Exploratory transcriptomic analysis of Staphylococcus aureus adaptation during polymicrobial interactions

Exploratory transcriptomic analysis of Staphylococcus aureus adaptation during polymicrobial interactions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploratory transcriptomic analysis of Staphylococcus aureus adaptation during polymicrobial interactions B. Nirmala, Yogendra Pratap Mathuria, Balram Ji Omar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7401018/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the microbial world, survival is not solitary. Staphylococcus aureus thrives or falters depending on its neighbors. This opportunistic pathogen frequently inhabits polymicrobial environments such as chronic wounds, implanted devices, and mucosal surfaces, where interspecies interactions shape its behavior and complicate treatment outcomes. In this study, we adopt an exploratory, systems-level approach to examine how S. aureus transcriptionally adapts during co-culture with three clinically relevant organisms: Pseudomonas aeruginosa , Candida albicans , and Enterococcus faecalis . RNA sequencing uncovered distinct ecological signatures: P. aeruginosa imposed a strongly antagonistic effect, driving global transcriptional repression, including silencing of virulence pathways; C. albicans promoted a synergistic response with activation of virulence, stress, and metabolic genes; while E. faecalis elicited a competitive program characterized by robust induction of the type VII secretion system, cytotoxic effectors, and biosynthetic functions. Rather than definitive mechanisms, these findings provide an exploratory map of interspecies transcriptional landscapes, generating hypotheses on how microbial neighbors modulate S. aureus biology and highlighting interspecies signaling as a potential target for managing polymicrobial infections. Biological sciences/Microbiology Biological sciences/Molecular biology Candida albicans Enterococcus faecalis Polymicrobial interactions Pseudomonas aeruginosa Transcriptomics Staphylococcus aureus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Microbes rarely live alone. The idea that microbes exist in isolation has been overturned by the discoveries revealing that most pathogens thrive in complex, polymicrobial communities, a concept dating back to the 17th century, when Antonie van Leeuwenhoek first described diverse ‘animalcules’ coexisting in dental plaque 1 . Polymicrobial infections represent a major clinical challenge due to the complex interspecies interactions that influence pathogen behavior, host response, and treatment outcomes 2 . When two microbes meet, they engage in molecular crosstalk through secreted factors, contact-dependent signaling, and competition for nutrients. These interactions can reprogram gene expression, modulate virulence, and reshape microbial physiology, in often unpredictable ways 3 . Among the diverse microbes implicated in such infections, Staphylococcus aureus stands out as a clinically significant, opportunistic pathogen. It is frequently isolated from biofilm-associated infections, including diabetic wounds, cystic fibrosis lungs, indwelling catheters, and prosthetic devices 4 – 7 . The presence of S. aureus in polymicrobial infections has been associated with increased morbidity, prolonged treatment duration, and an elevated mortality risk due to its ability to evade host immunity and resist antibiotics (Nair et al. 2014; Cheung et al. 2021; Howden et al. 2023). Yet, the extent to which these traits are shaped by cohabiting microbial species remains unexplored. Polymicrobial niches often harbor S. aureus alongside Gram-negative bacteria like Pseudomonas aeruginosa 10 , fungi such as Candida albicans 11 , and Gram-positive organisms like Enterococcus faecalis 12 . These interkingdom and intrakingdom interactions can shift S. aureus behavior toward suppression, synergy, or competition depending on the partner species and ecological context 13 , 14 . Such outcomes are not merely phenotypic but are driven by global transcriptional reprogramming that allows S. aureus to fine-tune virulence expression, metabolic adaptation, and stress responses. Transcriptional plasticity has emerged as a key survival strategy for S. aureus , enabling rapid adjustment of regulatory networks in response to fluctuating environmental and interspecies signals 15 . RNA sequencing offers an unprecedented opportunity to capture these global changes and elucidate how co-culture with diverse microbial partners remodels the gene expression landscape of S. aureus . While individual studies have examined binary interactions, systematic comparisons across multiple polymicrobial contexts remain scarce. Exploratory polymicrobial transcriptomics is critical as it enables researchers to capture unbiased, systems-level signatures of microbial crosstalk, beyond what reductionist single-interaction studies can reveal. Such exploratory mapping is critical to identify unexpected transcriptional trends, generate new hypotheses, and build a foundation for mechanistic investigations that directly inform treatment strategies for complex infections. Here, we take an exploratory approach to address this gap. We conducted a transcriptomic survey of S. aureus co-cultured with P. aeruginosa , C. albicans , and E. faecalis , representing taxonomically and functionally diverse neighbors. By integrating RNA sequencing with ultrastructural observations, we provide a systems-level view of S. aureus adaptation across polymicrobial settings. Rather than offering definitive mechanistic conclusions, this work generates testable hypotheses on how interspecies interactions influence S. aureus biology, laying the foundation for targeted strategies to mitigate polymicrobial infections. Methods Bacterial and fungal strains Clinical isolates of S. aureus , P. aeruginosa , E. faecalis , and C. albicans were obtained from the Bacteriology Laboratory, All India Institute of Medical Sciences (AIIMS), Rishikesh, India. Species identification was performed using standard biochemical tests and the VITEK 2 compact system (bioMérieux, Marcy-l’Étoile, France), and confirmed by MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA). The study protocol was approved by the Institutional Ethics Committee, AIIMS Rishikesh (Letter No. AIIMS/IEC/23/294). The study was conducted between January 2024 and May 2025, and all isolates were processed following institutional biosafety protocols. The S. aureus clinical isolate served as the focal organism for all mono- and co-culture experiments. Working stocks of each isolate were prepared from single colonies grown on tryptic soy agar (TSA; HiMedia, Mumbai, India) for bacteria, and Sabouraud dextrose agar (SDA; HiMedia) for C. albicans . Stocks were suspended in sterile 20% glycerol and stored at − 80°C for long-term preservation and at − 20°C for immediate storage. For routine use, working cultures were maintained on the same agar media at 4°C and subcultured when required. For experiments, cultures were revived on the same media and incubated at 37°C for 24 h. Liquid cultures for mono- and co-culture assays were grown in Luria–Bertani (LB) broth (HiMedia) at 37°C under static conditions for 24 h, using mid-log phase inocula standardized to an optical density at 600 nm (OD₆₀₀ ≈ 0.5). No antibiotics were included in co-cultures to avoid selective pressure. Culture purity was verified by colony morphology and Gram staining before each experiment. Polymicrobial co-culture setup Polymicrobial co-cultures were established to assess the influence of interacting partners on the global transcriptional activity of S. aureus . Clinical isolates of P. aeruginosa , C. albicans , and E. faecalis were used as interacting partners. All bacterial strains were maintained on tryptic soy agar (TSA; HiMedia) and C. albicans on Sabouraud dextrose agar (SDA; HiMedia) at 4°C for short-term storage. For experimental use, isolates were revived from storage onto their respective agar plates and incubated overnight at 37°C. Colonies from overnight cultures were resuspended in Luria–Bertani (LB) broth and adjusted to a 0.5 McFarland standard (~ 1.5 × 10⁸ CFU/mL). Equal volumes of S. aureus and the interacting partner were mixed in sterile test tubes and incubated statically at 37°C for 24 h. Monocultures of S. aureus prepared under identical conditions served as controls. After incubation, cultures were processed for downstream analyses, including scanning electron microscopy (SEM), RNA extraction, library preparation, and sequencing. SEM analysis Physical interactions between S. aureus and its co-culture partners were examined using SEM. Sterile glass coverslips were placed in the co-culture setup described above and incubated statically at 37°C for 24 h. Following incubation, coverslips were gently rinsed once by dipping in distilled water (~ 5 seconds) to remove non-adherent cells. Samples were fixed with 4% formaldehyde in 0.1 M phosphate-buffered saline (PBS) for 15–30 minutes, rinsed with PBS, and cross-linked with 2.5% glutaraldehyde in 0.1 M PBS for 2 hours at 4°C. After cross-linking, samples were rinsed with PBS and dehydrated through a graded ethanol series (50%, 70%, 80%, 90%, and 100%), each for 10 min. Dehydrated samples were subjected to critical point drying for 1 hour, mounted on stubs, and sputter-coated with gold using a Q150T S Plus turbomolecular pumped coater (Quorum Technologies Ltd., United Kingdom) 16 . The prepared samples were visualized using a field-emission scanning electron microscope (Gemini 560, Carl Zeiss Meditec AG, Germany), and representative images were captured for analysis of microbial associations. RNA extraction, library preparation, and sequencing Total RNA was extracted directly from each polymicrobial co-culture and the S. aureus monoculture at 24 hours post-inoculation using the HiPurA Total Bacterial RNA Isolation Kit (HiMedia Laboratories, Mumbai, India). RNA integrity was assessed on 1% agarose gel, and RNA concentration was measured using a Qubit® 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA libraries were prepared using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit (Illumina Inc., San Diego, CA, USA). Library fragment size distribution and quality were evaluated on the Agilent TapeStation 4150 system with High Sensitivity D1000 ScreenTape® (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on the Illumina NovaSeq X Plus platform (Illumina Inc., San Diego, CA, USA), generating paired-end 151 bp reads for transcriptomic analysis. Each condition was sequenced as a single biological replicate, with high coverage used to maximize transcriptome representation. Bioinformatics pipeline Raw reads from each condition were quality-checked with FastQC v0.12.1 and preprocessed using fastp v0.23.4 for adapter trimming and quality filtering. De novo transcriptome assembly was performed with Trinity v2.15.1, and assembly quality was assessed using assembly-stats v1.0.1. Transcript abundance was estimated with RSEM v1.3.3, and differential gene expression (DGE) analysis was carried out using edgeR v3.42.4. Genes were considered significantly differentially expressed if they exhibited an absolute log₂ fold change (|log₂FC| ≥ 1) and a false discovery rate (FDR < 0.05), adjusted using the Benjamini–Hochberg correction. Comparisons were made between each polymicrobial condition: S + P ( P. aeruginosa ), S + C ( C. albicans ), and S + E ( E. faecalis ), and the S. aureus monoculture. To ensure species specificity, predicted protein-coding sequences were aligned against the UniProt reviewed S. aureus database using BLAST v2.14.1, and only transcripts with ≥ 50% sequence similarity to S. aureus entries were retained. Coding regions were predicted with TransDecoder v5.5.0 and annotated against the UniProt reviewed database. Finally, annotated DEGs were subjected to Gene Ontology (GO) and KEGG pathway enrichment analyses to identify significantly enriched biological processes and pathways. Visualization and statistical analysis All downstream analyses were performed in Python (v3.10). Data handling and matrix operations were carried out using pandas (v2.0.3) and NumPy (v1.25.0). Differential expression outputs were visualized with matplotlib (v3.7.2) and seaborn (v0.12.2) to generate bar plots, scatter plots, volcano plots, and heatmaps, while Venn diagrams were produced with matplotlib-venn (v0.11.9). Dimensionality reduction and clustering were performed using scikit-learn (v1.3.0) and umap-learn (v0.5.3), whereas network visualizations were created with networkx (v3.1) and Sankey diagrams with plotly (v5.15.0). Enrichment outputs, including Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were plotted in Python using matplotlib and seaborn. Protein–protein interaction (PPI) networks were generated using the STRING web server (v12.0), and exported network views were incorporated for interpretation. All project files, including analysis scripts, pipelines, and processed datasets, are openly available in the GitHub repository (see Data Availability). Results and Discussion SEM analysis of microbial interactions SEM revealed distinct patterns of interaction between S. aureus and its co-culture partners ( P. aeruginosa , C. albicans , and E. faecalis ) (Fig. 1 ). In the monoculture control, S. aureus cells exhibited the characteristic grape-like clustering typical of staphylococcal growth, serving as a baseline for comparison with polymicrobial conditions (Fig. 1 d). In the S. aureus–P. aeruginosa co-culture, the cocci and rod-shaped cells were observed close but without extensive mixing. S. aureus appeared in scattered clusters, while P. aeruginosa maintained elongated arrangements surrounding these clusters (Fig. 1 b). This spatial separation suggests competitive coexistence, consistent with previous studies that describe inhibitory mechanisms employed by P. aeruginosa against S. aureus , including the secretion of biosurfactants and secondary metabolites that limit staphylococcal colonization 17 . Co-culture with C. albicans showed a different pattern. S. aureus cocci adhered extensively to the hyphal filaments of C. albicans , forming dense clusters along the fungal surface (Fig. 1 a). Such physical attachment has been widely documented as a hallmark of cross-kingdom biofilm formation, where fungal hyphae act as scaffolds that facilitate bacterial adhesion and persistence 18 . This structural arrangement highlights the potential for cooperative biofilm establishment and enhanced microbial resilience in polymicrobial infections involving C. albicans . In the S. aureus–E. faecalis co-culture, both species were found in compact aggregates, with S. aureus cells embedded in close contact with Enterococci (Fig. 1 c). The formation of such mixed clusters demonstrates that these organisms occupy shared spatial niches during co-culture. At the structural level, a close association may provide physical protection and stabilization within the biofilm matrix. Previous reports have noted that enterococcal interactions can alter staphylococcal growth and biofilm architecture 12 , 19 , suggesting that tight spatial clustering represents an adaptive arrangement shaped by polymicrobial pressures. Scale bars: 2–5 µm. RNA sequencing quality assessment RNA sequencing produced robust datasets across S. aureus monoculture and co-culture conditions. RNA extractions consistently yielded sufficient material for library preparation, with concentrations above the minimum requirement (≥ 25 ng/µl). Libraries exhibited uniform fragment sizes (411–431 bp) and concentrations (13.3–68.6 ng/µL), confirming the high quality of the input. Sequencing on the Illumina NovaSeq X Plus generated 82–113 million paired-end 151 bp reads per library, providing very high coverage for transcriptomic analysis. FASTQC initially detected adapter sequences and low-quality tails, which were successfully trimmed using fastp. Post-processing retained 97–99% of the reads across all libraries. Base quality was consistently high, with > 93% of bases above Q30 before filtering, improving to 96–97% after filtering, and mean Phred scores approaching 35. GC content across libraries ranged from 40–60%, consistent with expected variation in bacterial transcriptomes. Collectively, these QC metrics confirmed the reliability of the sequencing data and provided a strong foundation for downstream transcriptomic analysis. Global transcriptomic responses of S. aureus during polymicrobial co-culture Following quality control, differential expression analysis revealed partner-specific responses of S. aureus , with DEGs ranging from a few hundred to over a thousand. Co-culture with P. aeruginosa caused broad repression, while C. albicans and E. faecalis induced selective activation of virulence and metabolic pathways. These partner-specific responses are described in detail below. Pseudomonas aeruginosa induces global transcriptional suppression in Staphylococcus aureus during co-culture P. aeruginosa co-culture induced a global transcriptional shutdown in S. aureus , with 230 genes significantly downregulated and only one, hly (alpha-hemolysin), upregulated (log₂FC = + 0.48) (Fig. 2 a). Differentially expressed genes (DEGs) were identified using a stringent statistical threshold of |log₂ fold change| ≥ 1 and FDR < 0.05, ensuring high-confidence calls. This repression was illustrated by Venn and barplot analyses (Fig. 2 b-c). STRING network visualization (Fig. 2 e) revealed strong interconnectivity among the downregulated proteins, implicating core processes such as energy metabolism, translation, and regulatory pathways. In contrast, hly appeared as a solitary, unconnected node (Fig. 2 d), suggesting an independent, stress-induced regulatory response. Functional enrichment analyses (Fig. 2 f-g) identified suppression across critical metabolic and cellular pathways, including amino acid biosynthesis, carbohydrate metabolism, ribosome assembly, and membrane-associated functions. Genes encoding ribosomal proteins ( rplX, rpsD ) 20 and translation factors ( tuf, efp ) 21 were downregulated, indicating a protein synthesis shutdown likely driven by nutrient limitation or quorum-sensing interference from P. aeruginosa . Concurrent repression of TCA cycle enzymes ( mqo2 ) 22 , glycolytic genes ( ackA, ptsG ) 23 , and amino acid biosynthetic genes ( leuB , tyrA ) 24 reflects metabolic quiescence, consistent with reduced anabolic demand and respiratory inhibition by P. aeruginosa -derived molecules such as 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) 25 . Downregulation of stress response and chaperone genes ( dnaK, groEL ) 26 suggests impaired ability to respond to oxidative or envelope stress, possibly exacerbated by P. aeruginosa redox-active metabolites. Suppressed expression of staphyloxanthin biosynthesis genes ( crtQ, crtO, crtM )(B et al. 2024) likely compromises antioxidant defense, rendering S. aureus more susceptible to oxidative stress induced by P. aeruginosa -derived ROS. Silencing of key virulence factors ( spa, lukEv, clfA ) 28 and repression of global regulators ( sarZ, sigS, agrB ) 29 , 30 suggest upstream disruption of quorum-sensing and pathogenicity under competitive pressure. Downregulation of cell wall genes ( murG, lytN ) 31 may compromise envelope integrity, while suppression of nucleotide and cofactor metabolism genes ( purD, purN ) 32 , 33 reflects metabolic restraint. Notably, S. aureus downregulated metal ion transporters ( copA, nikD, norB ) 34 , likely in response to iron sequestration by P. aeruginosa siderophores such as pyoverdine. Finally, the absence of T7SS gene expression indicates complete suppression of interbacterial antagonism 35 , suggesting P. aeruginosa outcompetes and functionally disarms S. aureus . Despite this global repression, hly was uniquely upregulated in the P. aeruginosa co-culture. This gene encodes a stress-induced, independently regulated cytotoxin 36 , enabling S. aureus to retain minimal cytolytic potential even under widespread transcriptional silencing. Rather than indicating a full virulence program, this selective activation likely represents a compensatory survival response to polymicrobial stress. Gene-level visualizations (Fig. 3 a–e) demonstrated consistent repression across major functional modules, highlighting hly as a unique outlier. Heatmaps (Fig. 3 a–b) showed widespread downregulation across functional categories, while the circular barplot (Fig. 3 c) emphasized the predominance of suppressed transcripts. UMAP analysis (Fig. 3 e) revealed clustering of downregulated genes into distinct regulatory groups, with hly appearing as an isolated node. The Sankey diagram (Fig. 3 d), depicting the top DEGs involved in metabolism, stress response, and cell wall biogenesis, showed that all were directed into the downregulated category, further highlighting the extent of P. aeruginosa -mediated suppression. These findings indicate that P. aeruginosa enforces a coordinated and multifaceted transcriptional silencing of S. aureus , disrupting key metabolic, virulence, and stress response pathways. This repression reflects a competitive dominance strategy in polymicrobial environments, functionally disarming S. aureus and diminishing its ecological competitiveness. Elucidating the molecular basis of this suppression may inform the development of therapeutic approaches that harness or mimic interspecies antagonism. For example, P. aeruginosa -derived molecules such as HQNO, pyocyanin, or siderophore analogs could be repurposed or engineered to target S. aureus persistence mechanisms without promoting broad-spectrum resistance. Alternatively, disrupting S. aureus quorum-sensing or stress resilience pathways revealed to be suppressed in co-culture may offer synergistic strategies when combined with biofilm-targeted therapies. Such approaches represent a paradigm shift, from pathogen eradication to ecological modulation, offering precision tools for managing chronic, device-associated, or polymicrobial infections. Candida albicans induces transcriptional reprogramming and virulence activation in S. aureus during co-culture In contrast to the repressive effects of P. aeruginosa , co-culturing S. aureus with C. albicans elicited a bidirectional transcriptional response, with 64 genes significantly upregulated and 54 downregulated (Fig. 4 a), as illustrated by barplot and Venn analyses (Fig. 4 b-c). STRING-based interaction networks (Fig. 4 d-e) revealed that both gene sets formed interconnected modules, suggesting coordinated regulatory shifts. Functional enrichment analyses highlighted activation of amino acid biosynthesis, cofactor metabolism, carbohydrate processing, and siderophore pathways (KEGG, Fig. 4 f). GO terms (Fig. 4 g) emphasized ATP binding, metal ion binding, ribosomal structure, and membrane-associated components, indicating broad physiological adaptation. Upregulated genes reflected enhanced virulence, metabolic reprogramming, and stress tolerance. Virulence factors ( clfA , spa , lukEv , hly ) 37 suggested increased adhesion, immune evasion, and cytotoxicity, consistent with prior reports of S. aureus–C. albicans synergy during mucosal invasion. Oxidative stress defense genes ( sodM , sodA ) 38 were elevated, likely counteracting ROS generated by fungal metabolism or host immune activity. The induction of fermentative metabolism genes ( mqo2 , glcT ) 39 indicates a shift toward anaerobic energy production, reflecting hypoxic and nutrient-depleted biofilm conditions often shaped by C. albicans . Upregulation of membrane remodeling genes ( lytN ) 31 , anabolic ( aspS , argR ) 24 , and translational genes ( rpsL , rplX ) 20 suggests active growth and structural adaptation to hyphal invasion. Induced regulators ( mgrA , saeS ) 40 , 41 suggest global transcriptional activation. Elevated metal ion transporters ( copA , copB ) 34 likely reflect a response to iron limitation imposed by fungal siderophores. Conversely, downregulation of core metabolic ( ackA , glcU ) 42 , 43 , amino acid biosynthesis ( tyrS , glnA ) 24 , and stress response genes ( hslO , ctsR ) 44 , 45 suggests reduced energy flux, nutrient competition, and impaired oxidative stress adaptability in the shared niche. Downregulation of virulence and regulatory genes ( clfA , sarX ) 46 – 48 may reflect immune evasion or interkingdom tolerance. Suppressed DNA replication ( recU , dnaA ) 49 , 50 and cell division genes ( parC ) 51 genes indicate a shift toward quiescence. Repression of norB and norG suggests diminished efflux capacity, potentially increasing susceptibility to antimicrobial or metabolic stress during co-culture. Gene-level visualizations supported the observed transcriptional reprogramming. Heatmaps (Fig. 5 a-b) illustrated a clear contrast between upregulated and downregulated genes across key functional categories, indicating strong condition-specific expression shifts. The circular barplot (Fig. 5 c) emphasized the balanced distribution of regulatory changes, while UMAP clustering (Fig. 5 e) distinctly separated gene expression patterns into two polarized groups. The Sankey diagram (Fig. 5 d) mapped the top DEGs into major biological categories, highlighting the coordinated nature of the bidirectional response in S. aureus . Together, these results demonstrate that C. albicans induces a finely orchestrated transcriptional reprogramming in S. aureus , activating virulence, stress tolerance, and metabolic flexibility, while repressing growth-associated, biosynthetic, and oxidative defense pathways. This dual modulation likely reflects a survival strategy tailored to the polymicrobial niche, enabling S. aureus to persist under cooperative or competitive pressures imposed by C. albicans . The observed plasticity aligns with prior evidence of fungal–bacterial synergy in mucosal colonization, biofilms, and disseminated infections 52 . Importantly, the selective silencing of replication and redox defenses, alongside the activation of virulence, suggests a shift toward a quiescent yet pathogenic phenotype. These findings offer mechanistic insight into interkingdom interactions and identify potential vulnerabilities for therapeutic targeting in chronic or device-associated infections. Enterococcus faecalis activates type VII secretion and virulence expression in Staphylococcus aureus during co-culture Global transcriptomic profiling identified 1,576 DEGs in S. aureus during co-culture with E. faecalis , comprising 890 upregulated and 686 downregulated genes. The volcano plot (Fig. 6 a) highlights the distribution of significantly altered genes across a broad range of fold changes and p-values, while the bar plot and venn diagram (Fig. 6 b-c) confirm the relative proportions of gene regulation categories. STRING-based protein–protein interaction networks (Fig. 6 d-e) revealed tightly interconnected clusters among up- and downregulated DEGs, suggesting coordinated regulatory control during polymicrobial stress. Functional enrichment analysis (Fig. 6 f-g) showed that upregulated genes were enriched in amino acid biosynthesis, cofactor metabolism, and carbohydrate degradation pathways. GO molecular functions included ATP binding, metal ion binding, and stress response activities. At the same time, enriched cellular components such as membrane-associated and extracellular regions suggest enhanced environmental sensing, structural adaptation, and nutrient acquisition. S. aureus underwent extensive transcriptional reprogramming in response to E. faecalis , characterized by the upregulation of virulence, stress adaptation, metabolic plasticity, and interbacterial competition mechanisms. Upregulation of virulence genes ( spa , clfA , fnbB ) 37 , 53 supports enhanced adhesion, immune evasion, and biofilm formation. Elevated lukEv , hlgB , and hly 36 , 37 indicate a cytolytic phenotype, promoting tissue damage and competitive advantage. Notably, the activation of essA and splF indicated the type VII secretion system (T7SS) engagement 54 , a contact-dependent pathway in S. aureus that translocates toxic effectors into Gram-positive competitors to establish niche dominance. T7SS is selectively activated against structurally compatible bacteria such as E. faecalis , whose thick peptidoglycan layer enables effector delivery and physical sensing 55 . In contrast, P. aeruginosa , a Gram-negative bacterium, possesses an outer membrane that impedes T7SS activity and secretes diffusible inhibitors (e.g., HQNO, pyocyanin) 25 that suppress S. aureus transcriptionally, including T7SS components. Likewise, C. albicans lacks the structural features required for T7SS targeting and does not pose direct antibacterial aggression. Instead, S. aureus exhibits a synergistic transcriptional response to C. albicans , upregulating virulence and stress pathways without T7SS induction, consistent with their known mutual persistence in biofilms and mucosal niches 18 . This context-dependent specificity emphasizes a finely tuned defense system in S. aureus , selectively deployed against compatible bacterial threats. Stress adaptation was prominent, with upregulation of uvrA , recO , and lexA 50 , 56 , 57 indicating oxidative and DNA damage response to E. faecalis -derived ROS. Induced clpP , and cap5A 58 , 59 likely enhanced envelope stability and redox control. Adhesion-related genes ( atl , spa ) 28 , 60 were elevated, the concurrent upregulation of icaR 60 , a repressor of the icaADBC operon, and downregulation of icaD 60 ) suggest repression of polysaccharide biofilm, favoring a proteinaceous matrix phenotype. Metabolic reprogramming was marked by upregulation of ackA , and glcU 42 , 43 , 61 , indicating a shift between fermentative and respiratory modes. Elevated tyrA , and leuS 62 , 63 suggested increased anabolic demands. Upregulation of nikD , and kdpA 64 , 65 reflected active trace metal acquisition. Additionally, expression of mobile element genes ( tnpR , tnpC ) 66 and transcriptional regulators ( rpoB , and rpoE ) 67 suggested genomic plasticity and transcriptional adaptation under stress. Conversely, significant repression affected core biosynthetic, metabolic, and virulence pathways. Key glycolytic and TCA cycle genes ( gapA2 , glkA , mqo2 ) 22 , 68 , 69 were downregulated, indicating energy conservation. Suppression of purD 70 suggested reduced nucleotide synthesis, while atpF and ctaB 71 downregulation pointed to diminished respiration. Translational repression was evident from reduced rplU , and rpsL 20 , elongation factor efp 72 and tRNA synthetases ( pheS , ileS , lysS ) 73 . Downregulation of dnaA and recR 49 , 50 indicated cell cycle arrest and impaired DNA repair. Key virulence genes ( fnbA , hlgA ) 74 – 76 were downregulated, along with capsule and cell wall genes ( lytM , cls2 ) 31 , 77 ), indicating reduced structural virulence and immune evasion. Suppressed expression of staphyloxanthin genes (B et al. 2024) suggested weakened antioxidant defense. Downregulation of regulatory elements ( ctsR , rex ) 78 implied impaired environmental sensing, while decreased expression of division proteins ( ftsA , ezrA ) 79 supported a quiescent, metabolically restrained state under competitive pressure. Gene-level visualizations validated these findings. Heatmaps (Fig. 7 a-b) revealed distinct clusters of upregulated and downregulated genes, reflecting a coordinated transcriptional shift in response to polymicrobial co-culture. UMAP clustering and radial barplots (Fig. 7 c and 7 e) further illustrated a clear polarization of gene expression profiles, with minimal overlap between expression states. The Sankey diagram (Fig. 7 d) mapped major transcriptional changes across key functional categories, emphasizing the organized reprogramming of S. aureus under competitive stress. These data reveal that S. aureus mounts a dualistic response to E. faecalis co-culture, activating T7SS, virulence pathways, and biosynthesis, while concurrently silencing growth, translation, and energetically costly processes. This ecological trade-off prioritizes stress endurance, competitive fitness, and sessile adaptation over proliferation. Given the known secretion of bacteriocins and hydrogen peroxide by E. faecalis 19 , the transcriptional reprogramming in S. aureus likely reflects an integrated response to direct and indirect microbial competition. These findings emphasize the importance of interbacterial signaling in modulating pathogen behavior and suggest that disrupting communication pathways such as T7SS or quorum sensing may provide therapeutic avenues for managing polymicrobial infections. These findings not only reveal condition-specific transcriptional reprogramming in S. aureus but also offer translational insights into how microbial interactions could be harnessed therapeutically. By delineating the transcriptional logic governing S. aureus survival, virulence modulation, and stress adaptation in response to diverse microbial neighbors, this study provides a blueprint for targeted antimicrobial strategies that mimic or disrupt these ecological signals. Such approaches may inform novel treatments for polymicrobial infections, especially those associated with biofilms, chronic wounds, and implanted devices, by either potentiating suppressive interspecies effects (as seen with P. aeruginosa ) or neutralizing synergistic or modulatory virulence interactions (as observed with C. albicans and E. faecalis ). Furthermore, understanding these adaptive mechanisms enhances our ability to predict pathogen behavior in vivo and design precision interventions that shift microbial dynamics in favor of host recovery. However, this study has certain limitations. It presents a single-time-point, snapshot view of the transcriptomic landscape, without capturing dynamic transitions or temporal changes in gene expression. This study was designed as an exploratory, hypothesis-generating investigation to uncover broad transcriptional trends in S. aureus during polymicrobial co-culture. Therefore, RNA-seq was conducted without biological replicates or RT-qPCR validation, focusing instead on global expression patterns and shifts in functional categories. Whole transcriptome profiling was integrated to provide a broader, systems-level perspective and to serve as a resource for future studies, where individual genes can be validated in targeted experimental models. A graphical summary of the distinct interaction modes, antagonism with P. aeruginosa , synergy with C. albicans , and competitive response to E. faecalis , is depicted in Fig. 8 , highlighting condition-specific modulation of virulence, metabolism, adhesion, biofilm, and T7SS expression in S. aureus . This schematic illustrates the distinct gene expression profiles of S. aureus when co-cultured with C. albicans , P. aeruginosa , and E. faecalis . In the left panel, representing co-culture with C. albicans , S. aureus exhibits a synergistic interaction marked by upregulation of virulence and cell adhesion genes, while genes involved in T7SS secretion and metabolism are downregulated. In the centre panel, during co-culture with P. aeruginosa , an antagonistic response is observed, with global transcriptional repression affecting metabolism, biofilm formation, adhesion, and T7SS genes, indicating suppression of S. aureus pathogenic potential. In the right panel, co-culture with E. faecalis leads to a competitive interaction characterized by upregulation of T7SS, virulence factors, metabolic pathways, and adhesion genes, while biofilm-related genes are downregulated. Upregulated and downregulated functions are indicated by green (▲) and red (▼) arrows, respectively. Icons represent core functional themes such as T7SS, metabolism, biofilm, virulence, and adhesins. The central test tubes depict S. aureus co-cultured with each partner organism. Conclusion This study demonstrates that S. aureus exhibits distinct, context-dependent transcriptional responses when co-cultured with P. aeruginosa , C. albicans , and E. faecalis . While P. aeruginosa imposes a dominant suppressive effect, silencing metabolism, stress response, and virulence networks, C. albicans elicits a synergistic reprogramming marked by co-activation of virulence, metabolic flexibility, and redox tolerance. In contrast, E. faecalis triggers a defensive, antagonistic response characterized by activation of the T7SS, virulence regulators, and anabolic machinery, reflecting a competitive interbacterial strategy. These divergent expression profiles reflect tailored ecological adaptations: suppression under Gram-negative stress, cooperation with fungal partners, and aggression against Gram-positive competitors. These findings provide mechanistic insight into how S. aureus dynamically modulates its transcriptome to balance competition, coexistence, and persistence in polymicrobial environments, laying the foundation for precision microbiology strategies that target interspecies signaling and microbial crosstalk in complex infections. Designed as an exploratory, hypothesis-generating investigation, this study reveals global transcriptomic trends, offering a foundational framework for future functional dissection of virulence adaptation in polymicrobial infections. Declarations Author Contributions Nirmala B designed and performed the experiments, analyzed the data, and wrote the manuscript. Yogendra P Mathuria and Balram Ji Omar supervised the study and validated the findings. All authors reviewed and approved the final manuscript. Funding The authors declare that no funds, grants, or other support were received during the preparation of the manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data Availability The raw RNA-seq datasets generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1307151 . The processed data, including the complete normalized expression matrix and lists of differentially expressed genes (DEGs) for each co-culture condition, are provided in the Supplementary Dataset (Excel file) . This dataset includes sample metadata, expression values, and statistical outputs used for downstream analyses. The custom R scripts and visualization notebooks used for data processing and figure generation are openly available on GitHub at https://github.com/Nirmala-1997/Transcriptomics-Polymicrobial-regulation, ensuring full transparency and reproducibility. Acknowledgements We acknowledge the Department of Biotechnology, Ministry of Science and Technology, Government of India, for providing a DBT-SRF scholarship to B Nirmala. We also acknowledge the All India Institute of Medical Sciences (AIIMS) Rishikesh for providing infrastructural support for this study, and the Indian Institute of Technology (IIT) Roorkee for facilitating scanning electron microscopy (SEM) analysis. References Zhang, M., Whiteley, M. & Lewin, G. R. Polymicrobial Interactions of Oral Microbiota: a Historical Review and Current Perspective. mBio vol. 13 Preprint at (2022). https://doi.org/10.1128/mbio.00235-22 Mariani, F. & Galvan, E. M. Staphylococcus aureus in Polymicrobial Skinand Soft Tissue Infections: Impact of Inter-Species Interactionsin Disease Outcome. Antibiotics vol. 12 Preprint at (2023). https://doi.org/10.3390/antibiotics12071164 Yu, G., Ge, X., Li, W., Ji, L. & Yang, S. Interspecific cross-talk: The catalyst driving microbial biosynthesis of secondary metabolites. Biotechnology Advances vol. 76 Preprint at (2024). https://doi.org/10.1016/j.biotechadv.2024.108420 Rupp, M. et al. Polymicrobial infections and microbial patterns in infected nonunions - A descriptive analysis of 42 cases. BMC Infect. Dis 20 , (2020). Pouget, C. et al. Polymicrobial Biofilm Organization of Staphylococcus aureus and Pseudomonas aeruginosa in a Chronic Wound Environment. Int J. Mol. Sci 23 , (2022). Jean-Pierre, F., Vyas, A., Hampton, T. H. & Henson, M. A. O’toole, G. A. One versus many: Polymicrobial communities and the cystic fibrosis airway. mBio 12 , 1–7 (2021). Ramstedt, M. & Burmølle, M. Can multi-species biofilms defeat antimicrobial surfaces on medical devices? Current Opinion in Biomedical Engineering vol. 22 Preprint at (2022). https://doi.org/10.1016/j.cobme.2022.100370 Howden, B. P. et al. Staphylococcus aureus host interactions and adaptation. Nature Reviews Microbiology vol. 21 380–395 Preprint at (2023). https://doi.org/10.1038/s41579-023-00852-y Cheung, G. Y. C., Bae, J. S. & Otto, M. Pathogenicity and virulence of Staphylococcus aureus. Virulence vol. 12 547–569 Preprint at (2021). https://doi.org/10.1080/21505594.2021.1878688 Nguyen, A. T. & Oglesby-Sherrouse, A. G. Interactions between Pseudomonas aeruginosa and Staphylococcus aureus during co-cultivations and polymicrobial infections. Applied Microbiology and Biotechnology vol. 100 6141–6148 Preprint at (2016). https://doi.org/10.1007/s00253-016-7596-3 Harriott, M. M. & Noverr, M. C. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: Effects on antimicrobial resistance. Antimicrob. Agents Chemother. 53 , 3914–3922 (2009). Kao, P. H. N. et al. Enterococcus faecalis suppresses Staphylococcus aureus-induced NETosis and promotes bacterial survival in polymicrobial infections. FEMS Microbes 4 , (2023). Kulshrestha, A. & Gupta, P. Polymicrobial interaction in biofilm: mechanistic insights. Pathog Dis 80 , (2022). Eichelberger, K. R. & Cassat, J. E. Metabolic Adaptations During Staphylococcus aureus and Candida albicans Co-Infection. Frontiers in Immunology vol. 12 Preprint at (2021). https://doi.org/10.3389/fimmu.2021.797550 Dastgheyb, S. S. & Otto, M. Staphylococcal adaptation to diverse physiologic niches: An overview of transcriptomic and phenotypic changes in different biological environments. Future Microbiology vol. 10 –1995 Preprint at (1981). https://doi.org/10.2217/fmb.15.116 (2015). B, N. et al. A novel dual-staining method for cost-effective visualization and differentiation of microbial biofilms. Sci Rep 14 , (2024). Biswas, L. & Götz, F. Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis. Frontiers in Cellular and Infection Microbiology vol. 11 Preprint at (2022). https://doi.org/10.3389/fcimb.2021.824042 Jing, Q. et al. Staphylococcus aureus wraps around Candida albicans and synergistically escapes from Neutrophil extracellular traps. Front Immunol 15 , (2024). Nogueira Viçosa, G. et al. Impact of co-cultivation with Enterococcus faecalis over growth, enterotoxin production and gene expression of Staphylococcus aureus in broth and fresh cheeses. Int J. Food Microbiol 308 , (2019). Lyu, Z., Wilson, C. & Ling, J. Translational Fidelity during Bacterial Stresses and Host Interactions. Pathogens vol. 12 Preprint at (2023). https://doi.org/10.3390/pathogens12030383 Auburger, G., Key, J. & Gispert, S. The Bacterial ClpXP-ClpB Family Is Enriched with RNA-Binding Protein Complexes. Cells vol. 11 Preprint at (2022). https://doi.org/10.3390/cells11152370 Troitzsch, A. et al. Carbon source-dependent reprogramming of anaerobic metabolism in staphylococcus aureus. J Bacteriol 203 , (2021). Roux, A. E. et al. The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response. Microbiol Spectr 10 , (2022). Ashniev, G. A., Petrov, S. N., Iablokov, S. N. & Rodionov, D. A. Genomics-Based Reconstruction and Predictive Profiling of Amino Acid Biosynthesis in the Human Gut Microbiome. Microorganisms 10, (2022). Montagut, E. J. et al. An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates. Microbiol Spectr 10 , (2022). Liao, X., Chen, X., Sant’Ana, A. S., Feng, J. & Ding, T. Pre-Exposure of Foodborne Staphylococcus aureus Isolates to Organic Acids Induces Cross-Adaptation to Mild Heat. Microbiol Spectr 11 , (2023). B, N. & Omar, B. J. Enhancing Staphyloxanthin Synthesis in Staphylococcus aureus Using Innovative Agar Media Formulations. Cureus 10.7759/cureus.59892 (2024). Wang, M. & Zhang, Q. Characteristics of Virulence Genes of Clinically Isolated Staphylococci in Jingzhou Area. Contrast Media Mol Imaging (2022). (2022). Wen, Z. et al. Baohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ. Phytomedicine 130 , (2024). Yuan, L. et al. Exploring the potential of isorhapontigenin: attenuating Staphylococcus aureus virulence through MgrA-mediated regulation. mSphere 9, (2024). Wang, M., Buist, G. & van Dijl, J. M. Staphylococcus aureus cell wall maintenance – the multifaceted roles of peptidoglycan hydrolases in bacterial growth, fitness, and virulence. FEMS Microbiology Reviews vol. 46 Preprint at (2022). https://doi.org/10.1093/femsre/fuac025 Buvelot, H. et al. Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis. Front Immunol 12 , (2021). Gélinas, M., Museau, L., Milot, A. & Beauregard, P. B. The de Novo Purine Biosynthesis Pathway Is the Only Commonly Regulated Cellular Pathway during Biofilm Formation in TSB-Based Medium in Staphylococcus Aureus and Enterococcus Faecalis . (2021). https://journals.asm.org/journal/spectrum Hou, Z., Liu, L., Wei, J. & Xu, B. Progress in the Prevalence, Classification and Drug Resistance Mechanisms of Methicillin-Resistant Staphylococcus aureus. Infection and Drug Resistance vol. 16 3271–3292 Preprint at (2023). https://doi.org/10.2147/IDR.S412308 Cao, Z., Casabona, M. G., Kneuper, H., Chalmers, J. D. & Palmer, T. The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. Nat Microbiol 2 , (2016). Mohapatra, D., Das, Pattnaik, S. & Panda, S. In Vitro Detected hly II Cytotoxin in a Strain of Staphylococcus aureus (BM S-2) and Plant-Derived Aromatic Components: a Molecular Docking Study. Appl. Biochem. Biotechnol. 193 , 1639–1653 (2021). Wang, S. et al. A Practical Approach for Predicting Antimicrobial Phenotype Resistance in Staphylococcus aureus Through Machine Learning Analysis of Genome Data. Front Microbiol 13 , (2022). Martini, A. M., Alexander, S. A. & Khare, A. Mutations in the Staphylococcus aureus Global Regulator CodY confer tolerance to an interspecies redox-active antimicrobial. PLoS Genet. 21 , e1011610 (2025). Fuchs, S., Pané-Farré, J., Kohler, C., Hecker, M. & Engelmann, S. Anaerobic gene expression in Staphylococcus aureus. J. Bacteriol. 189 , 4275–4289 (2007). Liu, X., Wang, Y., Chang, W., Dai, Y. & Ma, X. AgrA directly binds to the promoter of vraSR and downregulates its expression in Staphylococcus aureus. Antimicrob Agents Chemother 68 , (2024). Wittekind, M. A., Briaud, P., Smith, J. L., Tennant, J. R. & Carroll, R. K. The Small Protein ScrA Influences Staphylococcus aureus Virulence-Related Processes via the SaeRS System. Microbiol Spectr 11 , (2023). Cordero, M. et al. The induction of natural competence adapts staphylococcal metabolism to infection. Nat Commun 13 , (2022). Li, M. et al. Mechanisms of mepA Overexpression and Membrane Potential Reduction Leading to Ciprofloxacin Heteroresistance in a Staphylococcus aureus Isolate. Int J. Mol. Sci 26 , (2025). Rossi, C. C. et al. Expression of the stress-response regulators CtsR and HrcA in the uropathogen Staphylococcus saprophyticus during heat shock. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 110 , 1105–1111 (2017). Yee, R., Feng, J., Wang, J., Chen, J. & Zhang, Y. Identification of Genes Regulating Cell Death in Staphylococcus aureus. Front Microbiol 10 , (2019). Xiao, Y. et al. Novel small-molecule compound YH7 inhibits the biofilm formation of Staphylococcus aureus in a sarX -dependent manner. mSphere 9, (2024). Bhattacharya, M., Scherr, T. D., Lister, J., Kielian, T. & Horswill, A. R. Extracellular adherence proteins reduce matrix porosity and enhance Staphylococcus aureus biofilm survival during prosthetic joint infection. Infect Immun 93 , (2025). Ma, Z. et al. The Recombinant Expression Proteins FnBP and ClfA From Staphylococcus aureus in Addition to GapC and Sip From Streptococcus agalactiae Can Protect BALB/c Mice From Bacterial Infection. Front Vet. Sci 8 , (2021). Barbuti, M. D., Myrbråten, I. S., Morales Angeles, D. & Kjos, M. The cell cycle of Staphylococcus aureus: An updated review. MicrobiologyOpen vol. 12 Preprint at (2023). https://doi.org/10.1002/mbo3.1338 Ha, K. P. & Edwards, A. M. DNA Repair in Staphylococcus aureus. Microbiology Mol. Biology Reviews 85 , (2021). Huynh, T. Q. et al. Genomic alterations involved in fluoroquinolone resistance development in Staphylococcus aureus. PLoS One 18 , (2023). Hu, Y. et al. Staphylococcus aureus synergized with Candida albicans to increase the pathogenesis and drug resistance in cutaneous abscess and peritonitis murine models. Pathogens 10 , (2021). Sun, H., Li, R. W., Wang, T. T. Y. & Ding, L. The Ligand Binding Domain of the Cell Wall Protein SraP Modulates Macrophage Apoptosis and Inflammatory Responses in Staphylococcus aureus Infections. Molecules 30 , (2025). Afzal, M., Vijay, A. K., Stapleton, F. & Willcox, M. D. P. Genomics of Staphylococcus aureus Strains Isolated from Infectious and Non-Infectious Ocular Conditions. Antibiotics 11, (2022). Garrett, S. R. & Palmer, T. The role of proteinaceous toxins secreted by Staphylococcus aureus in interbacterial competition. FEMS Microbes vol. 5 Preprint at (2024). https://doi.org/10.1093/femsmc/xtae006 Gibson, J. F. et al. Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species. PLoS Pathog 17 , (2021). Bastos, M. L. C. et al. What Do We Know About Staphylococcus aureus and Oxidative Stress? Resistance, Virulence, New Targets, and Therapeutic Alternatives. Toxics 13 , 390 (2025). Theis, T. J., Daubert, T. A., Kluthe, K. E., Brodd, K. L. & Nuxoll, A. S. Staphylococcus aureus persisters are associated with reduced clearance in a catheter-associated biofilm infection. Front Cell. Infect. Microbiol 13 , (2023). Wei, B. et al. Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP. Nat Commun 13 , (2022). Schwartbeck, B. et al. Various mutations in icaR, the repressor of the icaADBC locus, occur in mucoid Staphylococcus aureus isolates recovered from the airways of people with cystic fibrosis. Microbes Infect 26 , (2024). Bertrand, B. P. et al. Role of Staphylococcus aureus Formate Metabolism during Prosthetic Joint Infection. Infect Immun 19 , (2022). Heidarian, S., Guliaev, A., Nicoloff, H., Hjort, K. & Andersson, D. I. High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes. PLoS Biol 22 , (2024). Liao, Z. et al. Transcriptomic analyses reveal the potential antibacterial mechanism of citral against Staphylococcus aureus. Front Microbiol 14 , (2023). Ghssein, G. & Ezzeddine, Z. The Key Element Role of Metallophores in the Pathogenicity and Virulence of Staphylococcus aureus: A Review. Biology vol. 11 Preprint at (2022). https://doi.org/10.3390/biology11101525 Peng, H. et al. Transcriptomic Analysis Revealed Antimicrobial Mechanisms of Lactobacillus rhamnosus SCB0119 against Escherichia coli and Staphylococcus aureus. Int J. Mol. Sci 23 , (2022). Ji, X. et al. Tn560, a Novel Tn554 Family Transposon from Porcine Methicillin-Resistant Staphylococcus aureus ST398, Carries a Multiresistance Gene Cluster Comprising a Novel spc Gene Variant and the Genes lsa(E) and lnu(B). Antimicrobial Agents and Chemotherapy vol. 66 Preprint at (2022). https://doi.org/10.1128/aac.01947-21 Nielsen, T. K. et al. The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 68 , (2024). Pires, P. M., Santos, D., Calisto, F. & Pereira, M. The monotopic quinone reductases from Staphylococcus aureus. Biochim Biophys. Acta Bioenerg 1865 , (2024). De Backer, S. et al. Enzymes catalyzing the tca-and urea cycle influence the matrix composition of biofilms formed by methicillin-resistant staphylococcus aureus usa300. Microorganisms 6, (2018). Goncheva, M. I., Flannagan, R. S. & Heinrichs, D. E. De Novo Purine Biosynthesis Is Required for Intracellular Growth of Staphylococcus Aureus and for the Hypervirulence Phenotype of a PurR Mutant . (2020). https://journals.asm.org/journal/iai Xu, T. et al. Absence of protoheme IX farnesyltransferase CtaB causes virulence attenuation but enhances pigment production and persister survival in MRSA. Front Microbiol 7 , (2016). Zheng, X. et al. Expression of Staphylococcus aureus translation elongation factor P is regulated by a stress-inducible promotor. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiology 117 , (2024). Giegé, R. & Springer, M. Aminoacyl-tRNA Synthetases in the Bacterial World. EcoSal Plus 5 , (2012). Rahman, S. & Das, A. K. Staphylococcal superantigen-like protein 10 enhances the amyloidogenic biofilm formation in Staphylococcus aureus. BMC Microbiol 23 , (2023). Zhu, Z. et al. Molecular Characteristics and Pathogenicity of Staphylococcus aureus Exotoxins. International Journal of Molecular Sciences vol. 25 Preprint at (2024). https://doi.org/10.3390/ijms25010395 Rodrigues, R. A. et al. Comparative genomics study of Staphylococcus aureus isolated from cattle and humans reveals virulence patterns exclusively associated with bovine clinical mastitis strains. Front Microbiol 13 , (2022). Yamanashi, Y. et al. Effects of Growth Stage on the Characterization of Enterotoxin A-Producing Staphylococcus aureus-Derived Membrane vesicles. Microorganisms 10, (2022). Dmitriev, A. et al. The Intersection of the Staphylococcus aureus Rex and SrrAB Regulons: an Example of Metabolic Evolution That Maximizes Resistance to Immune Radicals. (2021). 10.1128/mBio Myrbråten, I. S. et al. SmdA is a Novel Cell Morphology Determinant in Staphylococcus aureus. mBio 13, (2022). Additional Declarations No competing interests reported. Supplementary Files SACvsSA.xlsx SAPvsSA.xlsx SAEvsSA.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7401018","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":508617844,"identity":"f4d3193f-d3f0-4d85-a8d0-91dabda4f43a","order_by":0,"name":"B. Nirmala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACxgY2ECUBYjY++ACk2NiJ18LcbDgDpIWZoD1sMAZ7mzQPiCakhbm9LfFx5Q6LaN0ZiW3SNr+2yfMxMzB++JiDx2E9xw4bnj0jkbvtRmKzdW7fbcM2ZgZmyZnb8GiZkd4m2dgG1tJ4O7fnNiNQCxszL34t7T+hWhqkLXtu2xOhJe0YI1RLkzTDj9uJhLX0HEuGOOzMw2bD3obbyW3MjM14/WLY3mb4sbGtLnfb8fSHD378uW07v7354IeP+LQ0wFgCCUA728A2N+BQDAHycBb/ASDxB6/iUTAKRsEoGKEAALgWVyN0MgkyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0005-9306-260X","institution":"Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, Uttarakhand, India","correspondingAuthor":true,"prefix":"","firstName":"B.","middleName":"","lastName":"Nirmala","suffix":""},{"id":508617845,"identity":"c3f08212-9819-4d80-a900-1f00305936e0","order_by":1,"name":"Yogendra Pratap Mathuria","email":"","orcid":"","institution":"Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, Uttarakhand, India","correspondingAuthor":false,"prefix":"","firstName":"Yogendra","middleName":"Pratap","lastName":"Mathuria","suffix":""},{"id":508617846,"identity":"bc1a7ac0-08b0-4bb0-9196-7f6c7ad7fd99","order_by":2,"name":"Balram Ji Omar","email":"data:image/png;base64,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","orcid":"","institution":"Department of Microbiology, All India Institute of Medical Sciences, Rishikesh-249203, Uttarakhand, India","correspondingAuthor":true,"prefix":"","firstName":"Balram","middleName":"Ji","lastName":"Omar","suffix":""}],"badges":[],"createdAt":"2025-08-18 15:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7401018/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7401018/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90405121,"identity":"c5415e7e-4d96-49c4-9086-a0e3422334d5","added_by":"auto","created_at":"2025-09-02 11:03:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":650162,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs confirming the polymicrobial association of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e with co-culture microbes. (a) \u003cem\u003eS. aureus + Candida albicans\u003c/em\u003e: yeast and hyphal forms of \u003cem\u003eC. albicans\u003c/em\u003eembedded with coccoid \u003cem\u003eS. aureus cells\u003c/em\u003e, forming dense polymicrobial clusters.\u003cbr\u003e\n(b) \u003cem\u003eS. aureus + Pseudomonas aeruginosa\u003c/em\u003e: rod-shaped \u003cem\u003eP. aeruginosa\u003c/em\u003einterspersed with S. aureus cells in mixed microcolonies.\u003cbr\u003e\n(c) \u003cem\u003eS. aureus + Enterococcus faecalis\u003c/em\u003e: compact aggregates of cocci indicating dual Gram-positive biofilm organization.\u003cbr\u003e\n(d) \u003cem\u003eS. aureus\u003c/em\u003e monoculture as control, showing characteristic coccoid clustering.\u003cbr\u003e\nScale bars: 2–5 μm.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/31fc938ed53e8a570d6b346d.jpeg"},{"id":90407567,"identity":"0ac102ca-e28c-4f81-9adc-c8035eb0ac46","added_by":"auto","created_at":"2025-09-02 11:27:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of \u003c/strong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003cstrong\u003e during co-culture with \u003c/strong\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Volcano plot showing differentially expressed genes (DEGs); green dot indicates the single upregulated gene (\u003cem\u003ehly\u003c/em\u003e), while red dots represent downregulated genes.\u003cbr\u003e\n(b) Venn diagram summarizing DEG distribution across upregulated, downregulated, and nonsignificant genes.\u003cbr\u003e\n(c) Bar plot depicting counts of upregulated and downregulated genes.\u003cbr\u003e\n(d) The STRING network view shows \u003cem\u003ehly\u003c/em\u003e as a disconnected, solitary node.\u003cbr\u003e\n(e) STRING interaction network of significantly downregulated genes, highlighting dense protein–protein interactions.\u003cbr\u003e\n(f) KEGG pathway enrichment analysis of downregulated genes; bars indicate gene count per pathway.\u003cbr\u003e\n(g) GO enrichment analysis categorized into biological processes (BP), molecular functions (MF), and cellular components (CC), with gene counts plotted per term.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/da661ed5e27230ed9e373687.jpeg"},{"id":90406432,"identity":"f0494e4c-3995-4ab6-b6d5-0bb0d3df2a58","added_by":"auto","created_at":"2025-09-02 11:11:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":377997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene-level visualization of \u003c/strong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003cstrong\u003e transcriptional response during co-culture with \u003c/strong\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003cstrong\u003e (S+P).\u003c/strong\u003e\u003cbr\u003e\n(a) Heatmap of normalized expression values for all differentially expressed genes (DEGs), showing widespread downregulation.\u003cbr\u003e\n(b) Focused heatmap of selected significantly downregulated genes involved in metabolism, stress response, and virulence.\u003cbr\u003e\n(c) A circular barplot highlights the dominance of transcriptional repression, with downregulated genes shown in pink and \u003cem\u003ehly\u003c/em\u003e as the only upregulated gene in green.\u003cbr\u003e\n(d) Sankey diagram mapping top-downregulated genes into functional categories such as nucleotide metabolism, energy production, and stress adaptation.\u003cbr\u003e\n(e) UMAP clustering of DEG expression profiles, showing coherent grouping of downregulated genes and \u003cem\u003ehly\u003c/em\u003e as a distant outlier.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/65f779c3d51f4af1e2e2065f.jpeg"},{"id":90405131,"identity":"4940a112-e2f3-4bca-b178-a2501b550d32","added_by":"auto","created_at":"2025-09-02 11:03:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of \u003c/strong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003cstrong\u003e during co-culture with \u003c/strong\u003e\u003cem\u003eCandida albicans (S+C)\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Volcano plot showing differentially expressed genes (DEGs), with upregulated genes in green and downregulated genes in red.\u003cbr\u003e\n(b) Bar plot showing the number of upregulated and downregulated genes.\u003cbr\u003e\n(c) Venn diagram summarizing DEG distribution across upregulated, downregulated, and nonsignificant categories.\u003cbr\u003e\n(d) STRING network of upregulated genes highlighting functional modules involved in virulence, stress response, and metabolism.\u003cbr\u003e\n(e) STRING network of downregulated genes showing scattered and loosely connected nodes.\u003cbr\u003e\n(f) KEGG pathway enrichment analysis shows major functional categories including amino acid biosynthesis, cofactor biosynthesis, and carbohydrate metabolism.\u003cbr\u003e\n(g) GO enrichment analysis of DEGs across biological processes (BP), molecular functions (MF), and cellular components (CC), revealing significant enrichment in ATP binding, DNA binding, and extracellular region components.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/ca34b355f043d1502e33734c.jpeg"},{"id":90405128,"identity":"de972adf-93ec-46e7-9ad9-7b7fa99e4a7f","added_by":"auto","created_at":"2025-09-02 11:03:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":367347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene-level visualization of \u003c/strong\u003eStaphylococcus aureus\u003cstrong\u003e transcriptomic reprogramming during co-culture with \u003c/strong\u003eCandida albicans\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Heatmap of log₂ fold change values for all differentially expressed genes (DEGs), showing bidirectional expression shifts.\u003cbr\u003e\n(b) Heatmap of the top 50 most upregulated and top 50 most downregulated genes, highlighting functional responses in virulence, metabolism, and stress adaptation.\u003cbr\u003e\n(c) Circular barplot displaying the relative proportion and magnitude of upregulated (green) and downregulated (pink) genes.\u003cbr\u003e\n(d) The Sankey diagram shows the top upregulated and downregulated genes mapped by expression category.\u003cbr\u003e\n(e) UMAP clustering of S. aureus gene expression profiles, showing clear separation between upregulated and downregulated gene groups during co-culture.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/86a15d8120ce3dba782dc424.jpeg"},{"id":90405149,"identity":"0d673314-4878-4621-b3b0-e5e9440f1233","added_by":"auto","created_at":"2025-09-02 11:03:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":271140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of \u003c/strong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003cstrong\u003e during co-culture with \u003c/strong\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Volcano plot of differentially expressed genes (DEGs), with upregulated genes in green and downregulated genes in red.\u003cbr\u003e\n(b) Venn diagram summarizing the distribution of DEGs across upregulated, downregulated, and nonsignificant categories.\u003cbr\u003e\n(c) Bar plot displaying the number of genes in each DEG category.\u003cbr\u003e\n(d) STRING interaction network of upregulated genes, showing functional clustering in pathways related to virulence, stress adaptation, and interbacterial competition.\u003cbr\u003e\n(e) STRING network of downregulated genes, depicting dense protein–protein connectivity associated with translation, metabolism, and cell division.\u003cbr\u003e\n(f) KEGG pathway enrichment analysis of all DEGs, showing the most significantly regulated biological processes, including amino acid biosynthesis, carbohydrate metabolism, and cofactor biosynthesis.\u003cbr\u003e\n(g) GO enrichment analysis categorized into biological processes (BP), molecular functions (MF), and cellular components (CC), highlighting enrichment in ATP binding, DNA binding, and membrane-associated components.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/91e36cfc1dac7ace2691ef3f.jpeg"},{"id":90405138,"identity":"c78ab97c-c183-4365-8a68-89ae87b84bdb","added_by":"auto","created_at":"2025-09-02 11:03:55","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":368733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene-level visualization of \u003c/strong\u003eStaphylococcus aureus\u003cstrong\u003e transcriptomic response during co-culture with \u003c/strong\u003eEnterococcus faecalis\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(a) Heatmap of log₂ fold change values for all differentially expressed genes (DEGs), highlighting a broad range of transcriptional modulation.\u003cbr\u003e\n(b) Heatmap of the top 50 most upregulated and top 50 most downregulated genes, revealing strong differential expression in stress response, virulence, and metabolic pathways.\u003cbr\u003e\n(c) Circular barplot representing the distribution and magnitude of gene regulation, with upregulated genes in green and downregulated genes in pink.\u003cbr\u003e\n(d) Sankey diagram mapping top DEGs into upregulated and downregulated categories.\u003cbr\u003e\n(e) UMAP clustering of gene expression profiles showing clear segregation between upregulated and downregulated gene groups under S+E condition.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/9e4ee050a5039a5cd1fa02e8.jpeg"},{"id":90406435,"identity":"2b0a0ba3-ee1b-4bea-8855-2bb93be5ae0b","added_by":"auto","created_at":"2025-09-02 11:11:55","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":197485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical summary of condition-specific transcriptional responses of \u003c/strong\u003eStaphylococcus aureus\u003cstrong\u003e during polymicrobial co-culture.\u003c/strong\u003e\u003cbr\u003e\nThis schematic illustrates the distinct gene expression profiles of S. aureus when co-cultured with C. albicans, P. aeruginosa, and E. faecalis. In the \u003cstrong\u003eleft panel\u003c/strong\u003e, representing co-culture with C. albicans, S. aureus exhibits a synergistic interaction marked by upregulation of virulence and cell adhesion genes, while genes involved in T7SS secretion and metabolism are downregulated. In the \u003cstrong\u003ecentre panel\u003c/strong\u003e, during co-culture with P. aeruginosa, an antagonistic response is observed, with global transcriptional repression affecting metabolism, biofilm formation, adhesion, and T7SS genes, indicating suppression of S. aureus pathogenic potential. In the \u003cstrong\u003eright panel\u003c/strong\u003e, co-culture with E. faecalis leads to a competitive interaction characterized by upregulation of T7SS, virulence factors, metabolic pathways, and adhesion genes, while biofilm-related genes are downregulated. Upregulated and downregulated functions are indicated by green (▲) and red (▼) arrows, respectively. Icons represent core functional themes such as T7SS, metabolism, biofilm, virulence, and adhesins. The central test tubes depict S. aureus co-cultured with each partner organism.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/0c9001f51a3d70332855ea89.jpeg"},{"id":99799282,"identity":"4800e4d8-5b51-4f56-bba6-5f537d9a61dc","added_by":"auto","created_at":"2026-01-08 13:49:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3669752,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/d92eae2d-aec9-4bc8-9d33-2efb7d8e1962.pdf"},{"id":90405123,"identity":"7aa2d7f2-21f5-492a-86b7-c811e17b0e53","added_by":"auto","created_at":"2025-09-02 11:03:55","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":318512,"visible":true,"origin":"","legend":"","description":"","filename":"SACvsSA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/3367b9369b3553324889bd03.xlsx"},{"id":90406429,"identity":"6ca2acfb-f05f-4fcd-b4ab-93ed2124a8a6","added_by":"auto","created_at":"2025-09-02 11:11:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":488619,"visible":true,"origin":"","legend":"","description":"","filename":"SAPvsSA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/2da9f763c802af43a5142649.xlsx"},{"id":90405133,"identity":"58108a3b-ac2d-4064-9935-5a699764e62e","added_by":"auto","created_at":"2025-09-02 11:03:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":494294,"visible":true,"origin":"","legend":"","description":"","filename":"SAEvsSA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7401018/v1/de0a897562707082f2394062.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploratory transcriptomic analysis of Staphylococcus aureus adaptation during polymicrobial interactions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobes rarely live alone. The idea that microbes exist in isolation has been overturned by the discoveries revealing that most pathogens thrive in complex, polymicrobial communities, a concept dating back to the 17th century, when Antonie van Leeuwenhoek first described diverse \u0026lsquo;animalcules\u0026rsquo; coexisting in dental plaque \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePolymicrobial infections represent a major clinical challenge due to the complex interspecies interactions that influence pathogen behavior, host response, and treatment outcomes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. When two microbes meet, they engage in molecular crosstalk through secreted factors, contact-dependent signaling, and competition for nutrients. These interactions can reprogram gene expression, modulate virulence, and reshape microbial physiology, in often unpredictable ways \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Among the diverse microbes implicated in such infections, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e stands out as a clinically significant, opportunistic pathogen. It is frequently isolated from biofilm-associated infections, including diabetic wounds, cystic fibrosis lungs, indwelling catheters, and prosthetic devices \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The presence of \u003cem\u003eS. aureus\u003c/em\u003e in polymicrobial infections has been associated with increased morbidity, prolonged treatment duration, and an elevated mortality risk due to its ability to evade host immunity and resist antibiotics (Nair et al. 2014; Cheung et al. 2021; Howden et al. 2023). Yet, the extent to which these traits are shaped by cohabiting microbial species remains unexplored.\u003c/p\u003e\u003cp\u003ePolymicrobial niches often harbor \u003cem\u003eS. aureus\u003c/em\u003e alongside Gram-negative bacteria like \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, fungi such as \u003cem\u003eCandida albicans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and Gram-positive organisms like \u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. These interkingdom and intrakingdom interactions can shift \u003cem\u003eS. aureus\u003c/em\u003e behavior toward suppression, synergy, or competition depending on the partner species and ecological context \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Such outcomes are not merely phenotypic but are driven by global transcriptional reprogramming that allows \u003cem\u003eS. aureus\u003c/em\u003e to fine-tune virulence expression, metabolic adaptation, and stress responses.\u003c/p\u003e\u003cp\u003eTranscriptional plasticity has emerged as a key survival strategy for \u003cem\u003eS. aureus\u003c/em\u003e, enabling rapid adjustment of regulatory networks in response to fluctuating environmental and interspecies signals \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. RNA sequencing offers an unprecedented opportunity to capture these global changes and elucidate how co-culture with diverse microbial partners remodels the gene expression landscape of \u003cem\u003eS. aureus\u003c/em\u003e. While individual studies have examined binary interactions, systematic comparisons across multiple polymicrobial contexts remain scarce.\u003c/p\u003e\u003cp\u003eExploratory polymicrobial transcriptomics is critical as it enables researchers to capture unbiased, systems-level signatures of microbial crosstalk, beyond what reductionist single-interaction studies can reveal. Such exploratory mapping is critical to identify unexpected transcriptional trends, generate new hypotheses, and build a foundation for mechanistic investigations that directly inform treatment strategies for complex infections.\u003c/p\u003e\u003cp\u003eHere, we take an exploratory approach to address this gap. We conducted a transcriptomic survey of \u003cem\u003eS. aureus\u003c/em\u003e co-cultured with \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e, representing taxonomically and functionally diverse neighbors. By integrating RNA sequencing with ultrastructural observations, we provide a systems-level view of \u003cem\u003eS. aureus\u003c/em\u003e adaptation across polymicrobial settings. Rather than offering definitive mechanistic conclusions, this work generates testable hypotheses on how interspecies interactions influence \u003cem\u003eS. aureus\u003c/em\u003e biology, laying the foundation for targeted strategies to mitigate polymicrobial infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eBacterial and fungal strains\u003c/p\u003e\u003cp\u003eClinical isolates of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e were obtained from the Bacteriology Laboratory, All India Institute of Medical Sciences (AIIMS), Rishikesh, India. Species identification was performed using standard biochemical tests and the VITEK 2 compact system (bioM\u0026eacute;rieux, Marcy-l\u0026rsquo;\u0026Eacute;toile, France), and confirmed by MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA). The study protocol was approved by the Institutional Ethics Committee, AIIMS Rishikesh (Letter No. AIIMS/IEC/23/294). The study was conducted between January 2024 and May 2025, and all isolates were processed following institutional biosafety protocols.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eS. aureus\u003c/em\u003e clinical isolate served as the focal organism for all mono- and co-culture experiments. Working stocks of each isolate were prepared from single colonies grown on tryptic soy agar (TSA; HiMedia, Mumbai, India) for bacteria, and Sabouraud dextrose agar (SDA; HiMedia) for \u003cem\u003eC. albicans\u003c/em\u003e. Stocks were suspended in sterile 20% glycerol and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for long-term preservation and at \u0026minus;\u0026thinsp;20\u0026deg;C for immediate storage. For routine use, working cultures were maintained on the same agar media at 4\u0026deg;C and subcultured when required.\u003c/p\u003e\u003cp\u003eFor experiments, cultures were revived on the same media and incubated at 37\u0026deg;C for 24 h. Liquid cultures for mono- and co-culture assays were grown in Luria\u0026ndash;Bertani (LB) broth (HiMedia) at 37\u0026deg;C under static conditions for 24 h, using mid-log phase inocula standardized to an optical density at 600 nm (OD₆₀₀ \u0026asymp; 0.5). No antibiotics were included in co-cultures to avoid selective pressure. Culture purity was verified by colony morphology and Gram staining before each experiment.\u003c/p\u003e\u003cp\u003ePolymicrobial co-culture setup\u003c/p\u003e\u003cp\u003ePolymicrobial co-cultures were established to assess the influence of interacting partners on the global transcriptional activity of \u003cem\u003eS. aureus\u003c/em\u003e. Clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e were used as interacting partners. All bacterial strains were maintained on tryptic soy agar (TSA; HiMedia) and \u003cem\u003eC. albicans\u003c/em\u003e on Sabouraud dextrose agar (SDA; HiMedia) at 4\u0026deg;C for short-term storage.\u003c/p\u003e\u003cp\u003eFor experimental use, isolates were revived from storage onto their respective agar plates and incubated overnight at 37\u0026deg;C. Colonies from overnight cultures were resuspended in Luria\u0026ndash;Bertani (LB) broth and adjusted to a 0.5 McFarland standard (~\u0026thinsp;1.5 \u0026times; 10⁸ CFU/mL). Equal volumes of \u003cem\u003eS. aureus\u003c/em\u003e and the interacting partner were mixed in sterile test tubes and incubated statically at 37\u0026deg;C for 24 h.\u003c/p\u003e\u003cp\u003eMonocultures of \u003cem\u003eS. aureus\u003c/em\u003e prepared under identical conditions served as controls. After incubation, cultures were processed for downstream analyses, including scanning electron microscopy (SEM), RNA extraction, library preparation, and sequencing.\u003c/p\u003e\u003cp\u003eSEM analysis\u003c/p\u003e\u003cp\u003ePhysical interactions between \u003cem\u003eS. aureus\u003c/em\u003e and its co-culture partners were examined using SEM. Sterile glass coverslips were placed in the co-culture setup described above and incubated statically at 37\u0026deg;C for 24 h. Following incubation, coverslips were gently rinsed once by dipping in distilled water (~\u0026thinsp;5 seconds) to remove non-adherent cells.\u003c/p\u003e\u003cp\u003eSamples were fixed with 4% formaldehyde in 0.1 M phosphate-buffered saline (PBS) for 15\u0026ndash;30 minutes, rinsed with PBS, and cross-linked with 2.5% glutaraldehyde in 0.1 M PBS for 2 hours at 4\u0026deg;C. After cross-linking, samples were rinsed with PBS and dehydrated through a graded ethanol series (50%, 70%, 80%, 90%, and 100%), each for 10 min. Dehydrated samples were subjected to critical point drying for 1 hour, mounted on stubs, and sputter-coated with gold using a Q150T S Plus turbomolecular pumped coater (Quorum Technologies Ltd., United Kingdom) \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe prepared samples were visualized using a field-emission scanning electron microscope (Gemini 560, Carl Zeiss Meditec AG, Germany), and representative images were captured for analysis of microbial associations.\u003c/p\u003e\u003cp\u003eRNA extraction, library preparation, and sequencing\u003c/p\u003e\u003cp\u003eTotal RNA was extracted directly from each polymicrobial co-culture and the \u003cem\u003eS. aureus\u003c/em\u003e monoculture at 24 hours post-inoculation using the HiPurA Total Bacterial RNA Isolation Kit (HiMedia Laboratories, Mumbai, India). RNA integrity was assessed on 1% agarose gel, and RNA concentration was measured using a Qubit\u0026reg; 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\u003cp\u003eRNA libraries were prepared using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit (Illumina Inc., San Diego, CA, USA). Library fragment size distribution and quality were evaluated on the Agilent TapeStation 4150 system with High Sensitivity D1000 ScreenTape\u0026reg; (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on the Illumina NovaSeq X Plus platform (Illumina Inc., San Diego, CA, USA), generating paired-end 151 bp reads for transcriptomic analysis. Each condition was sequenced as a single biological replicate, with high coverage used to maximize transcriptome representation.\u003c/p\u003e\u003cp\u003eBioinformatics pipeline\u003c/p\u003e\u003cp\u003eRaw reads from each condition were quality-checked with FastQC v0.12.1 and preprocessed using fastp v0.23.4 for adapter trimming and quality filtering. De novo transcriptome assembly was performed with Trinity v2.15.1, and assembly quality was assessed using assembly-stats v1.0.1. Transcript abundance was estimated with RSEM v1.3.3, and differential gene expression (DGE) analysis was carried out using edgeR v3.42.4. Genes were considered significantly differentially expressed if they exhibited an absolute log₂ fold change (|log₂FC| \u0026ge; 1) and a false discovery rate (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05), adjusted using the Benjamini\u0026ndash;Hochberg correction.\u003c/p\u003e\u003cp\u003eComparisons were made between each polymicrobial condition: S\u0026thinsp;+\u0026thinsp;P (\u003cem\u003eP. aeruginosa\u003c/em\u003e), S\u0026thinsp;+\u0026thinsp;C (\u003cem\u003eC. albicans\u003c/em\u003e), and S\u0026thinsp;+\u0026thinsp;E (\u003cem\u003eE. faecalis\u003c/em\u003e), and the \u003cem\u003eS. aureus\u003c/em\u003e monoculture. To ensure species specificity, predicted protein-coding sequences were aligned against the UniProt reviewed \u003cem\u003eS. aureus\u003c/em\u003e database using BLAST v2.14.1, and only transcripts with \u0026ge;\u0026thinsp;50% sequence similarity to \u003cem\u003eS. aureus\u003c/em\u003e entries were retained. Coding regions were predicted with TransDecoder v5.5.0 and annotated against the UniProt reviewed database. Finally, annotated DEGs were subjected to Gene Ontology (GO) and KEGG pathway enrichment analyses to identify significantly enriched biological processes and pathways.\u003c/p\u003e\u003cp\u003eVisualization and statistical analysis\u003c/p\u003e\u003cp\u003eAll downstream analyses were performed in Python (v3.10). Data handling and matrix operations were carried out using pandas (v2.0.3) and NumPy (v1.25.0). Differential expression outputs were visualized with matplotlib (v3.7.2) and seaborn (v0.12.2) to generate bar plots, scatter plots, volcano plots, and heatmaps, while Venn diagrams were produced with matplotlib-venn (v0.11.9). Dimensionality reduction and clustering were performed using scikit-learn (v1.3.0) and umap-learn (v0.5.3), whereas network visualizations were created with networkx (v3.1) and Sankey diagrams with plotly (v5.15.0). Enrichment outputs, including Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, were plotted in Python using matplotlib and seaborn. Protein\u0026ndash;protein interaction (PPI) networks were generated using the STRING web server (v12.0), and exported network views were incorporated for interpretation. All project files, including analysis scripts, pipelines, and processed datasets, are openly available in the GitHub repository (see Data Availability).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eSEM analysis of microbial interactions\u003c/p\u003e\n\u003cp\u003eSEM revealed distinct patterns of interaction between \u003cem\u003eS. aureus\u003c/em\u003e and its co-culture partners (\u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In the monoculture control, \u003cem\u003eS. aureus\u003c/em\u003e cells exhibited the characteristic grape-like clustering typical of staphylococcal growth, serving as a baseline for comparison with polymicrobial conditions (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eS. aureus\u0026ndash;P. aeruginosa\u003c/em\u003e co-culture, the cocci and rod-shaped cells were observed close but without extensive mixing. \u003cem\u003eS. aureus\u003c/em\u003e appeared in scattered clusters, while \u003cem\u003eP. aeruginosa\u003c/em\u003e maintained elongated arrangements surrounding these clusters (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). This spatial separation suggests competitive coexistence, consistent with previous studies that describe inhibitory mechanisms employed by \u003cem\u003eP. aeruginosa\u003c/em\u003e against \u003cem\u003eS. aureus\u003c/em\u003e, including the secretion of biosurfactants and secondary metabolites that limit staphylococcal colonization \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCo-culture with \u003cem\u003eC. albicans\u003c/em\u003e showed a different pattern. \u003cem\u003eS. aureus\u003c/em\u003e cocci adhered extensively to the hyphal filaments of \u003cem\u003eC. albicans\u003c/em\u003e, forming dense clusters along the fungal surface (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Such physical attachment has been widely documented as a hallmark of cross-kingdom biofilm formation, where fungal hyphae act as scaffolds that facilitate bacterial adhesion and persistence \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This structural arrangement highlights the potential for cooperative biofilm establishment and enhanced microbial resilience in polymicrobial infections involving \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eS. aureus\u0026ndash;E. faecalis\u003c/em\u003e co-culture, both species were found in compact aggregates, with \u003cem\u003eS. aureus\u003c/em\u003e cells embedded in close contact with \u003cem\u003eEnterococci\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). The formation of such mixed clusters demonstrates that these organisms occupy shared spatial niches during co-culture. At the structural level, a close association may provide physical protection and stabilization within the biofilm matrix. Previous reports have noted that enterococcal interactions can alter staphylococcal growth and biofilm architecture \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, suggesting that tight spatial clustering represents an adaptive arrangement shaped by polymicrobial pressures.\u003c/p\u003e\n\u003cp\u003eScale bars: 2\u0026ndash;5 \u0026micro;m.\u003c/p\u003e\n\u003cp\u003eRNA sequencing quality assessment\u003c/p\u003e\n\u003cp\u003eRNA sequencing produced robust datasets across \u003cem\u003eS. aureus\u003c/em\u003e monoculture and co-culture conditions. RNA extractions consistently yielded sufficient material for library preparation, with concentrations above the minimum requirement (\u0026ge;\u0026thinsp;25 ng/\u0026micro;l). Libraries exhibited uniform fragment sizes (411\u0026ndash;431 bp) and concentrations (13.3\u0026ndash;68.6 ng/\u0026micro;L), confirming the high quality of the input. Sequencing on the Illumina NovaSeq X Plus generated 82\u0026ndash;113 million paired-end 151 bp reads per library, providing very high coverage for transcriptomic analysis.\u003c/p\u003e\n\u003cp\u003eFASTQC initially detected adapter sequences and low-quality tails, which were successfully trimmed using fastp. Post-processing retained 97\u0026ndash;99% of the reads across all libraries. Base quality was consistently high, with \u0026gt;\u0026thinsp;93% of bases above Q30 before filtering, improving to 96\u0026ndash;97% after filtering, and mean Phred scores approaching 35. GC content across libraries ranged from 40\u0026ndash;60%, consistent with expected variation in bacterial transcriptomes. Collectively, these QC metrics confirmed the reliability of the sequencing data and provided a strong foundation for downstream transcriptomic analysis.\u003c/p\u003e\n\u003cp\u003eGlobal transcriptomic responses of \u003cem\u003eS. aureus\u003c/em\u003e during polymicrobial co-culture\u003c/p\u003e\n\u003cp\u003eFollowing quality control, differential expression analysis revealed partner-specific responses of \u003cem\u003eS. aureus\u003c/em\u003e, with DEGs ranging from a few hundred to over a thousand. Co-culture with \u003cem\u003eP. aeruginosa\u003c/em\u003e caused broad repression, while \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e induced selective activation of virulence and metabolic pathways. These partner-specific responses are described in detail below.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e induces global transcriptional suppression in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e during co-culture\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e co-culture induced a global transcriptional shutdown in \u003cem\u003eS. aureus\u003c/em\u003e, with 230 genes significantly downregulated and only one, \u003cem\u003ehly\u003c/em\u003e (alpha-hemolysin), upregulated (log₂FC\u0026thinsp;=\u0026thinsp;+\u0026thinsp;0.48) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). Differentially expressed genes (DEGs) were identified using a stringent statistical threshold of |log₂ fold change| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ensuring high-confidence calls. This repression was illustrated by Venn and barplot analyses (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb-c). STRING network visualization (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee) revealed strong interconnectivity among the downregulated proteins, implicating core processes such as energy metabolism, translation, and regulatory pathways. In contrast, \u003cem\u003ehly\u003c/em\u003e appeared as a solitary, unconnected node (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed), suggesting an independent, stress-induced regulatory response.\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analyses (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef-g) identified suppression across critical metabolic and cellular pathways, including amino acid biosynthesis, carbohydrate metabolism, ribosome assembly, and membrane-associated functions. Genes encoding ribosomal proteins (\u003cem\u003erplX, rpsD\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and translation factors (\u003cem\u003etuf, efp\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e were downregulated, indicating a protein synthesis shutdown likely driven by nutrient limitation or quorum-sensing interference from \u003cem\u003eP. aeruginosa\u003c/em\u003e. Concurrent repression of TCA cycle enzymes (\u003cem\u003emqo2\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, glycolytic genes (\u003cem\u003eackA, ptsG\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and amino acid biosynthetic genes (\u003cem\u003eleuB\u003c/em\u003e, \u003cem\u003etyrA\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e reflects metabolic quiescence, consistent with reduced anabolic demand and respiratory inhibition by \u003cem\u003eP. aeruginosa\u003c/em\u003e-derived molecules such as 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Downregulation of stress response and chaperone genes (\u003cem\u003ednaK, groEL\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e suggests impaired ability to respond to oxidative or envelope stress, possibly exacerbated by \u003cem\u003eP. aeruginosa\u003c/em\u003e redox-active metabolites. Suppressed expression of staphyloxanthin biosynthesis genes (\u003cem\u003ecrtQ, crtO, crtM\u003c/em\u003e)(B et al. 2024) likely compromises antioxidant defense, rendering \u003cem\u003eS. aureus\u003c/em\u003e more susceptible to oxidative stress induced by \u003cem\u003eP. aeruginosa\u003c/em\u003e-derived ROS.\u003c/p\u003e\n\u003cp\u003eSilencing of key virulence factors (\u003cem\u003espa, lukEv, clfA\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and repression of global regulators (\u003cem\u003esarZ, sigS, agrB\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e suggest upstream disruption of quorum-sensing and pathogenicity under competitive pressure. Downregulation of cell wall genes (\u003cem\u003emurG, lytN\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e may compromise envelope integrity, while suppression of nucleotide and cofactor metabolism genes (\u003cem\u003epurD, purN\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e reflects metabolic restraint. Notably, \u003cem\u003eS. aureus\u003c/em\u003e downregulated metal ion transporters (\u003cem\u003ecopA, nikD, norB\u003c/em\u003e) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, likely in response to iron sequestration by \u003cem\u003eP. aeruginosa\u003c/em\u003e siderophores such as pyoverdine. Finally, the absence of T7SS gene expression indicates complete suppression of interbacterial antagonism\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, suggesting \u003cem\u003eP. aeruginosa\u003c/em\u003e outcompetes and functionally disarms \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eDespite this global repression, hly was uniquely upregulated in the \u003cem\u003eP. aeruginosa\u003c/em\u003e co-culture. This gene encodes a stress-induced, independently regulated cytotoxin\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, enabling \u003cem\u003eS. aureus\u003c/em\u003e to retain minimal cytolytic potential even under widespread transcriptional silencing. Rather than indicating a full virulence program, this selective activation likely represents a compensatory survival response to polymicrobial stress.\u003c/p\u003e\n\u003cp\u003eGene-level visualizations (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;e) demonstrated consistent repression across major functional modules, highlighting \u003cem\u003ehly\u003c/em\u003e as a unique outlier. Heatmaps (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;b) showed widespread downregulation across functional categories, while the circular barplot (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) emphasized the predominance of suppressed transcripts. UMAP analysis (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee) revealed clustering of downregulated genes into distinct regulatory groups, with \u003cem\u003ehly\u003c/em\u003e appearing as an isolated node. The Sankey diagram (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed), depicting the top DEGs involved in metabolism, stress response, and cell wall biogenesis, showed that all were directed into the downregulated category, further highlighting the extent of \u003cem\u003eP. aeruginosa\u003c/em\u003e-mediated suppression.\u003c/p\u003e\n\u003cp\u003eThese findings indicate that \u003cem\u003eP. aeruginosa\u003c/em\u003e enforces a coordinated and multifaceted transcriptional silencing of \u003cem\u003eS. aureus\u003c/em\u003e, disrupting key metabolic, virulence, and stress response pathways. This repression reflects a competitive dominance strategy in polymicrobial environments, functionally disarming \u003cem\u003eS. aureus\u003c/em\u003e and diminishing its ecological competitiveness. Elucidating the molecular basis of this suppression may inform the development of therapeutic approaches that harness or mimic interspecies antagonism. For example, \u003cem\u003eP. aeruginosa\u003c/em\u003e-derived molecules such as HQNO, pyocyanin, or siderophore analogs could be repurposed or engineered to target \u003cem\u003eS. aureus\u003c/em\u003e persistence mechanisms without promoting broad-spectrum resistance. Alternatively, disrupting \u003cem\u003eS. aureus\u003c/em\u003e quorum-sensing or stress resilience pathways revealed to be suppressed in co-culture may offer synergistic strategies when combined with biofilm-targeted therapies. Such approaches represent a paradigm shift, from pathogen eradication to ecological modulation, offering precision tools for managing chronic, device-associated, or polymicrobial infections.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e induces transcriptional reprogramming and virulence activation in \u003cem\u003eS. aureus\u003c/em\u003e during co-culture\u003c/p\u003e\n\u003cp\u003eIn contrast to the repressive effects of \u003cem\u003eP. aeruginosa\u003c/em\u003e, co-culturing \u003cem\u003eS. aureus\u003c/em\u003e with \u003cem\u003eC. albicans\u003c/em\u003e elicited a bidirectional transcriptional response, with 64 genes significantly upregulated and 54 downregulated (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea), as illustrated by barplot and Venn analyses (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb-c). STRING-based interaction networks (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed-e) revealed that both gene sets formed interconnected modules, suggesting coordinated regulatory shifts.\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analyses highlighted activation of amino acid biosynthesis, cofactor metabolism, carbohydrate processing, and siderophore pathways (KEGG, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). GO terms (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg) emphasized ATP binding, metal ion binding, ribosomal structure, and membrane-associated components, indicating broad physiological adaptation.\u003c/p\u003e\n\u003cp\u003eUpregulated genes reflected enhanced virulence, metabolic reprogramming, and stress tolerance. Virulence factors (\u003cem\u003eclfA\u003c/em\u003e, \u003cem\u003espa\u003c/em\u003e, \u003cem\u003elukEv\u003c/em\u003e, \u003cem\u003ehly\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e suggested increased adhesion, immune evasion, and cytotoxicity, consistent with prior reports of \u003cem\u003eS. aureus\u0026ndash;C. albicans\u003c/em\u003e synergy during mucosal invasion. Oxidative stress defense genes (\u003cem\u003esodM\u003c/em\u003e, \u003cem\u003esodA\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e were elevated, likely counteracting ROS generated by fungal metabolism or host immune activity. The induction of fermentative metabolism genes (\u003cem\u003emqo2\u003c/em\u003e, \u003cem\u003eglcT\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e indicates a shift toward anaerobic energy production, reflecting hypoxic and nutrient-depleted biofilm conditions often shaped by \u003cem\u003eC. albicans\u003c/em\u003e. Upregulation of membrane remodeling genes (\u003cem\u003elytN\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, anabolic (\u003cem\u003easpS\u003c/em\u003e, \u003cem\u003eargR\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and translational genes (\u003cem\u003erpsL\u003c/em\u003e, \u003cem\u003erplX\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e suggests active growth and structural adaptation to hyphal invasion. Induced regulators (\u003cem\u003emgrA\u003c/em\u003e, \u003cem\u003esaeS\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e suggest global transcriptional activation. Elevated metal ion transporters (\u003cem\u003ecopA\u003c/em\u003e, \u003cem\u003ecopB\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e likely reflect a response to iron limitation imposed by fungal siderophores.\u003c/p\u003e\n\u003cp\u003eConversely, downregulation of core metabolic (\u003cem\u003eackA\u003c/em\u003e, \u003cem\u003eglcU\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, amino acid biosynthesis (\u003cem\u003etyrS\u003c/em\u003e, \u003cem\u003eglnA\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and stress response genes (\u003cem\u003ehslO\u003c/em\u003e, \u003cem\u003ectsR\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e suggests reduced energy flux, nutrient competition, and impaired oxidative stress adaptability in the shared niche.\u003c/p\u003e\n\u003cp\u003eDownregulation of virulence and regulatory genes (\u003cem\u003eclfA\u003c/em\u003e, \u003cem\u003esarX\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e may reflect immune evasion or interkingdom tolerance. Suppressed DNA replication (\u003cem\u003erecU\u003c/em\u003e, \u003cem\u003ednaA\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and cell division genes (\u003cem\u003eparC\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e genes indicate a shift toward quiescence. Repression of \u003cem\u003enorB\u003c/em\u003e and \u003cem\u003enorG\u003c/em\u003e suggests diminished efflux capacity, potentially increasing susceptibility to antimicrobial or metabolic stress during co-culture.\u003c/p\u003e\n\u003cp\u003eGene-level visualizations supported the observed transcriptional reprogramming. Heatmaps (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-b) illustrated a clear contrast between upregulated and downregulated genes across key functional categories, indicating strong condition-specific expression shifts. The circular barplot (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec) emphasized the balanced distribution of regulatory changes, while UMAP clustering (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee) distinctly separated gene expression patterns into two polarized groups. The Sankey diagram (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed) mapped the top DEGs into major biological categories, highlighting the coordinated nature of the bidirectional response in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTogether, these results demonstrate that \u003cem\u003eC. albicans\u003c/em\u003e induces a finely orchestrated transcriptional reprogramming in \u003cem\u003eS. aureus\u003c/em\u003e, activating virulence, stress tolerance, and metabolic flexibility, while repressing growth-associated, biosynthetic, and oxidative defense pathways. This dual modulation likely reflects a survival strategy tailored to the polymicrobial niche, enabling \u003cem\u003eS. aureus\u003c/em\u003e to persist under cooperative or competitive pressures imposed by \u003cem\u003eC. albicans\u003c/em\u003e. The observed plasticity aligns with prior evidence of fungal\u0026ndash;bacterial synergy in mucosal colonization, biofilms, and disseminated infections \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Importantly, the selective silencing of replication and redox defenses, alongside the activation of virulence, suggests a shift toward a quiescent yet pathogenic phenotype. These findings offer mechanistic insight into interkingdom interactions and identify potential vulnerabilities for therapeutic targeting in chronic or device-associated infections.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e activates type VII secretion and virulence expression in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e during co-culture\u003c/p\u003e\n\u003cp\u003eGlobal transcriptomic profiling identified 1,576 DEGs in \u003cem\u003eS. aureus\u003c/em\u003e during co-culture with \u003cem\u003eE. faecalis\u003c/em\u003e, comprising 890 upregulated and 686 downregulated genes. The volcano plot (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea) highlights the distribution of significantly altered genes across a broad range of fold changes and p-values, while the bar plot and venn diagram (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb-c) confirm the relative proportions of gene regulation categories. STRING-based protein\u0026ndash;protein interaction networks (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed-e) revealed tightly interconnected clusters among up- and downregulated DEGs, suggesting coordinated regulatory control during polymicrobial stress.\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analysis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef-g) showed that upregulated genes were enriched in amino acid biosynthesis, cofactor metabolism, and carbohydrate degradation pathways. GO molecular functions included ATP binding, metal ion binding, and stress response activities. At the same time, enriched cellular components such as membrane-associated and extracellular regions suggest enhanced environmental sensing, structural adaptation, and nutrient acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e underwent extensive transcriptional reprogramming in response to \u003cem\u003eE. faecalis\u003c/em\u003e, characterized by the upregulation of virulence, stress adaptation, metabolic plasticity, and interbacterial competition mechanisms. Upregulation of virulence genes (\u003cem\u003espa\u003c/em\u003e, \u003cem\u003eclfA\u003c/em\u003e, \u003cem\u003efnbB\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e supports enhanced adhesion, immune evasion, and biofilm formation. Elevated \u003cem\u003elukEv\u003c/em\u003e, \u003cem\u003ehlgB\u003c/em\u003e, and \u003cem\u003ehly\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e indicate a cytolytic phenotype, promoting tissue damage and competitive advantage. Notably, the activation of \u003cem\u003eessA\u003c/em\u003e and \u003cem\u003esplF\u003c/em\u003e indicated the type VII secretion system (T7SS) engagement\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, a contact-dependent pathway in \u003cem\u003eS. aureus\u003c/em\u003e that translocates toxic effectors into Gram-positive competitors to establish niche dominance. T7SS is selectively activated against structurally compatible bacteria such as \u003cem\u003eE. faecalis\u003c/em\u003e, whose thick peptidoglycan layer enables effector delivery and physical sensing\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eP. aeruginosa\u003c/em\u003e, a Gram-negative bacterium, possesses an outer membrane that impedes T7SS activity and secretes diffusible inhibitors (e.g., HQNO, pyocyanin)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e that suppress \u003cem\u003eS. aureus\u003c/em\u003e transcriptionally, including T7SS components. Likewise, \u003cem\u003eC. albicans\u003c/em\u003e lacks the structural features required for T7SS targeting and does not pose direct antibacterial aggression. Instead, \u003cem\u003eS. aureus\u003c/em\u003e exhibits a synergistic transcriptional response to \u003cem\u003eC. albicans\u003c/em\u003e, upregulating virulence and stress pathways without T7SS induction, consistent with their known mutual persistence in biofilms and mucosal niches\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This context-dependent specificity emphasizes a finely tuned defense system in \u003cem\u003eS. aureus\u003c/em\u003e, selectively deployed against compatible bacterial threats.\u003c/p\u003e\n\u003cp\u003eStress adaptation was prominent, with upregulation of \u003cem\u003euvrA\u003c/em\u003e, \u003cem\u003erecO\u003c/em\u003e, and \u003cem\u003elexA\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e indicating oxidative and DNA damage response to \u003cem\u003eE. faecalis\u003c/em\u003e-derived ROS. Induced \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003ecap5A\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e likely enhanced envelope stability and redox control. Adhesion-related genes (\u003cem\u003eatl\u003c/em\u003e, \u003cem\u003espa\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e were elevated, the concurrent upregulation of \u003cem\u003eicaR\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, a repressor of the \u003cem\u003eicaADBC\u003c/em\u003e operon, and downregulation of \u003cem\u003eicaD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e ) suggest repression of polysaccharide biofilm, favoring a proteinaceous matrix phenotype.\u003c/p\u003e\n\u003cp\u003eMetabolic reprogramming was marked by upregulation of \u003cem\u003eackA\u003c/em\u003e, and \u003cem\u003eglcU\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, indicating a shift between fermentative and respiratory modes. Elevated \u003cem\u003etyrA\u003c/em\u003e, and \u003cem\u003eleuS\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e suggested increased anabolic demands. Upregulation of \u003cem\u003enikD\u003c/em\u003e, and \u003cem\u003ekdpA\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e reflected active trace metal acquisition. Additionally, expression of mobile element genes (\u003cem\u003etnpR\u003c/em\u003e, \u003cem\u003etnpC\u003c/em\u003e) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and transcriptional regulators (\u003cem\u003erpoB\u003c/em\u003e, and \u003cem\u003erpoE\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e suggested genomic plasticity and transcriptional adaptation under stress.\u003c/p\u003e\n\u003cp\u003eConversely, significant repression affected core biosynthetic, metabolic, and virulence pathways. Key glycolytic and TCA cycle genes (\u003cem\u003egapA2\u003c/em\u003e, \u003cem\u003eglkA\u003c/em\u003e, \u003cem\u003emqo2\u003c/em\u003e) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e were downregulated, indicating energy conservation. Suppression of \u003cem\u003epurD\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e suggested reduced nucleotide synthesis, while \u003cem\u003eatpF\u003c/em\u003e and \u003cem\u003ectaB\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e downregulation pointed to diminished respiration. Translational repression was evident from reduced \u003cem\u003erplU\u003c/em\u003e, and \u003cem\u003erpsL\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, elongation factor efp \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e and tRNA synthetases (\u003cem\u003epheS\u003c/em\u003e, \u003cem\u003eileS\u003c/em\u003e, \u003cem\u003elysS\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Downregulation of \u003cem\u003ednaA\u003c/em\u003e and \u003cem\u003erecR\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e indicated cell cycle arrest and impaired DNA repair.\u003c/p\u003e\n\u003cp\u003eKey virulence genes (\u003cem\u003efnbA\u003c/em\u003e, \u003cem\u003ehlgA\u003c/em\u003e) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e were downregulated, along with capsule and cell wall genes (\u003cem\u003elytM\u003c/em\u003e, \u003cem\u003ecls2\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e ), indicating reduced structural virulence and immune evasion. Suppressed expression of staphyloxanthin genes (B et al. 2024) suggested weakened antioxidant defense. Downregulation of regulatory elements (\u003cem\u003ectsR\u003c/em\u003e, \u003cem\u003erex\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e implied impaired environmental sensing, while decreased expression of division proteins (\u003cem\u003eftsA\u003c/em\u003e, \u003cem\u003eezrA\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e supported a quiescent, metabolically restrained state under competitive pressure.\u003c/p\u003e\n\u003cp\u003eGene-level visualizations validated these findings. Heatmaps (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea-b) revealed distinct clusters of upregulated and downregulated genes, reflecting a coordinated transcriptional shift in response to polymicrobial co-culture. UMAP clustering and radial barplots (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee) further illustrated a clear polarization of gene expression profiles, with minimal overlap between expression states. The Sankey diagram (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed) mapped major transcriptional changes across key functional categories, emphasizing the organized reprogramming of \u003cem\u003eS. aureus\u003c/em\u003e under competitive stress.\u003c/p\u003e\n\u003cp\u003eThese data reveal that \u003cem\u003eS. aureus\u003c/em\u003e mounts a dualistic response to \u003cem\u003eE. faecalis\u003c/em\u003e co-culture, activating T7SS, virulence pathways, and biosynthesis, while concurrently silencing growth, translation, and energetically costly processes. This ecological trade-off prioritizes stress endurance, competitive fitness, and sessile adaptation over proliferation. Given the known secretion of bacteriocins and hydrogen peroxide by \u003cem\u003eE. faecalis\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the transcriptional reprogramming in \u003cem\u003eS. aureus\u003c/em\u003e likely reflects an integrated response to direct and indirect microbial competition. These findings emphasize the importance of interbacterial signaling in modulating pathogen behavior and suggest that disrupting communication pathways such as T7SS or quorum sensing may provide therapeutic avenues for managing polymicrobial infections.\u003c/p\u003e\n\u003cp\u003eThese findings not only reveal condition-specific transcriptional reprogramming in \u003cem\u003eS. aureus\u003c/em\u003e but also offer translational insights into how microbial interactions could be harnessed therapeutically. By delineating the transcriptional logic governing \u003cem\u003eS. aureus\u003c/em\u003e survival, virulence modulation, and stress adaptation in response to diverse microbial neighbors, this study provides a blueprint for targeted antimicrobial strategies that mimic or disrupt these ecological signals. Such approaches may inform novel treatments for polymicrobial infections, especially those associated with biofilms, chronic wounds, and implanted devices, by either potentiating suppressive interspecies effects (as seen with \u003cem\u003eP. aeruginosa\u003c/em\u003e) or neutralizing synergistic or modulatory virulence interactions (as observed with \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e). Furthermore, understanding these adaptive mechanisms enhances our ability to predict pathogen behavior in vivo and design precision interventions that shift microbial dynamics in favor of host recovery.\u003c/p\u003e\n\u003cp\u003eHowever, this study has certain limitations. It presents a single-time-point, snapshot view of the transcriptomic landscape, without capturing dynamic transitions or temporal changes in gene expression. This study was designed as an exploratory, hypothesis-generating investigation to uncover broad transcriptional trends in \u003cem\u003eS. aureus\u003c/em\u003e during polymicrobial co-culture. Therefore, RNA-seq was conducted without biological replicates or RT-qPCR validation, focusing instead on global expression patterns and shifts in functional categories. Whole transcriptome profiling was integrated to provide a broader, systems-level perspective and to serve as a resource for future studies, where individual genes can be validated in targeted experimental models.\u003c/p\u003e\n\u003cp\u003eA graphical summary of the distinct interaction modes, antagonism with \u003cem\u003eP. aeruginosa\u003c/em\u003e, synergy with \u003cem\u003eC. albicans\u003c/em\u003e, and competitive response to \u003cem\u003eE. faecalis\u003c/em\u003e, is depicted in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, highlighting condition-specific modulation of virulence, metabolism, adhesion, biofilm, and T7SS expression in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThis schematic illustrates the distinct gene expression profiles of \u003cem\u003eS. aureus\u003c/em\u003e when co-cultured with \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e. In the left panel, representing co-culture with \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e exhibits a synergistic interaction marked by upregulation of virulence and cell adhesion genes, while genes involved in T7SS secretion and metabolism are downregulated. In the centre panel, during co-culture with \u003cem\u003eP. aeruginosa\u003c/em\u003e, an antagonistic response is observed, with global transcriptional repression affecting metabolism, biofilm formation, adhesion, and T7SS genes, indicating suppression of \u003cem\u003eS. aureus\u003c/em\u003e pathogenic potential. In the right panel, co-culture with \u003cem\u003eE. faecalis\u003c/em\u003e leads to a competitive interaction characterized by upregulation of T7SS, virulence factors, metabolic pathways, and adhesion genes, while biofilm-related genes are downregulated. Upregulated and downregulated functions are indicated by green (▲) and red (▼) arrows, respectively. Icons represent core functional themes such as T7SS, metabolism, biofilm, virulence, and adhesins. The central test tubes depict \u003cem\u003eS. aureus\u003c/em\u003e co-cultured with each partner organism.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eS. aureus\u003c/em\u003e exhibits distinct, context-dependent transcriptional responses when co-cultured with \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e. While \u003cem\u003eP. aeruginosa\u003c/em\u003e imposes a dominant suppressive effect, silencing metabolism, stress response, and virulence networks, \u003cem\u003eC. albicans\u003c/em\u003e elicits a synergistic reprogramming marked by co-activation of virulence, metabolic flexibility, and redox tolerance. In contrast, \u003cem\u003eE. faecalis\u003c/em\u003e triggers a defensive, antagonistic response characterized by activation of the T7SS, virulence regulators, and anabolic machinery, reflecting a competitive interbacterial strategy. These divergent expression profiles reflect tailored ecological adaptations: suppression under Gram-negative stress, cooperation with fungal partners, and aggression against Gram-positive competitors. These findings provide mechanistic insight into how \u003cem\u003eS. aureus\u003c/em\u003e dynamically modulates its transcriptome to balance competition, coexistence, and persistence in polymicrobial environments, laying the foundation for precision microbiology strategies that target interspecies signaling and microbial crosstalk in complex infections. Designed as an exploratory, hypothesis-generating investigation, this study reveals global transcriptomic trends, offering a foundational framework for future functional dissection of virulence adaptation in polymicrobial infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eNirmala B designed and performed the experiments, analyzed the data, and wrote the manuscript. Yogendra P Mathuria and Balram Ji Omar supervised the study and validated the findings. All authors reviewed and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe raw RNA-seq datasets generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession number \u003cstrong\u003ePRJNA1307151\u003c/strong\u003e. The processed data, including the complete normalized expression matrix and lists of differentially expressed genes (DEGs) for each co-culture condition, are provided in the \u003cstrong\u003eSupplementary Dataset (Excel file)\u003c/strong\u003e. This dataset includes sample metadata, expression values, and statistical outputs used for downstream analyses. The custom R scripts and visualization notebooks used for data processing and figure generation are openly available on GitHub at https://github.com/Nirmala-1997/Transcriptomics-Polymicrobial-regulation, ensuring full transparency and reproducibility.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe acknowledge the Department of Biotechnology, Ministry of Science and Technology, Government of India, for providing a DBT-SRF scholarship to B Nirmala. We also acknowledge the All India Institute of Medical Sciences (AIIMS) Rishikesh for providing infrastructural support for this study, and the Indian Institute of Technology (IIT) Roorkee for facilitating scanning electron microscopy (SEM) analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang, M., Whiteley, M. \u0026amp; Lewin, G. R. Polymicrobial Interactions of Oral Microbiota: a Historical Review and Current Perspective. \u003cem\u003emBio\u003c/em\u003e vol. 13 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.00235-22\u003c/span\u003e\u003cspan address=\"10.1128/mbio.00235-22\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMariani, F. \u0026amp; Galvan, E. M. Staphylococcus aureus in Polymicrobial Skinand Soft Tissue Infections: Impact of Inter-Species Interactionsin Disease Outcome. \u003cem\u003eAntibiotics\u003c/em\u003e vol. 12 Preprint at (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antibiotics12071164\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics12071164\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu, G., Ge, X., Li, W., Ji, L. \u0026amp; Yang, S. Interspecific cross-talk: The catalyst driving microbial biosynthesis of secondary metabolites. \u003cem\u003eBiotechnology Advances\u003c/em\u003e vol. 76 Preprint at (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2024.108420\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2024.108420\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRupp, M. et al. Polymicrobial infections and microbial patterns in infected nonunions - A descriptive analysis of 42 cases. \u003cem\u003eBMC Infect. Dis\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePouget, C. et al. Polymicrobial Biofilm Organization of Staphylococcus aureus and Pseudomonas aeruginosa in a Chronic Wound Environment. \u003cem\u003eInt J. Mol. Sci\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJean-Pierre, F., Vyas, A., Hampton, T. H. \u0026amp; Henson, M. A. O\u0026rsquo;toole, G. A. One versus many: Polymicrobial communities and the cystic fibrosis airway. \u003cem\u003emBio\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 1\u0026ndash;7 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRamstedt, M. \u0026amp; Burm\u0026oslash;lle, M. Can multi-species biofilms defeat antimicrobial surfaces on medical devices? \u003cem\u003eCurrent Opinion in Biomedical Engineering\u003c/em\u003e vol. 22 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cobme.2022.100370\u003c/span\u003e\u003cspan address=\"10.1016/j.cobme.2022.100370\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHowden, B. P. et al. Staphylococcus aureus host interactions and adaptation. \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e vol. 21 380\u0026ndash;395 Preprint at (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-023-00852-y\u003c/span\u003e\u003cspan address=\"10.1038/s41579-023-00852-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheung, G. Y. C., Bae, J. S. \u0026amp; Otto, M. Pathogenicity and virulence of Staphylococcus aureus. \u003cem\u003eVirulence\u003c/em\u003e vol. 12 547\u0026ndash;569 Preprint at (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/21505594.2021.1878688\u003c/span\u003e\u003cspan address=\"10.1080/21505594.2021.1878688\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNguyen, A. T. \u0026amp; Oglesby-Sherrouse, A. G. Interactions between Pseudomonas aeruginosa and Staphylococcus aureus during co-cultivations and polymicrobial infections. \u003cem\u003eApplied Microbiology and Biotechnology\u003c/em\u003e vol. 100 6141\u0026ndash;6148 Preprint at (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-016-7596-3\u003c/span\u003e\u003cspan address=\"10.1007/s00253-016-7596-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarriott, M. M. \u0026amp; Noverr, M. C. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: Effects on antimicrobial resistance. \u003cem\u003eAntimicrob. Agents Chemother.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 3914\u0026ndash;3922 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKao, P. H. N. et al. Enterococcus faecalis suppresses Staphylococcus aureus-induced NETosis and promotes bacterial survival in polymicrobial infections. \u003cem\u003eFEMS Microbes\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKulshrestha, A. \u0026amp; Gupta, P. Polymicrobial interaction in biofilm: mechanistic insights. \u003cem\u003ePathog Dis\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEichelberger, K. R. \u0026amp; Cassat, J. E. Metabolic Adaptations During Staphylococcus aureus and Candida albicans Co-Infection. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e vol. 12 Preprint at (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2021.797550\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2021.797550\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDastgheyb, S. S. \u0026amp; Otto, M. Staphylococcal adaptation to diverse physiologic niches: An overview of transcriptomic and phenotypic changes in different biological environments. \u003cem\u003eFuture Microbiology\u003c/em\u003e vol. 10 \u0026ndash;1995 Preprint at (1981). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2217/fmb.15.116\u003c/span\u003e\u003cspan address=\"10.2217/fmb.15.116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB, N. et al. A novel dual-staining method for cost-effective visualization and differentiation of microbial biofilms. \u003cem\u003eSci Rep\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBiswas, L. \u0026amp; G\u0026ouml;tz, F. Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis. \u003cem\u003eFrontiers in Cellular and Infection Microbiology\u003c/em\u003e vol. 11 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fcimb.2021.824042\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2021.824042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJing, Q. et al. Staphylococcus aureus wraps around Candida albicans and synergistically escapes from Neutrophil extracellular traps. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNogueira Vi\u0026ccedil;osa, G. et al. Impact of co-cultivation with Enterococcus faecalis over growth, enterotoxin production and gene expression of Staphylococcus aureus in broth and fresh cheeses. \u003cem\u003eInt J. Food Microbiol\u003c/em\u003e \u003cb\u003e308\u003c/b\u003e, (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLyu, Z., Wilson, C. \u0026amp; Ling, J. Translational Fidelity during Bacterial Stresses and Host Interactions. \u003cem\u003ePathogens\u003c/em\u003e vol. 12 Preprint at (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pathogens12030383\u003c/span\u003e\u003cspan address=\"10.3390/pathogens12030383\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAuburger, G., Key, J. \u0026amp; Gispert, S. The Bacterial ClpXP-ClpB Family Is Enriched with RNA-Binding Protein Complexes. \u003cem\u003eCells\u003c/em\u003e vol. 11 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells11152370\u003c/span\u003e\u003cspan address=\"10.3390/cells11152370\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTroitzsch, A. et al. Carbon source-dependent reprogramming of anaerobic metabolism in staphylococcus aureus. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cb\u003e203\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoux, A. E. et al. The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAshniev, G. A., Petrov, S. N., Iablokov, S. N. \u0026amp; Rodionov, D. A. Genomics-Based Reconstruction and Predictive Profiling of Amino Acid Biosynthesis in the Human Gut Microbiome. \u003cem\u003eMicroorganisms\u003c/em\u003e 10, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMontagut, E. J. et al. An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiao, X., Chen, X., Sant\u0026rsquo;Ana, A. S., Feng, J. \u0026amp; Ding, T. Pre-Exposure of Foodborne Staphylococcus aureus Isolates to Organic Acids Induces Cross-Adaptation to Mild Heat. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB, N. \u0026amp; Omar, B. J. Enhancing Staphyloxanthin Synthesis in Staphylococcus aureus Using Innovative Agar Media Formulations. \u003cem\u003eCureus\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.59892\u003c/span\u003e\u003cspan address=\"10.7759/cureus.59892\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, M. \u0026amp; Zhang, Q. Characteristics of Virulence Genes of Clinically Isolated Staphylococci in Jingzhou Area. \u003cem\u003eContrast Media Mol Imaging\u003c/em\u003e (2022). (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWen, Z. et al. Baohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ. \u003cem\u003ePhytomedicine\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan, L. et al. Exploring the potential of isorhapontigenin: attenuating Staphylococcus aureus virulence through MgrA-mediated regulation. \u003cem\u003emSphere\u003c/em\u003e 9, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, M., Buist, G. \u0026amp; van Dijl, J. M. Staphylococcus aureus cell wall maintenance \u0026ndash; the multifaceted roles of peptidoglycan hydrolases in bacterial growth, fitness, and virulence. \u003cem\u003eFEMS Microbiology Reviews\u003c/em\u003e vol. 46 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/femsre/fuac025\u003c/span\u003e\u003cspan address=\"10.1093/femsre/fuac025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuvelot, H. et al. Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026eacute;linas, M., Museau, L., Milot, A. \u0026amp; Beauregard, P. B. \u003cem\u003eThe de Novo Purine Biosynthesis Pathway Is the Only Commonly Regulated Cellular Pathway during Biofilm Formation in TSB-Based Medium in Staphylococcus Aureus and Enterococcus Faecalis\u003c/em\u003e. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://journals.asm.org/journal/spectrum\u003c/span\u003e\u003cspan address=\"https://journals.asm.org/journal/spectrum\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHou, Z., Liu, L., Wei, J. \u0026amp; Xu, B. Progress in the Prevalence, Classification and Drug Resistance Mechanisms of Methicillin-Resistant Staphylococcus aureus. \u003cem\u003eInfection and Drug Resistance\u003c/em\u003e vol. 16 3271\u0026ndash;3292 Preprint at (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IDR.S412308\u003c/span\u003e\u003cspan address=\"10.2147/IDR.S412308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao, Z., Casabona, M. G., Kneuper, H., Chalmers, J. D. \u0026amp; Palmer, T. The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. \u003cem\u003eNat Microbiol\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohapatra, D., Das, Pattnaik, S. \u0026amp; Panda, S. In Vitro Detected hly II Cytotoxin in a Strain of Staphylococcus aureus (BM S-2) and Plant-Derived Aromatic Components: a Molecular Docking Study. \u003cem\u003eAppl. Biochem. Biotechnol.\u003c/em\u003e \u003cb\u003e193\u003c/b\u003e, 1639\u0026ndash;1653 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, S. et al. A Practical Approach for Predicting Antimicrobial Phenotype Resistance in Staphylococcus aureus Through Machine Learning Analysis of Genome Data. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartini, A. M., Alexander, S. A. \u0026amp; Khare, A. Mutations in the Staphylococcus aureus Global Regulator CodY confer tolerance to an interspecies redox-active antimicrobial. \u003cem\u003ePLoS Genet.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, e1011610 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuchs, S., Pan\u0026eacute;-Farr\u0026eacute;, J., Kohler, C., Hecker, M. \u0026amp; Engelmann, S. Anaerobic gene expression in Staphylococcus aureus. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e189\u003c/b\u003e, 4275\u0026ndash;4289 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, X., Wang, Y., Chang, W., Dai, Y. \u0026amp; Ma, X. AgrA directly binds to the promoter of vraSR and downregulates its expression in Staphylococcus aureus. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWittekind, M. A., Briaud, P., Smith, J. L., Tennant, J. R. \u0026amp; Carroll, R. K. The Small Protein ScrA Influences Staphylococcus aureus Virulence-Related Processes via the SaeRS System. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCordero, M. et al. The induction of natural competence adapts staphylococcal metabolism to infection. \u003cem\u003eNat Commun\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, M. et al. Mechanisms of mepA Overexpression and Membrane Potential Reduction Leading to Ciprofloxacin Heteroresistance in a Staphylococcus aureus Isolate. \u003cem\u003eInt J. Mol. Sci\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRossi, C. C. et al. Expression of the stress-response regulators CtsR and HrcA in the uropathogen Staphylococcus saprophyticus during heat shock. \u003cem\u003eAntonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol.\u003c/em\u003e \u003cb\u003e110\u003c/b\u003e, 1105\u0026ndash;1111 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYee, R., Feng, J., Wang, J., Chen, J. \u0026amp; Zhang, Y. Identification of Genes Regulating Cell Death in Staphylococcus aureus. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao, Y. et al. Novel small-molecule compound YH7 inhibits the biofilm formation of Staphylococcus aureus in a sarX -dependent manner. \u003cem\u003emSphere\u003c/em\u003e 9, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhattacharya, M., Scherr, T. D., Lister, J., Kielian, T. \u0026amp; Horswill, A. R. Extracellular adherence proteins reduce matrix porosity and enhance Staphylococcus aureus biofilm survival during prosthetic joint infection. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e, (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa, Z. et al. The Recombinant Expression Proteins FnBP and ClfA From Staphylococcus aureus in Addition to GapC and Sip From Streptococcus agalactiae Can Protect BALB/c Mice From Bacterial Infection. \u003cem\u003eFront Vet. Sci\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarbuti, M. D., Myrbr\u0026aring;ten, I. S., Morales Angeles, D. \u0026amp; Kjos, M. The cell cycle of Staphylococcus aureus: An updated review. \u003cem\u003eMicrobiologyOpen\u003c/em\u003e vol. 12 Preprint at (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mbo3.1338\u003c/span\u003e\u003cspan address=\"10.1002/mbo3.1338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHa, K. P. \u0026amp; Edwards, A. M. DNA Repair in Staphylococcus aureus. \u003cem\u003eMicrobiology Mol. Biology Reviews\u003c/em\u003e \u003cb\u003e85\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuynh, T. Q. et al. Genomic alterations involved in fluoroquinolone resistance development in Staphylococcus aureus. \u003cem\u003ePLoS One\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu, Y. et al. Staphylococcus aureus synergized with Candida albicans to increase the pathogenesis and drug resistance in cutaneous abscess and peritonitis murine models. \u003cem\u003ePathogens\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun, H., Li, R. W., Wang, T. T. Y. \u0026amp; Ding, L. The Ligand Binding Domain of the Cell Wall Protein SraP Modulates Macrophage Apoptosis and Inflammatory Responses in Staphylococcus aureus Infections. \u003cem\u003eMolecules\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAfzal, M., Vijay, A. K., Stapleton, F. \u0026amp; Willcox, M. D. P. Genomics of Staphylococcus aureus Strains Isolated from Infectious and Non-Infectious Ocular Conditions. \u003cem\u003eAntibiotics\u003c/em\u003e 11, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarrett, S. R. \u0026amp; Palmer, T. The role of proteinaceous toxins secreted by Staphylococcus aureus in interbacterial competition. \u003cem\u003eFEMS Microbes\u003c/em\u003e vol. 5 Preprint at (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/femsmc/xtae006\u003c/span\u003e\u003cspan address=\"10.1093/femsmc/xtae006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGibson, J. F. et al. Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBastos, M. L. C. et al. What Do We Know About Staphylococcus aureus and Oxidative Stress? Resistance, Virulence, New Targets, and Therapeutic Alternatives. \u003cem\u003eToxics\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 390 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTheis, T. J., Daubert, T. A., Kluthe, K. E., Brodd, K. L. \u0026amp; Nuxoll, A. S. Staphylococcus aureus persisters are associated with reduced clearance in a catheter-associated biofilm infection. \u003cem\u003eFront Cell. Infect. Microbiol\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei, B. et al. Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP. \u003cem\u003eNat Commun\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchwartbeck, B. et al. Various mutations in icaR, the repressor of the icaADBC locus, occur in mucoid Staphylococcus aureus isolates recovered from the airways of people with cystic fibrosis. \u003cem\u003eMicrobes Infect\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBertrand, B. P. et al. Role of Staphylococcus aureus Formate Metabolism during Prosthetic Joint Infection. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeidarian, S., Guliaev, A., Nicoloff, H., Hjort, K. \u0026amp; Andersson, D. I. High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes. \u003cem\u003ePLoS Biol\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiao, Z. et al. Transcriptomic analyses reveal the potential antibacterial mechanism of citral against Staphylococcus aureus. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhssein, G. \u0026amp; Ezzeddine, Z. The Key Element Role of Metallophores in the Pathogenicity and Virulence of Staphylococcus aureus: A Review. \u003cem\u003eBiology\u003c/em\u003e vol. 11 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biology11101525\u003c/span\u003e\u003cspan address=\"10.3390/biology11101525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng, H. et al. Transcriptomic Analysis Revealed Antimicrobial Mechanisms of Lactobacillus rhamnosus SCB0119 against Escherichia coli and Staphylococcus aureus. \u003cem\u003eInt J. Mol. Sci\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJi, X. et al. Tn560, a Novel Tn554 Family Transposon from Porcine Methicillin-Resistant Staphylococcus aureus ST398, Carries a Multiresistance Gene Cluster Comprising a Novel spc Gene Variant and the Genes lsa(E) and lnu(B). \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e vol. 66 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.01947-21\u003c/span\u003e\u003cspan address=\"10.1128/aac.01947-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNielsen, T. K. et al. The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePires, P. M., Santos, D., Calisto, F. \u0026amp; Pereira, M. The monotopic quinone reductases from Staphylococcus aureus. \u003cem\u003eBiochim Biophys. Acta Bioenerg\u003c/em\u003e \u003cb\u003e1865\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Backer, S. et al. Enzymes catalyzing the tca-and urea cycle influence the matrix composition of biofilms formed by methicillin-resistant staphylococcus aureus usa300. \u003cem\u003eMicroorganisms\u003c/em\u003e 6, (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoncheva, M. I., Flannagan, R. S. \u0026amp; Heinrichs, D. E. \u003cem\u003eDe Novo Purine Biosynthesis Is Required for Intracellular Growth of Staphylococcus Aureus and for the Hypervirulence Phenotype of a PurR Mutant\u003c/em\u003e. (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://journals.asm.org/journal/iai\u003c/span\u003e\u003cspan address=\"https://journals.asm.org/journal/iai\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu, T. et al. Absence of protoheme IX farnesyltransferase CtaB causes virulence attenuation but enhances pigment production and persister survival in MRSA. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng, X. et al. Expression of Staphylococcus aureus translation elongation factor P is regulated by a stress-inducible promotor. \u003cem\u003eAntonie van Leeuwenhoek Int. J. Gen. Mol. Microbiology\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGieg\u0026eacute;, R. \u0026amp; Springer, M. Aminoacyl-tRNA Synthetases in the Bacterial World. \u003cem\u003eEcoSal Plus\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahman, S. \u0026amp; Das, A. K. Staphylococcal superantigen-like protein 10 enhances the amyloidogenic biofilm formation in Staphylococcus aureus. \u003cem\u003eBMC Microbiol\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu, Z. et al. Molecular Characteristics and Pathogenicity of Staphylococcus aureus Exotoxins. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e vol. 25 Preprint at (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms25010395\u003c/span\u003e\u003cspan address=\"10.3390/ijms25010395\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRodrigues, R. A. et al. Comparative genomics study of Staphylococcus aureus isolated from cattle and humans reveals virulence patterns exclusively associated with bovine clinical mastitis strains. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamanashi, Y. et al. Effects of Growth Stage on the Characterization of Enterotoxin A-Producing Staphylococcus aureus-Derived Membrane vesicles. \u003cem\u003eMicroorganisms\u003c/em\u003e 10, (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDmitriev, A. et al. The Intersection of the Staphylococcus aureus Rex and SrrAB Regulons: an Example of Metabolic Evolution That Maximizes Resistance to Immune Radicals. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/mBio\u003c/span\u003e\u003cspan address=\"10.1128/mBio\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMyrbr\u0026aring;ten, I. S. et al. SmdA is a Novel Cell Morphology Determinant in Staphylococcus aureus. \u003cem\u003emBio\u003c/em\u003e 13, (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Candida albicans, Enterococcus faecalis, Polymicrobial interactions, Pseudomonas aeruginosa, Transcriptomics, Staphylococcus aureus","lastPublishedDoi":"10.21203/rs.3.rs-7401018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7401018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the microbial world, survival is not solitary. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e thrives or falters depending on its neighbors. This opportunistic pathogen frequently inhabits polymicrobial environments such as chronic wounds, implanted devices, and mucosal surfaces, where interspecies interactions shape its behavior and complicate treatment outcomes. In this study, we adopt an exploratory, systems-level approach to examine how \u003cem\u003eS. aureus\u003c/em\u003e transcriptionally adapts during co-culture with three clinically relevant organisms: \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e. RNA sequencing uncovered distinct ecological signatures: \u003cem\u003eP. aeruginosa\u003c/em\u003e imposed a strongly antagonistic effect, driving global transcriptional repression, including silencing of virulence pathways; \u003cem\u003eC. albicans\u003c/em\u003e promoted a synergistic response with activation of virulence, stress, and metabolic genes; while \u003cem\u003eE. faecalis\u003c/em\u003e elicited a competitive program characterized by robust induction of the type VII secretion system, cytotoxic effectors, and biosynthetic functions. Rather than definitive mechanisms, these findings provide an exploratory map of interspecies transcriptional landscapes, generating hypotheses on how microbial neighbors modulate \u003cem\u003eS. aureus\u003c/em\u003e biology and highlighting interspecies signaling as a potential target for managing polymicrobial infections.\u003c/p\u003e","manuscriptTitle":"Exploratory transcriptomic analysis of Staphylococcus aureus adaptation during polymicrobial interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-02 11:03:50","doi":"10.21203/rs.3.rs-7401018/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9240885-1b69-4068-9c17-c982522b003f","owner":[],"postedDate":"September 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54017365,"name":"Biological sciences/Microbiology"},{"id":54017366,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-01-08T10:09:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-02 11:03:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7401018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7401018","identity":"rs-7401018","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}