Although single-patient rooms are recommended for hospital design to reduce healthcare-associated infections, construction is costly, and supporting evidence remains limited. Most studies rely on pre/post time series analyses that cannot account for concurrent infection control and antimicrobial stewardship initiatives. To address this gap, we used comprehensive, hospital-wide whole-genome sequencing (WGS) surveillance to compare Staphylococcus aureus transmission rates in single- versus multi-bed rooms during routine clinical operations. Across two hospitals in an urban health system, ∼3,000 patients per month underwent active S. aureus surveillance using admission nasal swabs. Surveillance and clinical (blood, sputum, wound) isolates collected between October 2022 - December 2023 underwent WGS. Closely related isolates (< 20 single nucleotide polymorphisms) that were epidemiologically linked based on timestamped patient location data were classified as high-probability transmissions. We compared S. aureus transmission rates among single, two-bed, and four-bed rooms, all of which underwent identical cleaning and sporicidal disinfection protocols. Over 14-months, >5,000 isolates from 4,000 patients underwent WGS, 85% of which were from surveillance cultures. Of admissions, 21% were to single rooms, 59% to two-bedded rooms, and 20% to four-bedded rooms. Transmission risk was lowest in single rooms (0.5 transmissions per 1000 admissions), increased in two-bed rooms (1.2 per 1000 admissions), and was nearly ninefold higher in four-bed rooms compared to single rooms (4.4 transmissions per 1000 admissions). In this large WGS-based surveillance study, single and semi-private (two-bed) rooms were associated with substantially lower S. aureus transmission rates compared to four-bed rooms. These findings indicate that room occupancy is a modifiable factor in transmission risk and that standard infection control practices may be insufficient; therefore, innovative, continuous disinfection strategies are warranted. Ongoing work is adjusting for potential differences in patient populations across room types to refine estimates of the impact of room occupancy on transmission. All Authors: No reported disclosures

Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus detect SCCmec (Xpert) or the SCCmec–orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas–associated SCCmec variant, leading to identification of 64 variants from 45 patients—2% of 2432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.

Staphylococcus aureus colonization precedes invasive infection. Hospitalized patients with S. aureus colonization can transmit to other patients, but community-based S. aureus transmissions are not well characterized. We used comprehensive, hospital-wide whole genome sequencing (WGS) of S. aureus isolates coupled with demographic data to characterize community-based S. aureus transmission clusters. 8,567 S. aureus isolates from clinical and surveillance cultures collected between Oct 2022-Dec 2023 underwent WGS, with epidemiological and demographic data pulled from the electronic health record (EHR). Closely related isolates (< 20 single nucleotide polymorphisms) without in-hospital epidemiologic links underwent chart review, looking for community-based transmission clusters. Data was analyzed with Microsoft Excel. The five largest clusters of closely related isolates lacking in-hospital epidemiologic links were all methicillin-resistant (MRSA). Cluster 1 included 23 patients with clonal complex 8/USA300, 83% were male with mean age 41 years, with high rates of substance use (71%), HIV (52%), and self-reported men who have sex with men (MSM, 35%). Cluster 5 (8 patients, clonal complex 30) had similar demographics as cluster 1 but with higher rates of substance use (87%), with half of patients found down prior to admission. All clinical cultures in clusters 1 and 5 were from skin and soft tissue infections (SSTI). Patients in clusters 2 and 3 (n=27 combined, both clonal complex 5) all resided in Brooklyn, NY, with mean age 67-75 years, 75% had recent nursing home or group home exposure and were more likely to have bacteremia and no SSTI. Patients in cluster 4 (n=9, clonal complex 8) were children with a mean age of 2.5 years, 2/3 of whom had MRSA colonization detected while in the neonatal intensive care unit;1/3 had SSTI and were from Orthodox Jewish communities in Brooklyn. WGS of S. aureus isolates obtained in the hospital identified large community transmission clusters, highlighting a need to integrate community risk factors with hospital-based infection prevention strategies. Future interventions targeting risk profiles can enhance efforts to prevent hospital-acquired S. aureus infections. All Authors: No reported disclosures

Background: Staphylococcus aureus is a leading cause of healthcare-associated infections and is associated with high mortality. While decolonization has been effective in reducing methicillin-resistant S. aureus (MRSA) infections in adult and neonatal intensive care units (NICUs), little is known about the impact of decolonization on transmission of S. aureus. Here, we evaluated whether weekly screening and targeted decolonization reduce genomically defined transmission of S. aureus in a NICU. Methods: Infants admitted in 2022-2024 to the NICU at Tisch Hospital received weekly screening cultures of the nares, axilla and groin for MRSA and methicillin-susceptible S. aureus (MSSA). Colonized infants received topical decolonization with chlorhexidine 2% bathing and nasal, buttock, and umbilical mupirocin, with frequency and duration of decolonization based on postmenstrual age (gestational plus chronological age). Genome sequencing was used to identify transmission events, defined as genetically linked S. aureus isolates with <20 single nucleotide variants identified in two infants with overlapping stays. Transmission risk was analyzed using time-to-event (TTE) analyses, and the absolute risk reduction and number needed to treat (NNT) were estimated. Results: Among 1,597 screened infants, 188 (11.8%) were colonized with S. aureus (85.6% MSSA; 14.4% MRSA). Colonized infants had a median NICU length of stay of 96 days, and 39 (20.7%) were involved in at least one transmission event. Of these infants, 84.6% received full decolonization. Across the cohort, 389 conversions from colonization negative to positive were observed, including 97 recolonization events; 55 (56.7%) of these infants became colonized ? 3 times during their stay, indicating sustained exposure and transmission pressure. TTE modeling predicted a 30-day transmission risk of 0.6% if all patients were decolonized, and 8.9% risk if no patients were decolonized. This corresponds to a 30-day absolute risk reduction for transmission of 8.2% and an NNT of 12; decolonizing 12 infants prevented one S. aureus transmission. Conclusions: Genomically informed modeling indicates that weekly screening and targeted decolonization reduce S. aureus transmission in the NICU. Frequent recolonization supports the importance of weekly S. aureus screening and suggests that decolonization prevents infection partly by limiting spread to susceptible hosts, with implications for the spread of resistant strains. Future analyses incorporating time-varying eligibility and exposure will further refine these transmission estimates.

Staphylococcus aureus causes approximately 80% of skin and soft tissue infections (SSTIs). Collagen is the most abundant human extracellular matrix protein with critical roles in wound healing, and S. aureus encodes a collagen binding adhesin (Cna). The role of this protein during skin infections is unknown. Here we report that inability to bind collagen results in worsened pathology of intradermal δcna S. aureus infection. WT/Cna+ S. aureus showed reduced infection severity, aggregate formation, and significantly improved clearance of bacteria. Cna binds to the collagen-like domain of serum C1q protein to reduce its opsonophagocytic functions. We demonstrate that infection of C1qKO mice with WT bacteria show results similar to the δcna group. Conversely, inability to bind collagen resulted in an amplified inflammatory response caused in part by macrophage and neutrophil small molecule mediators released at the infection site (MMP-9, MMP-12, LTB4), resulting in increased immune cell infiltration and death.

Background:Resistance to mupirocin, a cornerstone of Staphylococcus aureus decolonization, is a recognized cause of decolonization failure. Its role in hospital transmission is unknown. Methods:We conducted genomic surveillance of >10,000 S. aureus isolates from adult patients at two urban hospitals where mupirocin decolonization is routine. Bacterial phenotypes and fitness were evaluated in vitro and in murine colonization models to interpret surveillance results. Results:Genome sequencing identified 475 hospital transmission events; conventional surveillance detected none. The plasmid-mediated resistance determinant mupA ( ileS2 ) was enriched eightfold in methicillin-resistant S. aureus (MRSA) relative to methicillin-susceptible strains. mupA was associated with nearly threefold greater hospital transmission, especially within healthcare-associated MRSA lineages. Surprisingly, multiple independently evolved inactivating mutations in the essential chromosomal gene ileS1 co-occurred with mupA , creating plasmid addiction in which mupA became indispensable for bacterial survival. Plasmid carriage activated the stringent response and reduced colonization fitness, but also conferred collateral tolerance to disinfectants such as ethanol and peroxide. Although addiction further reduced S. aureus fitness, it increased plasmid transfer, promoting spread despite these costs. Unexpectedly, we found a mupirocin-dependent vulnerability to isoleucine limitation, revealing a potential strategy to target mupA -mediated resistance. Conclusions:Hospital transmission of mupirocin-resistant MRSA is promoted by plasmids that create an evolutionary trap in which mupirocin use selects for bacterial dependence on costly resistance elements. These findings suggest that reducing mupirocin use alone is unlikely to eliminate resistance, underscore the need for genomic surveillance and resistance testing, and provide a framework for strategies to preserve mupirocin effectiveness. Major point:This work shows that mupirocin resistance promotes hospital transmission of MRSA, identifies a previously unappreciated mechanism of plasmid addiction, and exposes a collateral vulnerability. These findings underscore the need for genomic surveillance and provide a framework to preserve mupirocin effectiveness.

Background: Staphylococcus aureus is a leading cause of healthcare-associated infections and is associated with high mortality. While decolonization has been effective in reducing methicillin-resistant S. aureus (MRSA) infections in adult and neonatal intensive care units (NICUs), little is known about the impact of decolonization on transmission of S. aureus. Here, we evaluated whether weekly screening and targeted decolonization reduce genomically defined transmission of S. aureus in a NICU. Methods: Infants admitted in 2022-2024 to the NICU at Tisch Hospital received weekly screening cultures of the nares, axilla and groin for MRSA and methicillin-susceptible S. aureus (MSSA). Colonized infants received topical decolonization with chlorhexidine 2% bathing and nasal, buttock, and umbilical mupirocin, with frequency and duration of decolonization based on postmenstrual age (gestational plus chronological age). Genome sequencing was used to identify transmission events, defined as genetically linked S. aureus isolates with <20 single nucleotide variants identified in two infants with overlapping stays. Transmission risk was analyzed using time-to-event (TTE) analyses, and the absolute risk reduction and number needed to treat (NNT) were estimated. Results: Among 1,597 screened infants, 188 (11.8%) were colonized with S. aureus (85.6% MSSA; 14.4% MRSA). Colonized infants had a median NICU length of stay of 96 days, and 39 (20.7%) were involved in at least one transmission event. Of these infants, 84.6% received full decolonization. Across the cohort, 389 conversions from colonization negative to positive were observed, including 97 recolonization events; 55 (56.7%) of these infants became colonized ? 3 times during their stay, indicating sustained exposure and transmission pressure. TTE modeling predicted a 30-day transmission risk of 0.6% if all patients were decolonized, and 8.9% risk if no patients were decolonized. This corresponds to a 30-day absolute risk reduction for transmission of 8.2% and an NNT of 12; decolonizing 12 infants prevented one S. aureus transmission. Conclusions: Genomically informed modeling indicates that weekly screening and targeted decolonization reduce S. aureus transmission in the NICU. Frequent recolonization supports the importance of weekly S. aureus screening and suggests that decolonization prevents infection partly by limiting spread to susceptible hosts, with implications for the spread of resistant strains. Future analyses incorporating time-varying eligibility and exposure will further refine these transmission estimates.

Gastrointestinal (GI) colonization by methicillin-resistant Staphylococcus aureus (MRSA) is associated with a high risk of transmission and invasive disease in vulnerable populations. The immune and microbial factors that permit GI colonization remain unknown. Male sex is correlated with enhanced Staphylococcus aureus nasal carriage, skin and soft tissue infections, and bacterial sepsis. Here, we established a mouse model of sexual dimorphism during GI colonization by MRSA. Our results show that in contrast to male mice that were susceptible to persistent colonization, female mice rapidly cleared MRSA from the GI tract following oral inoculation in a manner dependent on the gut microbiota. This colonization resistance displayed by female mice was mediated by an increase in IL-17A+ CD4+ T cells (Th17) and dependent on neutrophils. Ovariectomy of female mice increased MRSA burden, but gonadal female mice that have the Y chromosome retained enhanced Th17 responses and colonization resistance. Our study reveals a novel intersection between sex and gut microbiota underlying colonization resistance against a major widespread pathogen.

Genomic surveillance of Staphylococcus aureus in hospitals usually focuses on clinical infections, missing transmissions from asymptomatic carriers and delaying detection and timely intervention. To address the issue, we performed whole-genome sequencing (WGS) on over 5,000 S. aureus isolates obtained from colonization screens at admission, in addition to standard clinical cultures, at two interconnected urban hospitals. By integrating genomic data with timestamped location information, we identified hundreds of transmissions missed by standard methods. However, nearly 70% of transmissions were detected during readmission after the index case had been discharged. This finding indicates that even with dense genomic sampling, real-time detection remains challenging due to asymptomatic carriage. Therefore, effective monitoring of nosocomial S. aureus transmission will likely require WGS and colonization sampling at both admission and discharge. The data also highlight patient- and strain-specific factors, including methicillin resistance, as predictors of S. aureus spread, which may enable cost-effective, targeted sequencing surveillance strategies.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present study shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a critical mechanism - epigenetic regulation of staphylococcal gene expression - by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Pathogens have evolved to be highly adapted to their natural host. Community-associated methicillin-resistant Staphylococcus aureus USA300, for instance, is a lineage responsible for the epidemic of skin and soft tissue infections (SSTIs) in humans. Owing to its human tropism, mechanisms that enabled the rise of USA300 as a major skin pathogen remain incompletely defined. By leveraging a rodent-adapted strain of S. aureus, we developed a natural model of SSTIs. We found that LukMF', a pore-forming leukocidin homolog to the human-specific LukSF-PV toxin, drives skin pathology in mice. LukMF' lyses neutrophils via the chemokine receptor CCR1, which in turn fuels inflammatory pathology and microbial survival within the infectious nidus. Ablation of CCR1, depletion of neutrophils, or vaccination with LukMF' all protected mice from skin pathology. Thus, these data support epidemiological studies linking leukocidins with human SSTIs and highlight the power of natural models to unearth potential targets to curtail infections.

Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N 6 -methyladenosine (m 6 A) at the level of individual RNAs. However, a lack of systematic benchmarking continues to raise questions regarding the sensitivity, specificity, and reproducibility of this method. To address this and to establish a best-practice workflow, we evaluated multiple Dorado versions using in vitro transcribed RNA and an m 6 A methyltransferase inhibitor as specificity controls. We established that stringent filtering is necessary to reduce false-positive calls and found strong concordance at high-stoichiometry sites when compared to an orthogonal m 6 A mapping method (GLORI). Further, by applying DRS to primary human fibroblasts and HD10.6 neurons, we uncovered cell type-specific differences in m 6 A stoichiometry, indicating a finely tuned epitranscriptomic regulation. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m 6 A epitranscriptome to fibroblasts and neurons.

The inflammasome plays multifaceted roles in cancer, but less is known about its function during premalignancy upon initial cell transformation. We report a homeostatic function of the inflammasome in suppressing malignant transformation through Ras inhibition. We identified increased hematopoietic stem cell (HSC) proliferation within the bone marrow of inflammasome-deficient mice. HSCs within an inflammasome-deficient stroma expressed a Ras signature associated with increased Ras pathway- and cancer-related transcripts and heightened levels of cytokine, chemokine and growth factor receptors. Stromal inflammasome deficiency established a poised Ras-dependent mitogenic state within HSCs, which fueled progeny B cell lymphomagenesis upon Myc deregulation in a spontaneous model of B cell lymphoma, and shortened its premalignant stage leading to faster onset of malignancy. Thus, the stromal inflammasome preserves tissue balance by restraining Ras to disrupt the most common oncogenic Myc–Ras cooperation and establish a natural defense against transition to malignancy. These findings should inform preventative therapies against hematological malignancies. Blander and colleagues report a homeostatic regulatory effect played by inflammasomes in the bone marrow stromal compartment that suppresses premalignant stages of lymphomagenesis.

Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N6-methyladenosine (m6A) at the level of individual RNAs. However, a lack of systematic benchmarking continues to raise questions regarding the sensitivity, specificity, and reproducibility of this method. To address this and to establish a best-practice workflow, we evaluated multiple Dorado versions using in vitro transcribed RNA and an m6A methyltransferase inhibitor as specificity controls. We established that stringent filtering is necessary to reduce false-positive calls and found strong concordance at high-stoichiometry sites when compared to an orthogonal m6A mapping method (GLORI). Further, by applying DRS to human primary fibroblasts and HD10.6 neurons, we uncovered cell type-specific differences in m6A stoichiometry, indicating a finely tuned epitranscriptomic regulation. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m6A epitranscriptome to fibroblasts and neurons.

Sulfasalazine is a prodrug known to be effective for the treatment of inflammatory bowel disease (IBD)-associated peripheral spondyloarthritis (pSpA), but the mechanistic role for the gut microbiome in regulating its clinical efficacy is not well understood. Here, treatment of 22 IBD-pSpA subjects with sulfasalazine identifies clinical responders with a gut microbiome enriched in Faecalibacterium prausnitzii and the capacity for butyrate production. Sulfapyridine promotes butyrate production and transcription of the butyrate synthesis gene but in F. prausnitzii in vitro, which is suppressed by excess folate. Sulfasalazine therapy enhances fecal butyrate production and limits colitis in wild-type and gnotobiotic mice colonized with responder, but not non-responder, microbiomes. F. prausnitzii is sufficient to restore sulfasalazine protection from colitis in gnotobiotic mice colonized with non-responder microbiomes. These findings reveal a mechanistic link between the efficacy of sulfasalazine therapy and the gut microbiome with the potential to guide diagnostic and therapeutic approaches for IBD-pSpA.

In Mycobacterium tuberculosis (Mtb), proteins that are posttranslationally modified with a prokaryotic ubiquitin-like protein (Pup) can be degraded by bacterial proteasomes. A single Pup-ligase and depupylase shape the pupylome, but the mechanisms regulating their substrate specificity are incompletely understood. Here, we identified a depupylation regulator, a protein called CoaX, through its copurification with the depupylase Dop. CoaX is a pseudopantothenate kinase that showed evidence of binding to pantothenate, an essential nutrient Mtb synthesizes, but not its phosphorylation. In a ∆coaX mutant, pantothenate synthesis enzymes including PanB, a substrate of the Pup-proteasome system (PPS), were more abundant than in the parental strain. In vitro, CoaX specifically accelerated depupylation of Pup~PanB, while addition of pantothenate inhibited this reaction. In culture, media supplementation with pantothenate decreased PanB levels, which required CoaX. Collectively, we propose CoaX regulates PanB abundance in response to pantothenate levels by modulating its vulnerability to proteolysis by Mtb proteasomes.

In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a Δ coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro , CoaX accelerated depupylation of Pup∼PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup∼PanB in response to pantothenate levels. One Sentence Summary A pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis .

With the development of sequencing technology and analytic tools, studying within-species variations enhances the understanding of microbial biological processes. Nevertheless, most existing methods designed for strain-level analysis lack the capability to concurrently assess both strain proportions and genome-wide single nucleotide variants (SNVs) across longitudinal metagenomic samples. In this study, we introduce LongStrain, an integrated pipeline for the analysis of large-scale metagenomic data from individuals with longitudinal or repeated samples. In LongStrain, we first utilize two efficient tools, Kraken2 and Bowtie2, for the taxonomic classification and alignment of sequencing reads, respectively. Subsequently, we propose to jointly model strain proportions and shared haplotypes across samples within individuals. This approach specifically targets tracking a primary strain and a secondary strain for each subject, providing their respective proportions and SNVs as output. With extensive simulation studies of a microbial community and single species, our results demonstrate that LongStrain is superior to two genotyping methods and two deconvolution methods across a majority of scenarios. Furthermore, we illustrate the potential applications of LongStrain in the real data analysis of The Environmental Determinants of Diabetes in the Young study and a gastric intestinal metaplasia microbiome study. In summary, the proposed analytic pipeline demonstrates marked statistical efficiency over the same type of methods and has great potential in understanding the genomic variants and dynamic changes at strain level. LongStrain and its tutorial are freely available online at https://github.com/BoyanZhou/LongStrain. IMPORTANCE:The advancement in DNA-sequencing technology has enabled the high-resolution identification of microorganisms in microbial communities. Since different microbial strains within species may contain extreme phenotypic variability (e.g., nutrition metabolism, antibiotic resistance, and pathogen virulence), investigating within-species variations holds great scientific promise in understanding the underlying mechanism of microbial biological processes. To fully utilize the shared genomic variants across longitudinal metagenomics samples collected in microbiome studies, we develop an integrated analytic pipeline (LongStrain) for longitudinal metagenomics data. It concurrently leverages the information on proportions of mapped reads for individual strains and genome-wide SNVs to enhance the efficiency and accuracy of strain identification. Our method helps to understand strains' dynamic changes and their association with genome-wide variants. Given the fast-growing longitudinal studies of microbial communities, LongStrain which streamlines analyses of large-scale raw sequencing data should be of great value in microbiome research communities.

Background: The frequency of Staphylococcus aureus transmission in hospitals is unknown: symptomatic infection may occur months after transmission and colonization, and infection prevention efforts rely on indirect measurements, rather than direct detection of transmission events. We implemented a hospital-based S. aureus screening program, combined with whole genome sequencing of S. aureus surveillance and clinical cultures and data extracted from the electronic health record, to identify S. aureus clonal complex-, patient- and location-specific factors associated with S. aureus transmission in our health system. Methods: Screening S. aureus cultures were obtained at admission by nasal swab for adults admitted to Medicine, Transplant, Oncology and intensive care, and weekly by swab of nares, axilla and groin for children admitted to intensive care and Oncology at NYU Langone Health in New York City. All methicillin-resistant S. aureus (MRSA) from screening and clinical (blood, wound, sputum) cultures and all methicillin-susceptible S. aureus (MSSA) from screening and blood cultures underwent whole genome sequencing. Isolates from distinct patients with < 2 0 single nucleotide pair differences were considered genetically related. Electronic health data was extracted for descriptive statistics and for spatiotemporal plots to assess plausible transmissions. We used REDCap electronic data capture tools hosted at NYU Grossman School of Medicine and SAS software for data analysis to evaluate S. aureus transmissions between November 2022 and November 2023. Results: We analyzed 8,567 S. aureus isolates: including 6,552 screening cultures, 1,008 blood cultures, and 1,007 clinical cultures. We found 424 plausible S. aureus hospital transmissions using sequencing and electronic health data. Screening cultures identified 75% of transmissions that would have otherwise been missed with blood and clinical cultures alone. The majority of positive screening cultures isolated MSSA, but the proportion of transmissions due to MSSA differed by age. In children, MSSA colonization accounted for 62% of transmissions. In adults, only 15% of transmissions were due to MSSA colonization, whereas MRSA colonization accounted for 56% of transmissions. Analysis of adult MRSA isolates by clonal complex found that 45% of transmissions were due to CC8, higher than the 17% among isolates agnostic of transmissions. Emergency departments and the neonatal intensive care unit had the highest number of transmissions. Patients involved in transmissions had longer lengths of stay and frequent hospitalizations. Conclusions: A S. aureus screening program, coupled with genome sequencing and electronic health data, can identify patient group, hospital locations and clonal complexes that are at high risk for S. aureus transmissions.