A simplified bacterial community found within the epidermis than at the epidermal surface of atopic dermatitis patients and healthy controls.

There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute to skin disorders, with atopic dermatitis (AD) routinely linked to increased Staphylococcus aureus (S. aureus) colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the 'core microbiome' of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status.

An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCCmec Cassette.

CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCCmec) type V(5C2&5), conferring ß-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCCmec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus, CRISPR-Cas is rare, but when present, it is located within the SCCmec element, which encodes resistance to methicillin and other ß-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCCmec elements in different species of non-S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus. Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.

Design and validation of Dolosigranulum pigrum specific PCR primers using the bacterial core genome.

Dolosigranulum pigrum-a lactic acid bacterium that is increasingly recognized as an important member of the nasal microbiome. Currently, there are limited rapid and low-cost options for confirming D. pigrum isolates and detecting D. pigrum in clinical specimens. Here we describe the design and validation of a novel PCR assay targeting D. pigrum that is both sensitive and specific. We designed a PCR assay targeting murJ, a single-copy core species gene identified through the analysis of 21 D. pigrum whole genome sequences. The assay achieved 100% sensitivity and 100% specificity against D. pigrum and diverse bacterial isolates and an overall 91.1% sensitivity and 100% specificity using nasal swabs, detecting D. pigrum at a threshold of 1.0 × 104 D. pigrum 16S rRNA gene copies per swab. This assay adds a reliable and rapid D. pigrum detection tool to the microbiome researcher toolkit investigating the role of generalist and specialist bacteria in the nasal environment.

All Staphylococcus aureus bacteraemia-inducing strains can cause infective endocarditis: Results of GWAS and experimental animal studies.

OBJECTIVES: We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB). METHODS: A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves. RESULTS: The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU. CONCLUSION: All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream.

Comparative genomics of Rothia species reveals diversity in novel biosynthetic gene clusters and ecological adaptation to different eukaryotic hosts and host niches.

Rothia species are understudied members of the phylum Actinobacteria and prevalent colonizers of the human and animal upper respiratory tract and oral cavity. The oral cavity, including the palatine tonsils, is colonized by a complex microbial community, which compete for resources, actively suppress competitors and influence host physiology. We analysed genomic data from 43 new porcine Rothia isolates, together with 112 publicly available draft genome sequences of Rothia isolates from humans, animals and the environment. In all Rothia genomes, we identified biosynthetic gene clusters predicted to produce antibiotic non-ribosomal peptides, iron scavenging siderophores and other secondary metabolites that modulate microbe-microbe and potentially microbe-host interactions. In vitro overlay inhibition assays corroborated the hypothesis that specific strains produce natural antibiotics. Rothia genomes encode a large number of carbohydrate-active enzymes (CAZy), with varying CAZy activities among the species found in different hosts, host niches and environments. These findings reveal competition mechanisms and metabolic specializations linked to ecological adaptation of Rothia species in different hosts.

Temporal and Spatial Variation of the Skin-Associated Bacteria from Healthy Participants and Atopic Dermatitis Patients.

Several factors have been shown to influence the composition of the bacterial communities inhabiting healthy skin, with variation between different individuals, differing skin depths, and body locations (spatial-temporal variation). Atopic dermatitis (AD) is a chronic skin disease also affecting the skin-associated bacterial communities. While the effects of AD have been studied on these processes individually, few have considered how AD disrupts the spatial-temporal variation of the skin bacteria as a whole (i.e., considered these processes simultaneously). Here, we characterized the skin-associated bacterial communities of healthy volunteers and lesional and nonlesional skin of AD patients by metabarcoding the universal V3-V4 16S rRNA region from tape strip skin samples. We quantified the spatial-temporal variation (interindividual variation, differing skin depths, multiple time points) of the skin-associated bacteria within healthy controls and AD patients, including the relative change induced by AD in each. Interindividual variation correlated with the bacterial community far more strongly than any other factors followed by skin depth and then AD status. There was no significant temporal variation found within either AD patients or healthy controls. The bacterial community was found to vary markedly according to AD severity, and between patients without and with filaggrin mutations. Therefore, future studies may benefit from sampling subsurface epidermal communities and considering AD severity and the host genome in understanding the role of the skin bacterial community within AD pathogenesis rather than considering AD as a presence-absence disorder. IMPORTANCE The bacteria associated with human skin may influence skin barrier function and the immune response. Previous studies have attempted to understand the factors that regulate the skin bacteria, characterizing the spatial-temporal variation of the skin bacteria within unaffected skin. Here, we quantified the effect of AD on the skin bacteria on multiple spatial-temporal factors simultaneously. Although significant community variation between healthy controls and AD patients was observed, the effects of AD on the overall bacterial community were relatively low compared to other measured factors. Results here suggest that changes in specific taxa rather than wholesale changes in the skin bacteria are associated with mild to moderate AD. Further studies would benefit from incorporating the complexity of AD into models to better understand the condition, including AD severity and the host genome, alongside microbial composition.

Skin Microbiome in Patients with Hand Eczema and Healthy Controls: A Three-week Prospective Study.

The pathogenesis of chronic hand eczema remains unclear. Insights into the skin microbiome in hand eczema and its potential relevance to disease severity may help to elucidate the underlying mechanisms of hand eczema. The aim of this study was to characterize the microbiome in patients with hand eczema and healthy controls. A 5-visit prospective study was conducted over a period of 3 weeks. At each visit, bacterial swabs were taken from the hands of patients with hand eczema and controls. The microbiome was examined using DNA extraction and 16S rRNA amplicon sequencing (V3-V4 regions). Fifty patients with hand eczema and 50 controls were included (follow-up rate=100%). The baseline bacterial α-diversity was reduced on the hands of patients with hand eczema compared with controls (effect size=-0.31; 95% confidence interval (95% CI) -0.50; -0.11; p = 0.003). The dysbiosis on the patients' hands was stable over the study period, was associated with disease severity, and was characterized by reduced bacterial diversity and different bacterial community compositions.

Changes in Skin and Nasal Microbiome and Staphylococcal Species Following Treatment of Atopic Dermatitis with Dupilumab.

Investigation of changes in the skin microbiome following treatment of atopic dermatitis (AD) with dupilumab may provide valuable insights into the skin microbiome as a therapeutic target. The aim of this study is to assess changes in the AD skin microbiome following treatment of AD with dupilumab (n = 27). E-swabs were collected from nose, lesional, and nonlesional skin before and after 16 weeks of dupilumab therapy, and the microbiome was analyzed by 16S rRNA and tuf gene sequencing. Data for 17 patients with milder disease receiving treatment with non-targeted therapies are also presented. The results show that both groups experienced clinical improvement (p < 0.001) following dupilumab therapy and that Shannon diversity increased and bacterial community structure changed. The relative abundance of the genus Staphylococcus (S.) and S. aureus decreased, while that of S. epidermidis and S. hominis increased. No significant changes were observed for patients receiving non-targeted treatments. The increases in S. epidermidis and S. hominis and the decrease in S. aureus correlated with clinical improvement. Furthermore, changes in S. hominis and S. epidermidis correlated inversely with S. aureus. In conclusion, treatment with dupilumab significantly changed the skin microbiome and decreased S. aureus. Our results suggest a favorable role of commensal staphylococci in AD.

Wearing Occlusive Gloves Increases the Density of Staphylococcus aureus in Patients with Hand Eczema.

Hand eczema is frequently colonized with Staphylococcus aureus. Some patients with hand eczema wear occlusive gloves regularly; however, the effect of this on the density of S. aureus is unexplored. The aim of this study is to examine the effect of occlusive gloves on the density of S. aureus sampled from the hands of patients with hand eczema. In an experimental set-up, patients with moderate-to-severe hand eczema wore an occlusive glove on one hand for 4 h with a 30-min break. Bacterial swabs were collected from the most severe eczema lesion on the hand before and immediately after glove exposure. S. aureus colony-forming units were counted and log-transformations used for comparison of before- and after-values. Among 30 patients, 19 (63%) were colonized with S. aureus. After glove occlusion S. aureus colony-forming units increased by a factor of 1.72 (p < 0.01). In conclusion, the density of sampled S. aureus on eczematous skin after prolonged wearing of occlusive gloves is greatly increased.

Staphylococcal Communities on Skin Are Associated with Atopic Dermatitis and Disease Severity.

The skin microbiota of atopic dermatitis (AD) patients is characterized by increased Staphylococcus aureus colonization, which exacerbates disease symptoms and has been linked to reduced bacterial diversity. Skin bacterial communities in AD patients have mostly been described at family and genus levels, while species-level characterization has been limited. In this study, we investigated the role of the bacteria belonging to the Staphylococcus genus using targeted sequencing of the tuf gene with genus-specific primers. We compared staphylococcal communities on lesional and non-lesional skin of AD patients, as well as AD patients with healthy controls, and determined the absolute abundance of bacteria present at each site. We observed that the staphylococcal community, bacterial alpha diversity, and bacterial densities were similar on lesional and non-lesional skin, whereas AD severity was associated with significant changes in staphylococcal composition. Increased S. aureus, Staphylococcus capitis, and Staphylococcus lugdunensis abundances were correlated with increased severity. Conversely, Staphylococcus hominis abundance was negatively correlated with severity. Furthermore, S. hominis relative abundance was reduced on AD skin compared to healthy skin. In conclusion, various staphylococcal species appear to be important for skin health.

Identification of cutaneous fungi and mites in adult atopic dermatitis: analysis by targeted 18S rRNA amplicon sequencing.

BACKGROUND: Atopic dermatitis (AD) patients have an altered skin bacterial community, with an abundance of Staphylococcus aureus associated with flares, highlighting that microbial organisms may be important for disease exacerbation. Despite strong evidence of association between bacterial skin colonisation and AD, very limited knowledge regarding the eukaryotic microbial community, including fungi and ectoparasites, in AD exists. In this study, we compared the skin and nasal eukaryotic microbial community between adult AD patients (n = 55) and non-AD healthy controls (n = 45) using targeted 18S rRNA amplicon sequencing. Analysis was based on the presence or absence of eukaryotic microorganisms. RESULTS: The cutaneous composition of the eukaryotic microbial community and the alpha-diversity differed significantly between AD patients and non-AD individuals, with increased species richness on AD skin. Alpha-diversity and beta-diversity were similar on lesional and non-lesional skin of patients. The ectoparasite Demodex folliculorum and the yeast Geotrichum candidum were significantly more prevalent on the skin of AD patients. The prevalence of D. folliculorum on lesional skin was greater among patients recently treated with topical corticosteroid. Malassezia was one of the most frequently detected genera at all sites, with M. globosa and M. restricta being the most prevalent. M. restricta was under represented in the anterior nares of AD patients as compared to the non-AD control population. CONCLUSION: Significant differences in the eukaryotic microbial communities were found between AD patients and non-AD individuals, with the most striking finding being the significantly overrepresentation of D. folliculorum on AD skin. Whether D. folliculorum can contribute to skin inflammation in AD needs further investigation.

Alteration of Bacterial Communities in Anterior Nares and Skin Sites of Patients Undergoing Arthroplasty Surgery: Analysis by 16S rRNA and Staphylococcal-Specific tuf Gene Sequencing.

The aim was to study alterations of bacterial communities in patients undergoing hip or knee arthroplasty to assess the impact of chlorhexidine gluconate soap decolonisation and systemic antibiotic prophylaxis. A Swedish multicentre, prospective collection of samples obtained from elective arthroplasty patients (n = 83) by swabbing anterior nares, skin sites in the groin and the site of planned surgery, before and after arthroplasty surgery, was analysed by 16S rRNA (V3-V4) gene sequencing and a complementary targeted tuf gene sequencing approach to comprehensively characterise alterations in staphylococcal communities. Significant reductions in alpha diversity was detected for both bacterial (p = 0.04) and staphylococcal (p = 0.03) groin communities after arthroplasty surgery with significant reductions in relative Corynebacterium (p = 0.001) abundance and Staphylococcus hominis (p = 0.01) relative staphylococcal abundance. In nares, significant reductions occurred for Staphylococcus hominis (p = 0.02), Staphylococcus haemolyticus (p = 0.02), and Staphylococcus pasteuri (p = 0.003) relative to other staphylococci. Staphylococcus aureus colonised 35% of anterior nares before and 26% after arthroplasty surgery. Staphylococcus epidermidis was the most abundant staphylococcal species at all sampling sites. No bacterial genus or staphylococcal species increased significantly after arthroplasty surgery. Application of a targeted tuf gene sequencing approach provided auxiliary staphylococcal community profiles and allowed species-level characterisation directly from low biomass clinical samples.

The Epidome - a species-specific approach to assess the population structure and heterogeneity of Staphylococcus epidermidis colonization and infection.

BACKGROUND: Although generally known as a human commensal, Staphylococcus epidermidis is also an opportunistic pathogen that can cause nosocomial infections related to foreign body materials and immunocompromized patients. Infections are often caused by multidrug-resistant (MDR) lineages that are difficult and costly to treat, and can have a major adverse impact on patients' quality of life. Heterogeneity is a common phenomenon in both carriage and infection, but present methodology for detection of this is laborious or expensive. In this study, we present a culture-independent method, labelled Epidome, based on an amplicon sequencing-approach to deliver information beyond species level on primary samples and to elucidate clonality, population structure and temporal stability or niche selection of S. epidermidis communities. RESULTS: Based on an assessment of > 800 genes from the S. epidermidis core genome, we identified genes with variable regions, which in combination facilitated the differentiation of phylogenetic clusters observed in silico, and allowed classification down to lineage level. A duplex PCR, combined with an amplicon sequencing protocol, and a downstream analysis pipeline were designed to provide subspecies information from primary samples. Additionally, a probe-based qPCR was designed to provide valuable absolute abundance quantification of S. epidermidis. The approach was validated on isolates representing skin commensals and on genomic mock communities with a sensitivity of < 10 copies/µL. The method was furthermore applied to a sample set of primary skin and nasal samples, revealing a high degree of heterogeneity in the S. epidermidis populations. Additionally, the qPCR showed a high degree of variation in absolute abundance of S. epidermidis. CONCLUSIONS: The Epidome method is designed for use on primary samples to obtain important information on S. epidermidis abundance and diversity beyond species-level to answer questions regarding the emergence and dissemination of nosocomial lineages, investigating clonality of S. epidermidis communities, population dynamics, and niche selection. Our targeted-sequencing method allows rapid differentiation and identification of clinically important nosocomial lineages in low-biomass samples such as skin samples.

Genomic analyses of Staphylococcus aureus clonal complex 45 isolates does not distinguish nasal carriage from bacteraemia.

Staphylococcus aureus is a colonizing opportunistic pathogen and a leading cause of bloodstream infection with high morbidity and mortality. S. aureus carriage frequency is reportedly between 20 and 40 % among healthy adults, with S. aureus colonization considered to be a risk factor for S. aureus bacteraemia. It is unknown whether a genetic component of the bacterium is associated with S. aureus bacteraemia in comparison to nasal carriage strains. Previous association studies primarily focusing on the clinical outcome of an S. aureus infection have produced conflicting results, often limited by study design challenged by sample collections and the clonal diversity of S. aureus. To date, no study has investigated whether genomic features separate nasal carriage isolates from S. aureus bacteraemia isolates within a single clonal lineage. Here we have investigated whether genomic features, including single-nucleotide polymorphisms (SNPs), genes, or kmers, distinguish S. aureus nasal carriage isolates from bacteraemia isolates that all belong to the same clonal lineage [clonal complex 45 (CC45)] using whole-genome sequencing (WGS) and a genome-wide association (GWA) approach. From CC45, 100 isolates (50 bacteraemia and 50 nasal carriage, geographically and temporally matched) from Denmark were whole-genome sequenced and subjected to GWA analyses involving gene copy number variation, SNPs, gene content, kmers and gene combinations, while correcting for lineage effects. No statistically significant association involving SNPs, specific genes, gene variants, gene copy number variation, or a combination of genes was identified that could distinguish bacteraemia isolates from nasal carriage isolates. The presented results suggest that all S. aureus nasal CC45 isolates carry the potential to cause invasive disease, as no core or accessory genome content or variations were statistically associated with invasiveness.

Evolution and Population Dynamics of Clonal Complex 152 Community-Associated Methicillin-Resistant Staphylococcus aureus.

Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe.IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

High persister cell formation by clinical Staphylococcus aureus strains belonging to clonal complex 30.

Bacterial persisters form a subpopulation of cells that survive lethal concentrations of antibiotics without being genetically different from the susceptible population. They are generally considered to be phenotypic variants that spontaneously have entered a dormant state with low ATP levels or reduced membrane potential. In Staphylococcus aureus, a serious opportunistic human pathogen, persisters are believed to contribute to chronic infections that are a major global healthcare problem. While S. aureus persisters have mostly been studied in laboratory strains, we have here investigated the ability of clinical strains to form persisters. For 44 clinical strains belonging to the major clonal complexes CC5, CC8, CC30 or CC45, we examined persister cell formation in stationary phase when exposed to 100 times the MIC of ciprofloxacin, an antibiotic that targets DNA replication. We find that while all strains are able to form persisters, those belonging to CC30 displayed on average 100-fold higher persister cell frequencies when compared to strains of other CCs. Importantly, there was no correlation between persister formation and the cellular ATP content of the individual strains, but the group of CC30 strains displayed slightly lower membrane potential compared to the non-CC30 group. CC30 strains have previously been associated with chronic and reoccuring infections and we hypothesize that there could be a correlation between lineage-specific characteristics displayed via in vitro persister assays and the observed clinical spectrum of disease.

Cohort description: The Danish Blood Donor Staphylococcus aureus Carriage Study.

PURPOSE: Staphylococcus aureus carriage poses an increased risk of S. aureus infection. The aim of this study was to investigate the colonization of S. aureus among healthy individuals and to establish a prospective cohort and biobank for research in the health consequences of colonization. POPULATION AND METHODS: The Danish Blood Donor S. aureus Carriage Study (DBDSaCS) was established in 2014. So far, a total of 6082 healthy participants have been included with nasal swabs and repeated swabs are performed at subsequent donations. Samples from the first 2217 participants were cultured using a two-step method to evaluate the effect of using enrichment broth. Furthermore, 262 participants were sampled from both the nares and the throat. All participants completed a questionnaire with self-reported health, anthropometric measurements, current smoking status, and physical activity. Plasma samples, nasal swab transport media, and S. aureus isolates were stored. RESULTS: The prevalence of S. aureus nasal colonization was 41%. The prevalence of colonization was higher in men (46%) than women (34%), lower for smokers, and decreased with increasing age (<25 years: 44% vs >55 years: 35%). In participants swabbed from the nose and throat, the prevalence of S. aureus colonization after enrichment was 55% with significantly higher prevalence in the throat (45%) than in the nose (40%). The use of an enrichment broth increased the proportion of S. aureus colonization. CONCLUSION: We describe a large and growing cohort of healthy individuals established to investigate predictors for S. aureus carriage and the health consequences of carriage. Multiple projects using data from DBDSaCS linked with Danish health registers, biomarkers, and genetic markers are ongoing. Results will be published in the coming years.