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:In principle, whole-genome sequencing of Staphylococcus aureus in hospitals is most effective when used prospectively, but its practical limits are unclear. METHODS:We performed large-scale genomic surveillance across two interconnected hospitals, sequencing 4,779 S. aureus isolates from admission screening and clinical cultures. Integration of genomic and epidemiologic data identified 361 transmission events undetected by standard surveillance. RESULTS:Despite dense sampling, most events (90%) were detected only at readmission, indicating that transmission escapes recognition during the index hospitalization. Serial or discharge screening is likely required to capture transmission. Detection depended on sampling: clinical isolates alone were insufficient, and nearly all events required screening and repeated sampling. CONCLUSIONS:Because such approaches are unlikely to scale if applied universally, surveillance must be targeted. Transmission concentrated in methicillin-resistant strains, and it increased when healthcare exposure coincided with hospital-associated strain lineage. These findings help define the limits of prospective genomic surveillance and provide a framework for targeted detection.

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.

Atopic dermatitis (AD) is a prevalent inflammatory skin disease with complex pathogenesis. Both skin and gut microbiota influence AD, with Staphylococcus aureus , in particular, exacerbating the disease. However, the relationship between S. aureus colonization in the gut and skin, and whether it affects AD, remains unclear. Using a combination of culture-based methods, microbiome analysis, and genome sequencing of S. aureus from multiple body sites of children with and without AD, we found that the gut represents a major reservoir of genetically diverse S. aureus that is transmitted to the skin, including mutants associated with worse disease. We validated this association between S. aureus gastrointestinal colonization and AD in an independent human cohort and demonstrated its direct effect on disease in an infantile AD mouse model, wherein S. aureus gastrointestinal colonization worsened skin inflammation. Overall, this study identifies a previously unrecognized S. aureus reservoir, with implications for microbiota-targeting therapies in AD. ### Competing Interest Statement The authors have declared no competing interest.

Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.

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.

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.

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.

Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment.

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 .