The similar high levels of cytokines seen in women with no history of HPV suggest that a certain level of inflammatory surveillance is required to maintain an HPV negative state. This data may inform therapies such as probiotics or pro-inflammatory agents for treatment of persistent HPV.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The microbiome plays an important role in human physiology. The composition of the human microbiome has been described at the phylum, class, genus, and species levels, however, it is largely unknown at the strain level. The importance of strain-level differences in microbial communities has been increasingly recognized in understanding disease associations. Current methods for identifying strain populations often require deep metagenomic sequencing and a comprehensive set of reference genomes. In this study, we developed a method, metagenomic multi-locus sequence typing (MG-MLST), to determine strain-level composition in a microbial community by combining high-throughput sequencing with multi-locus sequence typing (MLST). We used a commensal bacterium, Propionibacterium acnes, as an example to test the ability of MG-MLST in identifying the strain composition. Using simulated communities, MG-MLST accurately predicted the strain populations in all samples. We further validated the method using MLST gene amplicon libraries and metagenomic shotgun sequencing data of clinical skin samples. MG-MLST yielded consistent results of the strain composition to those obtained from nearly full-length 16S rRNA clone libraries and metagenomic shotgun sequencing analysis. When comparing strain-level differences between acne and healthy skin microbiomes, we demonstrated that strains of RT2/6 were highly associated with healthy skin, consistent with previous findings. In summary, MG-MLST provides a quantitative analysis of the strain populations in the microbiome with diversity and richness. It can be applied to microbiome studies to reveal strain-level differences between groups, which are critical in many microorganism-related diseases.

Four soil samples (SS-1—SS-4) isolated from semi-arid soils in Punjab, Pakistan were used as inocula for cowpea (V igna unguiculata L.) grown under salinity stress to analyze the composition of bacteria in the rhizosphere and within nodules through cultivation-dependent and cultivation-independent methods. Two cowpea varieties, 603 and the salt-tolerant CB 46, were each inoculated with four different native soil samples, and data showed that plants inoculated with soil samples SS-2 and SS-4 grew better than plants inoculated with soil samples SS-1 and SS-3. Bacteria were isolated from both soils and nodules, and 34 of the 51 original isolates tested positive for PGPR traits in plate assays with many exhibiting multiple plant growth-promoting properties. A number of isolates were positive for all PGPR traits tested. For the microbiome studies, environmental DNA (eDNA) was isolated from SS-1 and SS-4, which represented the extremes of the Pakistan soils to which the plants responded, and by 16S rRNA gene sequencing analysis were found to consist mainly of Actinobacteria, Firmicutes, and Proteobacteria. However, sequencing analysis of eDNA isolated from cowpea nodules established by the trap plants grown in the four Pakistan soils indicated that the nodule microbiome consisted almost exclusively of Proteobacterial sequences, particularly Bradyrhizobium . Yet, many other bacteria including Rhizobium , Mesorhizobium, Pseudomonas, as well as Paenibacillus , Bacillu s as well as non-proteobacterial genera were isolated from the nodules of soil-inoculated cowpea plants. This discrepancy between the bacteria isolated from cowpea nodules (Proteobacteria and non-Proteobacteria) versus those detected in the nodule microbiome (Proteobacteria) needs further study.

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.

This pilot study investigated the clinical and microbial changes that occur in patients undergoing orthodontic treatment using fixed appliances and clear aligners. The clear aligner group had significantly less plaque accumulation than the fixed appliances group, but no significant differences in gingival health were found. Next-generation sequencing of the 16S rRNA encoding gene revealed a shift in the oral microbiome from baseline with a unique microbial community discovered on the inner surface of clear aligners.

The mammalian heart switches its main metabolic substrate from glucose to fatty acids shortly after birth. This metabolic switch coincides with the loss of regenerative capacity in the heart. However, it is unknown whether glucose metabolism itself regulates heart regeneration. Here, we report that glucose metabolism is a determinant of regenerative capacity in the neonatal mammalian heart. Cardiac-specific overexpression of Glut1, the embryonic form of constitutively active glucose transporter, resulted in an increase in glucose uptake and concomitant glycogen storage in postnatal heart. Upon cryoinjury, Glut1 transgenic hearts showed higher regenerative capacity with less fibrosis than non-transgenic control hearts. Interestingly, flow cytometry analysis revealed two distinct populations of ventricular cardiomyocytes: Tnnt2-high and Tnnt2-low cardiomyocytes, the latter of which showed significantly higher mitotic activity in response to high intracellular glucose in Glut1 transgenic hearts. Metabolic profiling shows that Glut1-transgenic hearts have a significant increase in the glucose metabolites upon injury, and inhibition of the nucleotide biosynthesis abrogated the regenerative advantage of high intra-cardiomyocyte glucose level. Our data suggest that the increased in glucose metabolism promotes cardiac regeneration in neonatal mouse heart.

Type 2 diabetes mellitus (T2DM) is a systemic disease, predisposing patients to other inflammatory conditions including periodontitis. The subgingival microbiome, a key player in periodontitis pathogenesis, is not well characterized in T2DM population. To better understand whether the subgingival microbiome is different between T2DM and systemically healthy, nondiabetic (ND) subjects, we performed a longitudinal analysis of the subgingival microbiome in T2DM patients ( n = 15) compared with ND subjects ( n = 16). Using metagenomic shotgun sequencing, we investigated the microbiome in the healthy periodontal state, periodontitis state, and resolved state after treatment. We found that in the periodontitis state, the shift in the subgingival microbiome from the healthy state was less prominent in T2DM compared with ND subjects, yet the clinical signs of disease were similar for both. Furthermore, we revealed highly correlated presence of pathogenic species in relative abundance not only in the periodontitis state, but also in the healthy state in T2DM, suggesting an elevated risk of progression to periodontitis in this cohort. We further investigated the functional potentials of the subgingival microbiome and identified a set of microbial marker genes associated with the clinical states. These genes were significantly enriched in 21 pathways, some of which are associated with periodontitis and some potentially link T2DM and periodontitis. This study identified the longitudinal changes of the subgingival microbiome associated with periodontitis in T2DM and suggests that T2DM patients are more susceptible to shifts in the subgingival microbiome toward dysbiosis, potentially due to impaired host metabolic and immune regulation.

1. Environmental DNA (eDNA) metabarcoding is a promising method to monitor species and community diversity that is rapid, affordable, and non-invasive. Longstanding needs of the eDNA community are modular informatics tools, comprehensive and customizable reference databases, flexibility across high-throughput sequencing platforms, fast multilocus metabarcode processing, and accurate taxonomic assignment. As bioinformatics tools continue to improve, addressing each of these demands within a single bioinformatics toolkit is becoming a reality. 2. We present the modular metabarcode sequence toolkit Anacapa ( https://github.com/limey-bean/Anacapa/ ), which addresses the above needs, allowing users to build comprehensive reference databases and assign taxonomy to raw multilocus metabarcode sequence data A novel aspect of Anacapa is our database building module, Creating Reference libraries Using eXisting tools ( CRUX ), which generates comprehensive reference databases for specific user-defined metabarcode loci. The Quality Control and Dereplication module sorts and processes multiple metabarcode loci and processes merged, unmerged and unpaired reads maximizing recovered diversity. Followed by amplicon sequence variants (ASVs) detection using DADA2 . The Anacapa Classifier module aligns these ASVs to CRUX-generated reference databases using Bowtie2 . Taxonomy is assigned to ASVs with confidence scores using a Bayesian Lowest Common Ancestor ( BLCA ) method. The Anacapa Toolkit also includes an R package, ranacapa, for automated results exploration through standard biodiversity statistical analysis. 3. We performed a series of benchmarking tests to verify that the Anacapa Toolkit generates comprehensive reference databases that capture wide taxonomic diversity and that it can assign high-quality taxonomy to both MiSeq-length and Hi-Seq length sequence data. We demonstrate the value of the Anacapa Toolkit to assigning taxonomy to eDNA sequences from seawater samples from southern California including capability of this tool kit to process multilocus metabarcoding data. 4. The Anacapa Toolkit broadens the exploration of eDNA and assists in biodiversity assessment and management by generating metabarcode specific databases, processing multilocus data, retaining all read types, and expanding non-traditional eDNA targets. Anacapa software and source code are open and available in a virtual container to ease installation.

The mammalian heart switches its main metabolic substrate from glucose to fatty acids shortly after birth. This metabolic switch coincides with the loss of regenerative capacity in the heart. However, it is unknown whether glucose metabolism itself regulates heart regeneration. Here, we report that glucose metabolism is a determinant of regenerative capacity in the neonatal mammalian heart. Cardiac-specific overexpression of Glut1, the embryonic form of constitutively active glucose transporter, resulted in an increase in glucose uptake and concomitant glycogen storage in postnatal heart. Upon cryoinjury, Glut1 transgenic hearts showed higher regenerative capacity with less fibrosis than non-transgenic control hearts. Interestingly, flow cytometry analysis revealed two distinct populations of ventricular cardiomyocytes: Tnnt2-high and Tnnt2-low cardiomyocytes, the latter of which showed significantly higher mitotic activity in response to high intracellular glucose in Glut1 transgenic hearts. Metabolic profiling shows that Glut1-transgenic hearts have a significant increase in the glucose metabolites upon injury, and inhibition of the nucleotide biosynthesis abrogated the regenerative advantage of high intra-cardiomyocyte glucose level. Our data suggest that the increased in glucose metabolism promotes cardiac regeneration in neonatal mouse heart.

Skin micro-organisms play an important role in the disease progression of atopic dermatitis (AD). The skin pathogen, Staphylococcus aureus, in particular, methicillin-resistant S. aureus (MRSA), contributes to AD by producing virulence factors that enhance immune activation and skin barrier dysfunction (Schlievert et al., 2010Schlievert P.M. Strandberg K.L. Lin Y.C. Peterson M.L. Leung D.Y. Secreted virulence factor comparison between methicillin-resistant and methicillin-sensitive Staphylococcus aureus, and its relevance to atopic dermatitis.J Allergy Clin Immunol. 2010; 125: 39-49Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). While it is known that the relative abundance of S. aureus generally increases in AD lesions (Kong et al., 2012Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1099) Google Scholar, Shi et al., 2016Shi B. Bangayan N.J. Curd E. Taylor P.A. Gallo R.L. Leung D.Y. et al.The skin microbiome is different in pediatric versus adult atopic dermatitis.J Allergy Clin Immunol. 2016; 138: 1233-1236Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), it is not clear whether the skin microbiome composition in MRSA-colonized skin differs from methicillin-sensitive S. aureus (MSSA)−colonized skin. It is important to understand the potential differences in the composition of skin commensal bacteria, as they play a critical role in reducing the growth of pathogenic S. aureus and thus the risk of AD (Kennedy et al., 2017Kennedy E.A. Connolly J. Hourihane J.O. Fallon P.G. McLean W.H. Murray D. et al.Skin microbiome before development of atopic dermatitis: early colonization with commensal staphylococci at 2 months is associated with a lower risk of atopic dermatitis at 1 year.J Allergy Clin Immunol. 2017; 139: 166-172Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, Nakatsuji et al., 2017Nakatsuji T. Chen T.H. Narala S. Chun K.A. Two A.M. Yun T. et al.Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis.Sci Transl Med. 2017; 9: 378Crossref Scopus (552) Google Scholar). In this study, we screened for the presence of MRSA and MSSA strains on the skin in a cohort of 339 AD patients, including 169 young children (2−12 years) and 170 teenagers (13−17 years) and adults (18−74 years). This study was approved by the Institutional Review Boards at National Jewish Health in Denver and University of California, Los Angeles. Written informed consent was obtained from all participants. As the skin microbiome of teenagers is closer to adults than young children (Shi et al., 2016Shi B. Bangayan N.J. Curd E. Taylor P.A. Gallo R.L. Leung D.Y. et al.The skin microbiome is different in pediatric versus adult atopic dermatitis.J Allergy Clin Immunol. 2016; 138: 1233-1236Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), we combined teenagers and adults into one group in the analysis. We collected skin swabs from lesional skin and adjacent normal-appearing non-lesional skin of the volar forearm from each AD patient. Using a culture-based assay (Supplementary Material online), MSSA was detected on the lesional skin of 199 (58.7%) AD patients (95 young children and 104 teenagers/adults), while MRSA was found in 23 (6.8%) AD patients (15 young children and 8 teenagers/adults). The detection frequency of MSSA and MRSA strains in pediatric patients of our cohort is consistent with previous studies (Suh et al., 2008Suh L. Coffin S. Leckerman K.H. Gelfand J.M. Honig P.J. Yang A.C. Methicillin-resistant Staphylococcus aureus colonization of children with atopic dermatitis.Pediatr Dermatol. 2008; 25: 528-534Crossref PubMed Scopus (65) Google Scholar). S. aureus was not detected at the lesional site of the remaining 117 (34.5%) AD patients. Among the 23 AD patients carrying MRSA on the lesional skin, 11 were also carrying MRSA at the adjacent nonlesional site. In addition to the culture-based assay, we compared the microbiome of MRSA- versus MSSA-colonized AD lesional skin in a subgroup of these subjects. The microbiome composition of 128 of the 339 AD patients (Supplementary Table S1 online), including 19 MRSA carriers, 70 MSSA carriers, and 39 patients without S. aureus detected in the culture-based assay, was profiled using the culture-independent 16S ribosomal RNA gene sequencing analysis (Supplementary Figure S1). The negative S. aureus culture results of 39 patients were corroborated by 16S ribosomal RNA gene sequencing analysis, which showed a significantly low relative abundance (<1%) of S. aureus in the microbiome of these subjects. We found that the microbial diversity (alpha diversity) was significantly decreased in MRSA-colonized than MSSA-colonized lesional skin (P = 0.001) (Supplementary Figure S2 online). The relative abundance of S. aureus was significantly higher on MRSA-colonized (52.5% ± 9.3%) than MSSA-colonized (25.0% ± 4.1%) lesional skin (P = 0.017) (Figure 1a), suggesting that MRSA plays a more significant role in reducing the skin microbiome diversity than MSSA. Our previous study demonstrated that the prevalent skin commensals, Streptococcus, Propionibacterium, and Corynebacterium, were significantly decreased in relative abundance on AD skin compared to healthy skin (Shi et al., 2016Shi B. Bangayan N.J. Curd E. Taylor P.A. Gallo R.L. Leung D.Y. et al.The skin microbiome is different in pediatric versus adult atopic dermatitis.J Allergy Clin Immunol. 2016; 138: 1233-1236Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In this study, we found that the relative abundances of Streptococcus and Propionibacterium were significantly decreased on MSSA-colonized lesional skin (both P < 1E-3) when compared to the skin with no S. aureus detected in culture (Figures 1d, 1e). We found a significant further reduction in Streptococcus and Propionibacterium on MRSA-colonized lesional skin compared to MSSA-colonized lesional skin (P = 0.023 and P = 0.007, respectively). The relative abundance of Corynebacterium was significantly decreased on MRSA-colonized lesional skin as well (P < 1E-3) when compared to MSSA-colonized lesional skin (Figure 1f). Staphylococcus epidermidis has been considered a skin commensal, however, its role in AD is unclear. Previous studies reported an increase in abundance of S. epidermidis on lesional skin of pediatric AD, which may suggest a compensatory mechanism of S. epidermidis to control pathogens (Byrd et al., 2017Byrd A.L. Deming C. Cassidy S.K.B. Harrison O.J. Ng W.I. Conlan S. et al.Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis.Sci Transl Med. 2017; 9: eaal4651Crossref PubMed Scopus (282) Google Scholar, Kong et al., 2012Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1099) Google Scholar). In the current study, we found that the relative abundance of S. epidermidis was not significantly changed in MRSA colonization (Figure 1b). Other commensal Staphylococcus species, including coagulase-negative Staphylococcus with antimicrobial activity, such as Staphylococcus hominis, are commonly found on healthy skin but rarely on AD skin (Nakatsuji et al., 2017Nakatsuji T. Chen T.H. Narala S. Chun K.A. Two A.M. Yun T. et al.Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis.Sci Transl Med. 2017; 9: 378Crossref Scopus (552) Google Scholar). In this study, we found that the relative abundance of S. hominis was significantly decreased on MSSA-colonized lesional skin compared to non−S. aureus skin (P < 0.004) and further decreased on MRSA-colonized lesional skin (P < 0.004) (Figure 1c), with the same trend as skin commensals Streptococcus and Propionibacterium. We also compared the microbiome between MRSA and MSSA colonization at the non-lesional site. Nine of the 19 MRSA carriers had MRSA detected not only on the lesional skin but also on the adjacent nonlesional site. On the nonlesional site, we found that compared to MSSA colonization, MRSA colonization was not associated with a significant increase of S. aureus (P = 0.25). The relative abundances of Propionibacterium (P = 0.018) and S. hominis (P = 0.013) decreased significantly, but other commensals did not change significantly (Figures 1a−1f). Overall, our data suggest that compared to MSSA, MRSA colonization is associated with a significantly greater decrease in relative abundance of skin commensal bacteria. Previous studies have shown that the skin microbiome varies by age in both healthy individuals (Oh et al., 2012Oh J. Conlan S. Polley E.C. Segre J.A. Kong H.H. Shifts in human skin and nares microbiota of healthy children and adults.Genome Med. 2012; 4: 77Crossref PubMed Scopus (225) Google Scholar) and AD patients (Shi et al., 2016Shi B. Bangayan N.J. Curd E. Taylor P.A. Gallo R.L. Leung D.Y. et al.The skin microbiome is different in pediatric versus adult atopic dermatitis.J Allergy Clin Immunol. 2016; 138: 1233-1236Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In our cohort of 128 AD patients, 59 (46%) were young children and 69 (54%) are teenagers/adults. Among the 19 AD patients carrying MRSA at the lesional site (Supplementary Table S2 online), we found that MRSA colonization shifted the microbiome composition in both age groups with an increased relative abundance of S. aureus and decreased relative abundances of skin commensals except for S. epidermidis (Supplementary Figure S3 online). To identify correlated interactions among the bacteria in the AD skin microbiome, we calculated the Pearson correlation coefficients among the prevalent skin commensals and S. aureus based on their relative abundances on the lesional skin. To avoid false positives, correlations were not counted in samples that had no S. aureus detected in the culture. We found that the relative abundances of the prevalent skin commensals mentioned were negatively correlated with S. aureus, suggesting an antagonistic relationship between the skin commensals and this AD pathogen. We compared the correlations between each skin commensal and S. aureus on MRSA- versus MSSA-colonized lesional skin. We found that in teenager/adult patients, the skin commensals, Streptococcus, Propionibacterium, Corynebacterium, S. hominis, and S. epidermidis, had greater inverse correlations with S. aureus on MRSA-colonized skin compared to MSSA-colonized skin. In young children, only S. epidermidis and S. hominis showed greater inverse correlations with S. aureus on MRSA-colonized skin (Supplementary Figure S3). Previous studies have identified the skin commensals Streptococcus, Propionibacterium, and Corynebacterium with inhibitory activities against S. aureus in vitro (Bessesen et al., 2015Bessesen M.T. Kotter C.V. Wagner B.D. Adams J.C. Kingery S. Benoit J.B. et al.MRSA colonization and the nasal microbiome in adults at high risk of colonization and infection.J Infect. 2015; 71: 649-657Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Shu et al., 2013Shu M. Wang Y. Yu J. Kuo S. Coda A. Jiang Y. et al.Fermentation of Propionibacterium acnes, a commensal bacterium in the human skin microbiome, as skin probiotics against methicillin-resistant Staphylococcus aureus.PLoS One. 2013; 8e55380Crossref PubMed Scopus (173) Google Scholar). S. epidermidis is able to protect the skin from S. aureus infection through microbe−microbe interactions, as well as microbe−host interactions by enhancing immune responses to inhibit the growth of S. aureus (Iwase et al., 2010Iwase T. Uehara Y. Shinji H. Tajima A. Seo H. Takada K. et al.Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization.Nature. 2010; 465: 346-349Crossref PubMed Scopus (619) Google Scholar, Lai et al., 2010Lai Y. Cogen A.L. Radek K.A. Park H.J. Macleod D.T. Leichtle A. et al.Activation of TLR2 by a small molecule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections.J Invest Dermatol. 2010; 130: 2211-2221Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The role of skin commensals in AD, in particular when MRSA colonizes the skin, needs future investigations. We also investigated the influences of other confounding factors. In our cohort, disease severity was not significantly associated with MRSA colonization (Supplementary Table S2). Within the patients with severe AD (n = 71), we found that the microbial diversity was lower in MRSA-colonized than MSSA-colonized lesional skin (P = 0.002, Supplementary Figure S4 online). The number of patients with moderate AD and MRSA colonization (n = 2) in our cohort was too low to allow us to perform a similar analysis. Additionally, to exclude the influence of medications on the skin microbiome, AD patients were excluded if they received medications, including antibiotic treatment within 7 days of sampling (more details in Supplementary Material). In summary, our study suggests that MRSA colonization on AD skin correlates with a more profound change in the composition of commensal bacteria than MSSA colonization. The prevalent commensal bacteria had significant reductions in relative abundance in MSSA-colonized lesional skin compared to no S. aureus colonization and an even more significant decrease on MRSA-colonized lesional skin (Figures 1b−1f). Future studies are needed to better understand the mechanisms of potential microbial competition between skin commensals and MRSA in AD pathogenesis. Importantly, the maturation stage of host immune responses is an important factor, given the observation of differences in the prevalence of MRSA colonization between age groups in our cohort (Supplementary Table S2). Our current findings determined by 16S ribosomal RNA sequencing data may limit the representation of the absolute cell counts of microbial species and the taxonomic composition at the strain level. Additionally, our skin samples were collected from volar forearm of AD patients. Future studies are needed to investigate the influence of MRSA colonization on the skin microbiome at other body sites. Our findings suggest, however, that antibiotic treatment in AD patients with MRSA infection may not have long-term benefit due to its antimicrobial against of beneficial bacteria and, therefore, lacks a therapeutic strategy to promote commensal bacteria recolonization of the skin. This highlights the need to develop new approaches, such as probiotics or prebiotics, that replenish skin commensals as a means of combating skin infection and preventing MRSA relapse (Nakatsuji et al., 2017Nakatsuji T. Chen T.H. Narala S. Chun K.A. Two A.M. Yun T. et al.Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis.Sci Transl Med. 2017; 9: 378Crossref Scopus (552) Google Scholar). The authors state no conflict of interest. This work was partially funded by National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (grant U19 AI117673, NIH/National Center for Research Resources grant UL1 RR025780, NIH/National Institute of General Medical Sciences R01GM099530, and The Edelstein Family Chair of Pediatric Allergy-Immunology at National Jewish Health. We thank the Clinical and Translational Research Center nurses for their assistance in performing this protocol. We also thank Jack Hu from Rho Federal Systems Division Inc for his help on statistical analysis. Download .pdf (.65 MB) Help with pdf files Supplementary Data

Background: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. Results: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. Conclusions: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Pediatric and adult atopic dermatitis (AD) have different disease manifestations. The skin microbiome is thought to be critical in driving disease development. Whether the skin microbiome in young AD children is different from adults is unknown. We collected swabs from lesional and non-lesional skin of the volar forearm of 128 AD patients and 68 healthy subjects. We compared the skin microbiome of AD patients with healthy individuals in different age groups (2-12 and 13-62) using 16S rRNA gene sequencing. We analyzed correlations between the microbiome and age and investigated gene functions encoded in microbial genomes. We found that the healthy skin microbiome was significantly different between young children and adults in microbial diversity and in relative abundance of prevalent bacterial genera. Compared to the diverse microbial community on healthy skin, AD skin microbiome was dominated by Staphylococcus species at all ages. Importantly however, shifts in the AD microbiome compared to the healthy microbiome were different between young children and adults. We identified distinct clusters of childhood-associated (represented by Streptococcus), adult-associated (Propionibacterium and Corynebacterium), and AD-associated skin bacteria. By analyzing 46 genomes representing major species in the clusters, we further identified specific functional profiles among these clusters. Childhood-associated skin bacteria Streptococcus are replaced by adult-associated lipophilic commensals that associate with sebum production at puberty. Pathways unique to Propionibacterium and Corynebacterium, including porphyrin and chlorophyll metabolism, may provide additional protection for skin health in adults. Our findings suggest that pediatric and adult AD are driven by different microbial influences.

Atopic dermatitis (AD) is the most common inflammatory skin disease in the general population. The disease prevalence is associated with age. AD often starts in early childhood, affecting 15% to 30% of children.1Bieber T. Atopic dermatitis.N Engl J Med. 2008; 358: 1483-1494Crossref PubMed Scopus (1557) Google Scholar However, up to 70% of children with AD show clearing of the disease or a spontaneous remission around puberty, even in patients with filaggrin mutations. AD can persist or start in adulthood. However, the prevalence of AD in adults is only approximately 3%. Although multiple factors contribute to AD pathogenesis, skin micro-organisms are critical in driving disease development. Shifts in the skin microbiome were observed during disease progression of pediatric AD.2Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1099) Google Scholar In adult AD, however, the skin microbiome is not well characterized. Comparisons of the skin microbiome among age groups of patients with AD and healthy controls will help delineate age differences in the microbial pathogenesis of this disease. In this study, we recruited 128 patients with AD: 59 young children (age, 2-12 years), 13 teenagers (age, 13-17 years), and 56 adults (age, 18-62 years). A cohort of 68 age-matched nonatopic healthy controls (age, 3-59 years) was also enrolled, which includes 13 young children, 10 teenagers, and 45 adults (see Table E1 in this article's Online Repository at www.jacionline.org). We collected 2 swab samples from the volar forearm of each patient with AD, one from lesional skin and one from adjacent normal-appearing nonlesional skin. One swab sample of the volar forearm was collected from each healthy subject. In total, 324 samples were analyzed using 16S ribosomal RNA (rRNA) gene sequencing. After data cleaning, on average 32,311 paired-end 16S rRNA sequences were obtained for each sample. Our data provided sufficient sequencing depths at the species level as indicated by the rarefaction curve (see Fig E1 in this article's Online Repository at www.jacionline.org). This large study cohort and the high sequencing coverage enabled us to robustly identify differences in the skin microbiome between age groups in healthy individuals and patients with AD. In the skin microbiome of healthy individuals, we identified 7 prevalent bacterial phyla and 20 genera (Fig 1, A). Four of the genera (Propionibacterium, Corynebacterium, Staphylococcus, and Streptococcus) were present in 90% or more of the healthy subjects, with 5% or more relative abundance in at least 1 age group. Among the 15 species identified (see Fig E2 in this article's Online Repository at www.jacionline.org), Propionibacterium acnes, Staphylococcus epidermidis, and Streptococcus mitis/oralis/pneumoniae/sanguinis were the most prevalent species in healthy subjects, accounting for 98.5% of total Propionibacterium species, 46.6% of total Staphylococcus species, and 32.5% of total Streptococcus species in relative abundance. To determine whether the stages of the human physical development have a significant effect on the skin microbiome,3Oh J. Conlan S. Polley E.C. Segre J.A. Kong H.H. Shifts in human skin and nares microbiota of healthy children and adults.Genome Med. 2012; 4: 77Crossref PubMed Scopus (225) Google Scholar we compared the microbiome among the age groups. We found that the healthy skin microbiome was significantly more diverse in young children than in adults (alpha diversity, P = .01), and was distinct between the 2 age groups as indicated by beta diversity (ANOSIM, P = .009) (Fig 2, A). At the genus level, Streptococcus, Granulicatella, Gemella, Rothia, and Haemophilus were more abundant in young children, whereas Propionibacterium, Corynebacterium, Staphylococcus, Lactobacillus, Finegoldia, and Anaerococcus were more abundant in adults (see Table E2 in this article's Online Repository at www.jacionline.org). At the species level, Streptococcus salivarius/thermophilus/vestibularis was more abundant in young children (P = .045) (see Table E3 in this article's Online Repository at www.jacionline.org), whereas P acnes and S epidermidis were more abundant in adults (P = .01 and P < 1 × 10−5, respectively) (Fig 1, B). Staphylococcus aureus was detected in 20.6% of the healthy subjects, but had very low relative abundance (<1%) in all age groups. Increased host sebum production and changes in the skin structure at and after puberty may facilitate the colonization and growth of lipophilic bacteria Propionibacterium and Corynebacterium,4Hannigan G.D. Grice E.A. Microbial ecology of the skin in the era of metagenomics and molecular microbiology.Cold Spring Harb Perspect Med. 2013; 3: a015362Crossref Scopus (70) Google Scholar which replace Streptococcus and become dominant in adulthood. Teenagers are in transition from young children to adults in physical development, and their skin microbiome is in transition as well, with a higher similarity to adults than to young children,3Oh J. Conlan S. Polley E.C. Segre J.A. Kong H.H. Shifts in human skin and nares microbiota of healthy children and adults.Genome Med. 2012; 4: 77Crossref PubMed Scopus (225) Google Scholar reflected in the representative micro-organisms (Fig 1, B) and the overall microbial community structure (Fig 2, A). In subsequent analyses, we combined teenagers and adults into one group and compared with young children. Similar to the healthy skin microbiome, in the AD skin microbiome we identified significant differences between young children and adults-teenagers (beta diversity, ANOSIM, P < .001) (Fig 2, A). In AD nonlesional skin, the microbiome diversity was significantly higher in young children than in adults-teenagers (alpha diversity, P = .036). The 20 prevalent genera identified in healthy controls were also detected in most of the patients with AD (Fig 1, C). Eight of the genera were significantly different in relative abundance between young children and adults-teenagers in both lesional and nonlesional skin (see Table E4 in this article's Online Repository at www.jacionline.org). The age differences were consistent with those observed in healthy controls. The microbiome differences in lesional and nonlesional skin were identified previously in patients with AD5Flores G.E. Seite S. Henley J.B. Martin R. Zelenkova H. Aguilar L. et al.Microbiome of affected and unaffected skin of patients with atopic dermatitis before and after emollient treatment.J Drugs Dermatol. 2014; 13: 1365-1372PubMed Google Scholar; however, it was unclear whether the differences were associated with age. In this study, we found that the microbiome diversity was significantly decreased in lesional skin compared with nonlesional skin in both young children (P < .001) and adults-teenagers (P = .013). In both age groups, Staphylococcus was significantly more abundant in lesional skin (P ≤ .012) and was also more abundant in nonlesional skin compared with healthy skin (P < .003), suggesting that nonlesional skin is susceptible to pathogen colonization and is at risk to progress toward diseased state. In contrast, skin commensals Streptococcus and Propionibacterium were observed in lower relative abundance in lesional skin compared with nonlesional skin and in nonlesional skin compared with healthy skin, but their changes were specific to young children and adults-teenagers, respectively. To better understand the bacterial associations with age in the skin microbiome, we calculated correlations among the 20 prevalent genera on the basis of their relative abundances in patients with AD and healthy controls. Three distinct bacterial clusters were identified: adult-associated, childhood-associated, and AD-associated (Fig 2, B). Most of the bacterial organisms were clustered in either adult-associated group or in childhood-associated group (see Tables E2-E5 in this article's Online Repository at www.jacionline.org), suggesting that age differences in the skin microbiome may be attributed to shifts in skin micro-organisms that are coordinated with each other in abundance during host maturation. AD-associated cluster consisted of only Staphylococcus. We identified multiple species within the major genera. Except for S aureus, species within each genus were positively correlated in relative abundance (see Fig E3 in this article's Online Repository at www.jacionline.org). S aureus was inversely correlated with other species, including those from the same genus, suggesting an antagonistic relationship between S aureus and skin commensals. We further investigated differences in the gene functions encoded in the genomes of age-specific and AD-associated skin bacteria. We analyzed 46 genomes of 13 major species found in our cohort (see this article's Methods section in the Online Repository at www.jacionline.org). A total of 1910 KEGG orthologous groups (KO genes) were identified, 833 of which were unique to 1 of the 3 skin bacterial clusters (see Fig E4 and Table E6 in this article's Online Repository at www.jacionline.org). Among the 316 KO genes unique to AD-associated cluster, 63 KO genes were S aureus–specific, involved in disease-associated pathways including S aureus infection and bacterial invasion of epithelial cells. Among the 517 KO genes specific to skin commensals, 113 were unique to childhood-associated Streptococcus spp and 404 were unique to adult-associated P acnes and Corynebacterium spp. It has been suggested that Streptococcus can inhibit S aureus growth by producing hydrogen peroxide,6Regev-Yochay G. Trzcinski K. Thompson C.M. Malley R. Lipsitch M. Interference between Streptococcus pneumoniae and Staphylococcus aureus: in vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae.J Bacteriol. 2006; 188: 4996-5001Crossref PubMed Scopus (150) Google Scholar while adult-associated commensals Propionibacterium and Corynebacterium harbor genes involved in porphyrin metabolism and can reduce S aureus infection.7Orenstein A. Klein D. Kopolovic J. Winkler E. Malik Z. Keller N. et al.The use of porphyrins for eradication of Staphylococcus aureus in burn wound infections.FEMS Immunol Med Microbiol. 1997; 19: 307-314Crossref PubMed Scopus (81) Google Scholar In addition, metabolites of adult-associated skin commensals can decrease skin pH and enhance antimicrobial activities, thus suppressing adherence and growth of S aureus in human keratinocytes.4Hannigan G.D. Grice E.A. Microbial ecology of the skin in the era of metagenomics and molecular microbiology.Cold Spring Harb Perspect Med. 2013; 3: a015362Crossref Scopus (70) Google Scholar, 8Shu M. Wang Y. Yu J. Kuo S. Coda A. Jiang Y. et al.Fermentation of Propionibacterium acnes, a commensal bacterium in the human skin microbiome, as skin probiotics against methicillin-resistant Staphylococcus aureus.PLoS One. 2013; 8: e55380Crossref PubMed Scopus (173) Google Scholar, 9Wang Y. Dai A. Huang S. Kuo S. Shu M. Tapia C.P. et al.Propionic acid and its esterified derivative suppress the growth of methicillin-resistant Staphylococcus aureus USA300.Benef Microbes. 2014; 5: 161-168Crossref PubMed Scopus (40) Google Scholar In summary, we identified significant differences in the AD skin microbiome between young children and adults-teenagers. Among many other factors that we examined, including host factors, clinical parameters, disease history, and history of concomitant medications (see Table E7 in this article's Online Repository at www.jacionline.org), we found that the microbiome was also correlated with disease severity in AD lesional skin. This is consistent with previous observations that the skin microbiome changed with disease progression.2Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1099) Google Scholar Although AD pathogenic factors drive the disease development, age-specific skin commensals possess various potentials in defending against pathogens and maintaining skin health at different development stages. Our findings, from a new perspective of the skin microbiome, may partly explain the age differences in AD. The sequence data from this study have been deposited to NCBI BioProject accession number 268694. We thank Joanne Streib, Gayle Spears, and Caroline Bronchick for their invaluable assistance in the processing and collection of skin swabs as well as recruitment of study subjects into this Atopic Dermatitis Research Network protocol. We also thank Keli Artis, Denise Babineau, and Alice Lail from Rho Federal Systems Division, Inc, for their help on this study. Download .doc (.08 MB) Help with doc files Online Repository text Download .pdf (.04 MB) Help with pdf files Fig E1 Download .pdf (.13 MB) Help with pdf files Fig E2 Download .pdf (.04 MB) Help with pdf files Fig E3 Download .pdf (.06 MB) Help with pdf files Fig E4 Download .xlsx (.01 MB) Help with xlsx files Table E1 Download .xlsx (.01 MB) Help with xlsx files Table E2 Download .xlsx (.01 MB) Help with xlsx files Table E3 Download .xlsx (.01 MB) Help with xlsx files Table E4 Download .xlsx (.01 MB) Help with xlsx files Table E5 Download .xlsx (.12 MB) Help with xlsx files Table E6 Download .xlsx (.01 MB) Help with xlsx files Table E7

Studies have emphasized the importance of disease-associated microorganisms in perturbed communities, however, the protective roles of commensals are largely under recognized and poorly understood. Using acne as a model disease, we investigated the determinants of the overall virulence property of the skin microbiota when disease- and health-associated organisms coexist in the community. By ultra-deep metagenomic shotgun sequencing, we revealed higher relative abundances of propionibacteria and Propionibacterium acnes phage in healthy skin. In acne patients, the microbiome composition at the species level and at P. acnes strain level was more diverse than in healthy individuals, with enriched virulence-associated factors and reduced abundance of metabolic synthesis genes. Based on the abundance profiles of the metagenomic elements, we constructed a quantitative prediction model, which classified the clinical states of the host skin with high accuracy in both our study cohort (85%) and an independent sample set (86%). Our results suggest that the balance between metagenomic elements, not the mere presence of disease-associated strains, shapes the overall virulence property of the skin microbiota. This study provides new insights into the microbial mechanism of acne pathogenesis and suggests probiotic and phage therapies as potential acne treatments to modulate the skin microbiota and to maintain skin health.