Environmental and Chemical Modulation of Staphylococcus aureus Newman Biofilm Formation

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Ragab, Ahmed R. El‐Sheakh, Shokri M. Shafik This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7714339/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Abstract Staphylococcus aureus biofilm formation enhances survival on host tissues and medical devices. This study tested how oxidative stress (H₂O₂), pH (5–9), NaCl (0–10%), and human serum (5–50%) affect Newman strain biofilm and key genes ( icaA , icaD , sarA ). Biofilm was quantified by crystal violet assays, and gene expression measured by quantitative real-time PCR. Biofilm biomass was quantified using crystal violet staining assays under various environmental conditions. Statistical significance was determined using ANOVA with post-hoc analysis ( p < 0.05). Hydrogen peroxide induced a dose-dependent reduction in biomass, with significant repression of icaA, icaD , and sarA expression at 3% H₂O₂ (≤ 22.8%, p < 0.01). Similarly, deviations from neutral pH markedly impaired biofilm formation, with acidic (pH 5) and alkaline (pH 9) conditions reducing biomass by 34.6% and 41.7%, respectively, accompanied by strong downregulation of biofilm-associated genes ( p < 0.001). In contrast, NaCl exerted a biphasic effect: mild osmotic stress (1.25% and 5%) enhanced biofilm biomass (up to 154.2%) and gene expression ( icaA 160.55%, icaD 168.18%, sarA 149.8%, p < 0.01), whereas higher concentrations (≥ 10%) restored expression to near-control levels. Serum exposure produced a threshold-dependent response, with low concentrations (5–10%) slightly enhancing gene expression (~ 110%), while higher concentrations (20–50%) significantly repressed both biomass and transcription, with profound inhibition observed at 50% ( icaA 12.94%, icaD 10.88%, sarA 12.79%, p < 0.001). Environmental stressors modulate Staphylococcus aureus biofilm formation in a dose-dependent manner via regulation of the ica operon and sarA , offering molecular insights that may guide strategies for biofilm control. Staphylococcus aureus Newman Environmental factors Oxidative stress ica operon Gene expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Key Points Oxidative stress (H₂O₂) dose-dependently inhibits S. aureus Newman biofilms. Mild NaCl levels enhance biofilm formation via upregulation of ica and sarA . High serum concentrations (≥20%) suppress biofilm biomass and gene expression. Introduction Biofilm formation represents a fundamental survival strategy employed by Staphylococcus aureus , enabling bacterial persistence on medical devices, chronic wounds, and host tissues (Peng et al. 2022 ). These complex microbial communities provide protection against antimicrobial agents and host immune responses, contributing significantly to the burden of healthcare-associated infections and treatment failures (Ciofu et al. 2022 ). The clinical significance of Staph. aureus biofilms is underscored by their association with device-related infections, chronic osteomyelitis, and persistent bacteremia, conditions that often require prolonged antibiotic therapy and surgical intervention (Pietrocola et al. 2022 ; Di Domenico, Oliva, and Guembe 2022 ). The molecular architecture of Staph. aureus biofilms is governed by a sophisticated regulatory network involving multiple genetic determinants, containing icaA and icaD , that mediate polysaccharide intercellular adhesion (PIA) result and sarA , a key global regulator of biofilm production (Gowrishankar et al. 2016 ; Liu et al. 2024 ).Environmental factors such as oxidative stress, pH alterations, and osmotic environments are noted to influence biofilm development (Goller and Romeo 2008 ). Hydrogen peroxide, a primary reactive oxygen species, is produced by neutrophils and macrophages as part of the innate immune response and has been shown to influence biofilm formation in various bacterial species (da Cruz Nizer et al. 2024 ). pH changes can alter bacterial adherence characteristics and exopolysaccharide (EPS) production, thereby modulating biofilm integrity (Chaggar et al. 2022 ). Acidic conditions may arise in infected wounds due to bacterial metabolism and tissue hypoxia, while alkaline environments can result from certain therapeutic interventions (Wang et al. 2024 ). Similarly, osmotic stress induced by saline concentrations influences biofilm stability, as Staph. aureus frequently encounters variable salinity levels in wound environments and healing implants (Tonon et al. 2020 ). Additionally, serum components, containing proteins and immune factors, can either improve or prevent biofilm composition depending on their concentration (Wuren et al. 2014 ). Despite these known environmental cues, the transcriptional responses of Staph. aureus biofilms to such stresses are poorly understood. Staph. aureus Newman was originally isolated from a case of human osteomyelitis and has since become a widely used lab model for biofilm studies due to its well-characterized genome and virulence traits, including prophages and pathogenicity islands that underpin its behavior in infection models (Baba et al. 2008 ). Recent transcriptomic comparisons across multiple Staph. aureus strains have revealed that global gene expression during biofilm formation varies substantially by strain, marking Newman as particularly notable for its unique regulatory dynamics (Tomlinson, Malof, and Shaw 2021 ). Moreover, experimental evolution of Newman under selective conditions identified new mutations (e.g., in manA , narH , fruB ) capable of enhancing biofilm formation, underlining the strain’s adaptive complexity and relevance for modeling stress- or mutation-driven biofilm behavior (Long et al. 2023). Despite the recognized importance of environmental factors in biofilm development, comprehensive studies examining the coordinated effects of multiple stressors on both phenotypic biofilm formation and underlying gene expression in Staph. aureus Newman are limited. Furthermore, the dose-dependent relationships between environmental stressors and biofilm responses have not been systematically characterized, limiting our understanding of clinically relevant exposure levels. This study aimed to characterize the dose-dependent effects of hydrogen peroxide, pH variation, saline concentration, and human serum on Staph. aureus Newman biofilm formation, elucidate the corresponding changes in expression of key biofilm-associated genes ( icaA , icaD , and sarA ), identify optimal conditions for biofilm inhibition or enhancement, and provide molecular insights that may inform the development of environment-based biofilm control strategies. Materials and Methods Bacterial Strain and Culture Conditions The standard strain of Staph. aureus Newman (ATCC 25904) was obtained from Microbiology Department, Faculty of Pharmacy, National Mansoura University. The strain was routinely cultured on Tryptic Soy Agar (Oxoid, UK) and incubated at 37°C for 18–24 hours under aerobic conditions. The bacterial culture was diluted to 1:100 in TSB to obtain a standardized inoculum. Biofilm Formation Assay Biofilm formation was quantified using the crystal violet staining method as previously described with modifications (Stepanovic et al. 2007 ). Briefly, S. aureus cultures were inoculated into 96-well microtiter plates containing 200 µL of TSB supplemented with the respective test conditions. Plates were incubated at 37°C for 24 to 72 hours. After incubation, planktonic cells were removed by washing with phosphate-buffered saline (PBS) (pH 7.4), and adherent biofilms were fixed with 99% methanol for 30 minutes. Biofilms were stained with 0.1% crystal violet (Sigma-Aldrich, USA) for 15 minutes, followed by three washes with PBS. The bound crystal violet was solubilized using 33% glacial acetic acid (Adwic, Egypt), and absorbance was measured at 570 nm using a microplate reader (BioTek, USA). Experimental Conditions Effect of Hydrogen Peroxide To assess the impact of oxidative stress on biofilm formation, hydrogen peroxide (H₂O₂; Sigma-Aldrich, USA) was added to TSB at final concentrations of 0.5%, 1%, 2%, and 3%. The control group contained TSB without H₂O₂. These concentrations were selected based on previous studies investigating the oxidative stress response in S. aureus biofilms, with adaptations specific to the current experimental model (Kaplan 2011 ). Biofilm formation was measured after 72 hours of incubation. Effect of pH Variation To evaluate the influence of pH on biofilm development, the pH of TSB was adjusted to 5, 7, and 9 using sterile 1M HCl or NaOH (Sigma-Aldrich, USA). While previous research has shown that acidic and alkaline conditions affect biofilm integrity and gene expression (Tan 2014 ), the selected pH values were optimized in the present study to capture a broad physiological range. Cultures were incubated for 24 hours prior to biofilm quantification. Effect of Saline Concentration To examine the effect of osmotic stress, NaCl was added to TSB at final concentrations of 1.25%, 2.5%, 5%, and 10%, with standard TSB (0.9% NaCl) used as a control. These concentrations were guided by prior findings indicating that salinity alters biofilm production (Beckingsale 2008 ), though specific salt levels were optimized for the current strain and conditions. Biofilm formation was assessed after 24 hours. Effect of Serum Concentration Human serum (Sigma-Aldrich, USA) was added to TSB at final concentrations of 5%, 10%, 20%, and 50% to investigate the influence of host-derived factors on biofilm formation. Serum-free TSB served as the control. Previous studies have demonstrated that serum components can modulate biofilm formation (Skovdal et al. 2021 ); however, the concentration range used here was tailored to assess dose-dependent effects in vitro. Biofilms were quantified after 24 hours. Genotypic Analysis by Quantitative Real-Time PCR (qRT-PCR) RNA Extraction and cDNA Synthesis Total RNA was extracted from biofilm-forming Staph. aureus cells using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. RNA purity and concentration were determined using a Nanodrop Spectrophotometer (Thermo Fisher, USA). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). qRT-PCR Amplification qRT-PCR was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA) with SYBR Green Master Mix in a 20 µL reaction mixture containing 10 µL SYBR Green Master Mix , 1 µL of each primer (10 µM), 2 µL cDNA, and 6 µL nuclease-free water. The thermal cycling conditions included an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of amplification (95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds). A melting curve analysis was conducted to verify amplification specificity. Target Genes and Primers The expression of five key biofilm-associated genes was analyzed as shown in Table 1 : Table 1 Primers used for the 3 genes responsible for biofilm formation in Staphylococcus aureus Newman strain in addition for primers of house keeping genes Gene Forward Primer (5’-3’) Reverse Primer (5’-3’) Reference icaA ACA CTT GCT GGC GCA GTC AA TCT GGA ACC AAC ATC CAA CA (Mirzaee, Najar Peerayeh, and Ghasemian 2014) icaD GAA CCG CTT GCC ATG TGT TG GCT TGA CCA TGT TGC GTA ACC (Namvar et al. 2013 ) sarA TCT TGT TAA TGC ACA ACA ACG TGT TTG CTT CAG TGA TTC GTT (Sambanthamoorthy, Smeltzer, and Elasri 2006) 16 s rRNA TGT CGT GAG ATG TTG GG CGA TTC CAG CTT CAT GT (Lee et al. 2013) Relative gene expression was calculated using the 2^(-ΔΔCt) method, normalizing target gene expression to 16 s rRNA (Livak and Schmittgen 2001). 2.5. Statistical Analysis All experiments were performed in quadruplicate. Data was analyzed using GraphPad Prism 5 (GraphPad Software, USA). One-way ANOVA followed by Tukey’s post hoc test was used to compare experimental conditions. Results were presented as mean ± standard deviation (SD), with p < 0.05 considered statistically significant. Results Effect of Hydrogen Peroxide on Biofilm Formation Exposure to increasing concentrations of hydrogen peroxide (H₂O₂) resulted in a statistically significant, dose-dependent reduction in biofilm biomass ( p < 0.001). The mean OD₅₇₀ values decreased from 1.00 ± 0.00 in the control to 0.18 ± 0.04 at 1%, 0.15 ± 0.05at 2%, and 0.14 ± 0.02 at 3% H₂O₂, representing 81%, 84%, and 85% reductions, respectively [Figure 1 A]. Additionally, the genotypic analysis revealed a dose-dependent downregulation of icaA, icaD , and sarA under hydrogen peroxide (H₂O₂) stress compared to the untreated control. At 1% H₂O₂, icaA and icaD showed moderate repression (56.76% and 50.24%, respectively), while sarA expression was relatively less affected (63.03%). Increasing concentration to 2% further reduced icaA and icaD to 37.19% and 30.35%, with sarA maintaining slightly higher expression (41.75%). At 3% H₂O₂, all three genes were markedly repressed, with icaA (19.38%) and icaD (16.01%) showing the greatest decline, and sarA retaining relatively higher expression (22.8%) ( p < 0.01) [Figure 1 B]. Effect of pH on Biofilm Formation Biofilm formation was significantly affected by the pH of the growth medium. The highest biofilm biomass was observed at neutral pH 7 (OD₅₇₀ = 1.00 ± 0.00). In contrast, both acidic (pH 5) and alkaline (pH 9) conditions led to a significant reduction in biomass by 33.1% and 42.5%, respectively ( p < 0.01) [Figure 2 A]. In accordance with phenotypic results of the effect of pH on biofilm formation, the gene expression analysis under different pH conditions demonstrated that both acidic (pH 5) and alkaline (pH 9) environments significantly reduced the transcription of icaA, icaD , and sarA compared to the neutral pH 7 control (100%). At pH 5, icaA (51.76%) and icaD (46.65%) showed moderate repression, while sarA decreased to 40.61% ( p < 0.001). Exposure to alkaline stress (pH 9) resulted in a stronger downregulation, with icaA (39.23%), icaD (34.15%), and sarA (30.78%) showing marked reductions ( p < 0.001) [Figure 2 B]. Effect of Saline Concentration on Biofilm Formation Biofilm formation was significantly influenced by sodium chloride (NaCl) concentration. The maximum biomass was recorded at 1.25% NaCl, reaching 171.06% ± 0.03% relative to the control ( p < 0.01). A moderate increase was observed at 5% NaCl (133% ± 0.03), while biofilm biomass returned to near-control levels at 2.5% and 10% NaCl (~ 97%) as shown in [Figure 3 A]. The expressions of icaA, icaD , and sarA were differentially modulated by increasing NaCl concentrations compared to the 0% control (100%). At 1.25% NaCl, all three genes were markedly upregulated, with icaA (160.55%), icaD (168.18%), and sarA (149.8%) showing significant induction ( p < 0.01), suggesting that mild osmotic stress stimulates biofilm-associated gene expression. At 2.5% NaCl, expression levels stabilized near control values ( icaA 103.53%, icaD 100%, sarA 100%), indicating an adaptive balance. Exposure to 5% NaCl again enhanced transcription, with icaA (123.11%), icaD (123.11%), and sarA (114.87%) showing moderate but significant upregulation ( p < 0.05). At the highest concentration tested (10% NaCl), expression returned close to baseline, with icaA and icaD at 103.53% and sarA at 107.18% [Figure 3 B]. Collectively, these findings indicate that NaCl induces a biphasic transcriptional response, where mild to moderate osmotic stress promotes ica and sarA expression, potentially enhancing biofilm formation, while extreme salinity drives expression back to near basal levels, reflecting stress adaptation and regulatory control. Effect of Serum Concentration on Biofilm Formation Biofilm biomass exhibited a concentration-dependent response to human serum. The highest formation was recorded at 5% serum (100.9 ± 0.007%) and 10% serum (99.5 ± 0.005%), both statistically comparable to the control. A moderate reduction was observed at 20% serum (92.8 ± 0.006%) as shown in [Figure 4A], while a significant inhibition was noted at 50% serum, where biofilm biomass dropped to 4.86 ± 0.007% ( p < 0.001). The expressions of icaA, icaD , and sarA was influenced in a concentration-dependent manner by serum exposure compared with the 0% control (100%). At low serum levels (5–10%), a slight upregulation was observed, with icaA (110.96% at 5%, 108.45% at 10%), icaD (110.96% at both 5% and 10%), and sarA (107.18% at 5%, 107.7% at 10%) showing modest but consistent increases ( p < 0.05). However, at 20% serum, expression declined significantly, with icaA (81.23%), icaD (73.2%), and sarA (84.09%) all showing suppression ( p < 0.01). At the highest tested concentration (50%), a marked downregulation was observed, with icaA (12.94%), icaD (10.88%), and sarA (12.79%) exhibiting profound repression ( p < 0.001) [Figure 4B]. These results suggest that while low serum concentrations may transiently enhance biofilm-associated gene expression, higher serum levels strongly inhibit transcription, indicating a threshold-dependent regulatory effect of host factors on Staph. aureus biofilm genotypic response. Discussion Biofilm formation by Staph. aureus is a key factor contributing to its pathogenicity and antibiotic resistance, allowing bacterial survival in hostile environments, including medical devices and host tissues (Stewart and Franklin 2008). This study investigated the effects of oxidative stress (hydrogen peroxide), pH variation, saline concentration, and serum levels on Staph. aureus biofilm formation, using both phenotypic (crystal violet assay) and genotypic (qRT-PCR) approaches. The results demonstrate that these environmental factors significantly influence biofilm development and the expression of key biofilm-associated genes ( icaA , icaD , and sarA ), providing deeper insights into potential strategies for biofilm control. Staph. aureus Newman strain has its well-characterized genome, clinical relevance, and robust biofilm machinery including icaA , icaD , and sarA . Previous research has indicated that Newman exhibits unique responses to stress, particularly under iron-limited conditions, where biofilm formation is regulated by Emp and Eap proteins (Yin et al. 2017a). Moreover, genetic studies have shown that the Newman strain has a point mutation in the saeS gene affecting its biofilm phenotype, further supporting the need to study its environmental adaptability (Cue et al. 2015 ). Unlike prior studies that focused on single stressors or solely on phenotypic biofilm quantification, our research utilized a dual approach combining crystal violet staining with qRT-PCR analysis of core biofilm genes. This approach allowed for a deeper understanding of the biofilm regulatory mechanisms in response to multiple clinically relevant environmental challenges. Such an integrated analysis has not been systematically applied to the Newman strain, thus filling a critical gap in the literature and providing a comprehensive model for environmental modulation of biofilm formation. The results showed that hydrogen peroxide significantly reduced biofilm biomass in a dose-dependent manner, with 3% H₂O₂ leading to the highest inhibition ( p < 0.001). This was accompanied by a significant downregulation of icaA and icaD expression, which are essential for polysaccharide intercellular adhesion (PIA) production, a major component of Staph. aureus biofilms (Marques et al. 2021 ). Hydrogen peroxide acts as an oxidative stressor, damaging bacterial DNA, proteins, and membranes, thereby impairing biofilm integrity (Baldeck and Marquis 2008 ). These findings are consistent with previous studies that demonstrated H₂O₂-mediated inhibition of S. aureus biofilm formation and suppression of the ica operon (Liu et al. 2017 ). Given the potent anti-biofilm effects of hydrogen peroxide, its use in wound care, disinfectants, and medical device sterilization may be an effective strategy for controlling Staph. aureus infections (Lineback et al. 2018 ). Biofilm formation was highest at neutral pH and significantly reduced under acidic (pH 5) and alkaline (pH 9) conditions ( p < 0.01). Acidic environments disrupt bacterial cell wall stability and metabolic activity, leading to decreased biofilm production (O'Leary et al. 2015 ). Conversely, alkaline conditions may interfere with protein stability and enzyme function, reducing bacterial adhesion (Nostro et al. 2012 ). These findings align with previous reports indicating that Staph. aureus biofilms are highly sensitive to pH fluctuations (Tango et al. 2018 ). The ability to manipulate pH could serve as an adjunct strategy for biofilm control in medical and industrial settings (Nostro et al. 2012 ). pH-responsive polymeric nanomaterials can enhance antibacterial activity against biofilm cells, potentially addressing biofilm-related microbial infections (Jeong et al. 2023). The observed variations in Staph. aureus biofilm formation under different NaCl concentrations align with existing research highlighting the influence of environmental factors on biofilm development. Studies have demonstrated that NaCl can significantly impact biofilm formation in Staph. aureus . For instance, research indicates that biofilm production increases with NaCl concentrations up to a certain threshold, beyond which it plateaus or decreases. This multiphasic response suggests the involvement of specific regulatory pathways that mediate biofilm formation in response to varying NaCl levels (Lim et al. 2004 ).​ The production of polysaccharide intercellular adhesin (PIA), an essential component of the biofilm matrix, depends on the ica operon. The expression of these genes can be influenced by environmental factors such as NaCl. Studies have shown that NaCl can activate the ica operon, leading to increased PIA production and enhanced biofilm formation (Agarwal and Jain 2013 ).​ The concurrent upregulation of sarA , a global regulator of biofilm-associated genes, further supports its role in biofilm adaptation under saline stress. However, at 10% NaCl, gene expression and biofilm formation decline, indicating an inhibitory threshold beyond which salt disrupts biofilm regulatory networks. These findings align with previous research highlighting the influence of NaCl on Staph. aureus biofilm development, emphasizing its role in modulating virulence and persistence in saline environments (Islam, Ross, and Marten 2015 ). In our study, biofilm biomass was highest at 5% serum, comparable to the control, but significantly inhibited at 50% serum, suggesting the presence of inhibitory components at higher concentrations. The serum component affects the transcription of biofilm-related genes, such as the intercellular adhesin gene icaA in Staph. aureus , suggesting a regulatory role in biofilm formation. Additionally, serum proteases like plasma kallikrein and plasmin have been found to cleave bacterial adhesins, preventing biofilm formation (Abraham and Jefferson 2010 ; Arenas et al. 2021 ). It is found that Serum provides a dual effect on Staph. aureus biofilm formation depending on its concentration. Low serum concentrations (5–10%) is associated with upregulation of adhesion-related genes and biofilm inducers like sarA and icaA , which promotes biofilm formation by increasing bacterial aggregation and enhancing adherence to host proteins such as fibronectin and fibrinogen (Yin et al. 2017b). However, at higher concentrations (around 50%), serum introduces inhibitory factors, which are potentially low molecular weight components or immune-related molecules that suppress biofilm formation although it supports planktonic growth (Abraham and Jefferson 2010 ).This dual actions reflects clinical scenarios as in case of bloodstream infections, initial adhesion with immune evasion may occur in areas with low serum content such as tissues., while higher serum levels in circulation could inhibit biofilm maturation, possibly selecting for strains or conditions that overcome these inhibitory effects (Abraham and Jefferson 2010 ; Yin et al. 2017b). These findings focus on the complex interplay between host environment and Staph. aureus biofilm dynamics, with important effects for infection persistence and treatment strategies. Our study emphasizes the importance of biofilm-associated genes in the alternation of the biomass-related transcription profiles of Staph. aureus . In addition to planktonic growth of the bacteria, several genes involved in cellular adhesion, production of extracellular matrix and adaptation to metabolic states exhibit altered expression, mostly upregulation, causing significant transcriptional reprogramming in biofilm-embedded cells. For instance, a global gene regulator such as sarA in addition to some additional gene regulators such as agr system are responsible for expression of various downstream effects, including extracellular protease production and intracellular adhesion. Thus, it affects both biomass accumulation and biofilm production (Beenken et al. 2004 ; Humeniuk et al. 2023 ; Syed et al. 2024 ). Additionally, the expression of ica operon, which is responsible for synthesis of polysaccharide intracellular adhesin (PIA) and expression of genes capable of autolysis such as atlA, are linked closely to stability and maturation of biofilm resulting in firms biomass structure (Beenken et al. 2004 ; Liang et al. 2023 ; Boles et al. 2010 ). Environmental factors such as oxidative stress, pH fluctuations, osmotic changes, and serum-derived factors are induced by Staph. aureus and transduced into genetic regulation through complex networks involving two-component systems, global regulators, and small RNAs (Liu, Zhang, and Ji 2020 ). These environmental cues can affect the activity of sarA , a global regulator that influences biofilm formation by modulating the expression of ica operon, which encodes vital enzymes for polysaccharide intercellular adhesin (PIA) production (Peng et al. 2022 ). Adjustment of PIA synthesis and biofilm architecture in response to different stressors can be done by either upregulation or downregulation of icaA and icaD , which are mainly influenced by sarA as a global regulator for biofilm formation. The complex dynamicity of biofilm formation of Staph. aureus can enhance its survival and persistence, especially in hostile environments such as those encountered during infection or on medical devices (Wu et al. 2021 ; Peng et al. 2022 ; Liu, Zhang, and Ji 2020 ). The findings of the research have significant therapeutic and industrial applications as they shed light on Staph. aureus biofilm development is modulated. In clinical settings, biofilm-associated infections pose challenges in wound care, bloodstream infections, and medical device-related complications due to their resistance to antibiotics and host immune responses. The observed inhibition of biofilm formation at higher hydrogen peroxide concentrations (≥ 3%) suggests potential applications in antimicrobial therapies and hospital disinfection protocols and this align with (Lineback et al. 2018 ). Controlling the formation of biofilms is essential from an industrial aspect in the manufacturing of biomedical devices, water treatment, and food processing. Understanding the effects of oxidative stress, salinity, and pH on biofilm formation could assist in developing antibiofilm coatings, improve industrial cleaning techniques, and design improved sanitation protocols for preventing microbiological contamination (Shineh et al. 2023). Limitation and future directions: This study is limited using a single laboratory strain under in-vitro conditions, which may not reflect the genetic diversity of clinical or environmental isolates or the complexity of in-vivo biofilms. Future work should include multiple clinical strains, explore combined environmental stresses (e.g., immune factors, nutrient limitation), and test anti-biofilm strategies in in-vivo models to bridge laboratory findings with clinical applications. Conclusion This study demonstrates that Staphylococcus aureus biofilm formation is highly sensitive to environmental and host-related stresses. Hydrogen peroxide and non-neutral pH conditions significantly reduced biomass and downregulated icaA , icaD , and sarA , with polysaccharide-associated genes most affected. Sodium chloride induced a biphasic response, where mild osmotic stress enhanced biofilm formation, but extreme salinity normalized expression to basal levels. Serum exerted a threshold effect, with low concentrations slightly stimulating gene expression and high concentrations strongly suppressing biofilm development. Together, these results indicate to different but related stress-mediated processes that control the regulation of biofilms and offer possible directions for anti-biofilm therapies. Declarations ORCID [0000-0002-5193-9662] 1 , [0000-0003-2745-3811] 2 , [0000-0002-1147-336X] 3 Competing Interests The authors declare no competing interests Ethics Approval Not applicable. The study did not involve human participants or animal experiments; only a standard laboratory strain of Staphylococcus aureus Newman (ATCC 25904) was used Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors Author Contribution Conceptualization: Ahmed R. Ragab; Methodology: Ahmed R. Ragab, Ahmed R. El-Sheakh; Formal analysis and investigation: Ahmed R. Ragab; Writing – original draft preparation: Ahmed R. Ragab; Writing – review and editing: Ahmed R. El-Sheakh, Shokri M. Shafik; Funding acquisition: Not applicable; Resources: Shokri M. Shafik; Supervision: Shokri M. Shafik. All authors have read and approved the final manuscript. Data Availability All data generated or analysed during this study are included in this published article. Additional raw datasets are available from the corresponding author upon reasonable request References Abraham N, Jefferson K (2010) A low molecular weight component of serum inhibits biofilm formation in Staphylococcus aureus. Microb Pathog 49 6:388–391 Agarwal A, Jain A (2013) Glucose & sodium chloride induced biofilm production & ica operon in clinical isolates of staphylococci. Indian J Med Res 138:262–266 Arenas J, Zalán Szabó J, Van Der Wal C, Maas T, Riaz T, Tønjum, Tommassen J (2021) Serum proteases prevent bacterial biofilm formation: role of kallikrein and plasmin. 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Infect Immun, 91: e0053822 Marques V, Santos H, Santos T, Melo D, Coelho S, Coelho I, Souza M (2021) 'Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis'. Pesquisa Vet Brasileira, 41 Mirzaee M, Peerayeh SN, Abdol-Majid G (2014) Detection of icaABCD genes and biofilm formation in clinical isolates of methicillin resistant Staphylococcus aureus. Iran J Pathol 9:257–262 Namvar AE, Asghari B, Ezzatifar F, Azizi G, Lari AR (2013) Detection of the intercellular adhesion gene cluster (ica) in clinical Staphylococcus aureus isolates. GMS Hyg Infect Control 8:Doc03 Nostro A, Cellini L, Di Giulio M, D’Arrigo M, Marino A, Blanco A, Favaloro A, Cutroneo G, Bisignano G (2012) 'Effect of alkaline pH on staphylococcal biofilm formation', Apmis , 120 O'Leary D, McCabe E, McCusker M, Martins M, Fanning S, Duffy G (2015) Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104. Int J Food Microbiol 206:7–16 Peng Q, Tang X, Dong W, Ning Sun, and, Yuan W (2022) 'A Review of Biofilm Formation of Staphylococcus aureus and Its Regulation Mechanism'. Antibiotics, 12 Pietrocola G, Campoccia D, Motta C, Montanaro L, Arciola CR, Speziale P (2022) 'Colonization and Infection of Indwelling Medical Devices by Staphylococcus aureus with an Emphasis on Orthopedic Implants'. 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Monash University Tango C, Nkufi S, Akkermans M, Hussain, Imran Khan J, Van Impe Y-G, Jin, Oh D (2018) 'Modeling the effect of pH, water activity, and ethanol concentration on biofilm formation of Staphylococcus aureus', Food microbiology , 76: 287 – 95 Tomlinson BR, Malof ME, Shaw LN (2021) 'A global transcriptomic analysis of Staphylococcus aureus biofilm formation across diverse clonal lineages'. Microb Genom, 7 Tonon C, Panariello B, Spolidorio D, Gossweiler A, Duarte S (2020) 'Anti-biofilm effect of ozonized physiological saline solution on peri-implant-related biofilm'. J Periodontol Wang Y, Miao F, Bai J, Wang Z, Qin W (2024) An observational study of the pH value during the healing process of diabetic foot ulcer. J Tissue Viability 33:208–214 Wu S, Zhang J, Peng Q, Liu Y, Lei L, Zhang H (2021) 'The Role of Staphylococcus aureus YycFG in Gene Regulation, Biofilm Organization and Drug Resistance', Antibiotics , 10 Wuren T, Takahito Toyotome M, Yamaguchi A, Takahashi-Nakaguchi Y, Muraosa M, Yahiro D-N, Wang A, Watanabe H, Taguchi, Kamei K (2014) Effect of serum components on biofilm formation by Aspergillus fumigatus and other Aspergillus species. Jpn J Infect Dis 67 3:172–179 Yin S, Jiang B, Huang G, Gong Y, You B, Yang Z, Chen Y, Chen J, Yuan Z, Li M, Hu F, Zhao Y, Peng Y 2017a. 'Burn Serum Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress'. Front Microbiol, 8: 1191 Yin S, Jiang B, Huang G, Gong Y, You B, Yang Z, Chen Y, Chen J, Yuan Z, Li M, Hu F, Zhao Y, and Yizhi Peng. 2017b. 'Burn Serum Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress'. Front Microbiol, 8 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Applied Microbiology and Biotechnology → 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-7714339","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":535359615,"identity":"3baa5f21-9bdc-46e7-aa70-ec59782fa8f4","order_by":0,"name":"Ahmed R. Ragab","email":"data:image/png;base64,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","orcid":"","institution":"Mansoura National University","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"R.","lastName":"Ragab","suffix":""},{"id":535359616,"identity":"22d1a30e-b49c-4cac-84d9-9c6073183b39","order_by":1,"name":"Ahmed R. El‐Sheakh","email":"","orcid":"","institution":"Mansoura National University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"R.","lastName":"El‐Sheakh","suffix":""},{"id":535359617,"identity":"cd293d39-5361-4753-a6da-b666e1bcd67a","order_by":2,"name":"Shokri M. 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(A) Biofilm biomass measured by crystal violet assay at OD₅₇₀. (B) Relative gene expression of icaA, icaD and sarA determined by qRT-PCR normalized to 16S rRNA. Error bars indicate SD (n=3 biological replicates).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714339/v1/3edbf8c306a8ae07b3c16bd1.jpeg"},{"id":94729420,"identity":"1875bbd1-d07c-4201-bd80-e2756addb98c","added_by":"auto","created_at":"2025-10-30 07:04:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312218,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH on S. aureus Newman biofilm. (A) Biomass quantification using crystal violet assay. (B) Relative gene expression of of icaA, icaD and sarA normalized to 16S rRNA. Data shown as mean ± SD, n=3. Statistical analysis\u003cstrong\u003e \u003c/strong\u003eby one-way ANOVA with Tukey post hoc test; *p\u0026lt;0.05, **p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714339/v1/069ccdc0e13a95a7ed23b2d1.jpeg"},{"id":94698813,"identity":"89ec2cd1-b11b-474a-8084-0e075740d275","added_by":"auto","created_at":"2025-10-29 19:03:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":371264,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NaCl on S. aureus Newman biofilm. (A) Phenotypic biomass measurement. (B) Relative gene expression of icaA, icaD and sarA. Data are mean ± SD, n=3 biological replicates. Statistical analysis\u003cstrong\u003e \u003c/strong\u003eby one-way ANOVA with Tukey post hoc test; *p\u0026lt;0.05, **p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714339/v1/99d8d385baac2ff190269293.jpeg"},{"id":94729526,"identity":"481ad302-7ebf-4f2d-aa19-a9ba1b47a94e","added_by":"auto","created_at":"2025-10-30 07:05:06","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":395392,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of human serum on S. aureus Newman biofilm. (A) Biofilm biomass by crystal violet staining. (B) Relative gene expression of icaA, icaD and sarA relative to 16S rRNA. Mean ± SD, n=3. Statistical analysis\u003cstrong\u003e \u003c/strong\u003eby one-way ANOVA with Tukey post hoc test; *p\u0026lt;0.05, **p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7714339/v1/88d05bb910686194fff23563.jpeg"},{"id":105754923,"identity":"f36ddb66-2b0b-4980-860b-108c20e65124","added_by":"auto","created_at":"2026-03-30 16:23:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2132516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7714339/v1/5d5bb35d-d722-4783-8d70-f22e89138dff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Environmental and Chemical Modulation of Staphylococcus aureus Newman Biofilm Formation","fulltext":[{"header":"Key Points","content":"\u003cul\u003e\n \u003cli\u003eOxidative stress (H₂O₂) dose-dependently inhibits \u003cem\u003eS. aureus\u003c/em\u003e Newman biofilms.\u003c/li\u003e\n \u003cli\u003eMild NaCl levels enhance biofilm formation via upregulation of \u003cem\u003eica\u0026nbsp;\u003c/em\u003eand \u003cem\u003esarA\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eHigh serum concentrations (\u0026ge;20%) suppress biofilm biomass and gene expression.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eBiofilm formation represents a fundamental survival strategy employed by \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, enabling bacterial persistence on medical devices, chronic wounds, and host tissues (Peng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These complex microbial communities provide protection against antimicrobial agents and host immune responses, contributing significantly to the burden of healthcare-associated infections and treatment failures (Ciofu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The clinical significance of \u003cem\u003eStaph. aureus\u003c/em\u003e biofilms is underscored by their association with device-related infections, chronic osteomyelitis, and persistent bacteremia, conditions that often require prolonged antibiotic therapy and surgical intervention (Pietrocola et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Di Domenico, Oliva, and Guembe \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe molecular architecture of \u003cem\u003eStaph. aureus\u003c/em\u003e biofilms is governed by a sophisticated regulatory network involving multiple genetic determinants, containing \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaD\u003c/em\u003e, that mediate polysaccharide intercellular adhesion (PIA) result and \u003cem\u003esarA\u003c/em\u003e, a key global regulator of biofilm production (Gowrishankar et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Environmental factors such as oxidative stress, pH alterations, and osmotic environments are noted to influence biofilm development (Goller and Romeo \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Hydrogen peroxide, a primary reactive oxygen species, is produced by neutrophils and macrophages as part of the innate immune response and has been shown to influence biofilm formation in various bacterial species (da Cruz Nizer et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). pH changes can alter bacterial adherence characteristics and exopolysaccharide (EPS) production, thereby modulating biofilm integrity (Chaggar et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Acidic conditions may arise in infected wounds due to bacterial metabolism and tissue hypoxia, while alkaline environments can result from certain therapeutic interventions (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, osmotic stress induced by saline concentrations influences biofilm stability, as \u003cem\u003eStaph. aureus\u003c/em\u003e frequently encounters variable salinity levels in wound environments and healing implants (Tonon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, serum components, containing proteins and immune factors, can either improve or prevent biofilm composition depending on their concentration (Wuren et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite these known environmental cues, the transcriptional responses of \u003cem\u003eStaph. aureus\u003c/em\u003e biofilms to such stresses are poorly understood. \u003cem\u003eStaph. aureus\u003c/em\u003e Newman was originally isolated from a case of human osteomyelitis and has since become a widely used lab model for biofilm studies due to its well-characterized genome and virulence traits, including prophages and pathogenicity islands that underpin its behavior in infection models (Baba et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Recent transcriptomic comparisons across multiple \u003cem\u003eStaph. aureus\u003c/em\u003e strains have revealed that global gene expression during biofilm formation varies substantially by strain, marking Newman as particularly notable for its unique regulatory dynamics (Tomlinson, Malof, and Shaw \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, experimental evolution of Newman under selective conditions identified new mutations (e.g., in \u003cem\u003emanA\u003c/em\u003e, \u003cem\u003enarH\u003c/em\u003e, \u003cem\u003efruB\u003c/em\u003e) capable of enhancing biofilm formation, underlining the strain\u0026rsquo;s adaptive complexity and relevance for modeling stress- or mutation-driven biofilm behavior (Long et al. 2023).\u003c/p\u003e\u003cp\u003eDespite the recognized importance of environmental factors in biofilm development, comprehensive studies examining the coordinated effects of multiple stressors on both phenotypic biofilm formation and underlying gene expression in \u003cem\u003eStaph. aureus\u003c/em\u003e Newman are limited. Furthermore, the dose-dependent relationships between environmental stressors and biofilm responses have not been systematically characterized, limiting our understanding of clinically relevant exposure levels. This study aimed to characterize the dose-dependent effects of hydrogen peroxide, pH variation, saline concentration, and human serum on \u003cem\u003eStaph. aureus\u003c/em\u003e Newman biofilm formation, elucidate the corresponding changes in expression of key biofilm-associated genes (\u003cem\u003eicaA\u003c/em\u003e, \u003cem\u003eicaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e), identify optimal conditions for biofilm inhibition or enhancement, and provide molecular insights that may inform the development of environment-based biofilm control strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial Strain and Culture Conditions\u003c/h2\u003e\u003cp\u003eThe standard strain of \u003cem\u003eStaph. aureus\u003c/em\u003e Newman (ATCC 25904) was obtained from Microbiology Department, Faculty of Pharmacy, National Mansoura University. The strain was routinely cultured on Tryptic Soy Agar (Oxoid, UK) and incubated at 37\u0026deg;C for 18\u0026ndash;24 hours under aerobic conditions. The bacterial culture was diluted to 1:100 in TSB to obtain a standardized inoculum.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBiofilm Formation Assay\u003c/h3\u003e\n\u003cp\u003eBiofilm formation was quantified using the crystal violet staining method as previously described with modifications (Stepanovic et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Briefly, \u003cem\u003eS. aureus\u003c/em\u003e cultures were inoculated into 96-well microtiter plates containing 200 \u0026micro;L of TSB supplemented with the respective test conditions. Plates were incubated at 37\u0026deg;C for 24 to 72 hours. After incubation, planktonic cells were removed by washing with phosphate-buffered saline (PBS) (pH 7.4), and adherent biofilms were fixed with 99% methanol for 30 minutes. Biofilms were stained with 0.1% crystal violet (Sigma-Aldrich, USA) for 15 minutes, followed by three washes with PBS. The bound crystal violet was solubilized using 33% glacial acetic acid (Adwic, Egypt), and absorbance was measured at 570 nm using a microplate reader (BioTek, USA).\u003c/p\u003e\n\u003ch3\u003eExperimental Conditions\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Hydrogen Peroxide\u003c/h2\u003e\u003cp\u003eTo assess the impact of oxidative stress on biofilm formation, hydrogen peroxide (H₂O₂; Sigma-Aldrich, USA) was added to TSB at final concentrations of 0.5%, 1%, 2%, and 3%. The control group contained TSB without H₂O₂. These concentrations were selected based on previous studies investigating the oxidative stress response in \u003cem\u003eS. aureus\u003c/em\u003e biofilms, with adaptations specific to the current experimental model (Kaplan \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Biofilm formation was measured after 72 hours of incubation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffect of pH Variation\u003c/h3\u003e\n\u003cp\u003eTo evaluate the influence of pH on biofilm development, the pH of TSB was adjusted to 5, 7, and 9 using sterile 1M HCl or NaOH (Sigma-Aldrich, USA). While previous research has shown that acidic and alkaline conditions affect biofilm integrity and gene expression (Tan \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), the selected pH values were optimized in the present study to capture a broad physiological range. Cultures were incubated for 24 hours prior to biofilm quantification.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Saline Concentration\u003c/h2\u003e\u003cp\u003eTo examine the effect of osmotic stress, NaCl was added to TSB at final concentrations of 1.25%, 2.5%, 5%, and 10%, with standard TSB (0.9% NaCl) used as a control. These concentrations were guided by prior findings indicating that salinity alters biofilm production (Beckingsale \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), though specific salt levels were optimized for the current strain and conditions. Biofilm formation was assessed after 24 hours.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffect of Serum Concentration\u003c/h3\u003e\n\u003cp\u003eHuman serum (Sigma-Aldrich, USA) was added to TSB at final concentrations of 5%, 10%, 20%, and 50% to investigate the influence of host-derived factors on biofilm formation. Serum-free TSB served as the control. Previous studies have demonstrated that serum components can modulate biofilm formation (Skovdal et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); however, the concentration range used here was tailored to assess dose-dependent effects in vitro. Biofilms were quantified after 24 hours.\u003c/p\u003e\n\u003ch3\u003eGenotypic Analysis by Quantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNA Extraction and cDNA Synthesis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from biofilm-forming \u003cem\u003eStaph. aureus\u003c/em\u003e cells using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer\u0026rsquo;s instructions. RNA purity and concentration were determined using a Nanodrop Spectrophotometer (Thermo Fisher, USA). Complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eqRT-PCR Amplification\u003c/h2\u003e\u003cp\u003e\u003cem\u003eqRT-PCR was performed using a StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems, USA) with SYBR Green Master Mix in a 20 \u0026micro;L reaction mixture containing 10 \u0026micro;L SYBR Green Master Mix\u003c/em\u003e, 1 \u0026micro;L of each primer (10 \u0026micro;M), 2 \u0026micro;L cDNA, and 6 \u0026micro;L nuclease-free water. The thermal cycling conditions included an initial denaturation at 95\u0026deg;C for 10 minutes, followed by 40 cycles of amplification (95\u0026deg;C for 15 seconds, 60\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds). A melting curve analysis was conducted to verify amplification specificity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTarget Genes and Primers\u003c/h2\u003e\u003cp\u003eThe expression of five key biofilm-associated genes was analyzed as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e:\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers used for the 3 genes responsible for biofilm formation in Staphylococcus aureus Newman strain in addition for primers of house keeping genes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward Primer (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse Primer (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eicaA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACA CTT GCT GGC GCA GTC AA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTCT GGA ACC AAC ATC CAA CA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Mirzaee, Najar Peerayeh, and Ghasemian 2014)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eicaD\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGAA CCG CTT GCC ATG TGT TG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCT TGA CCA TGT TGC GTA ACC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Namvar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003esarA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCT TGT TAA TGC ACA ACA ACG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGT TTG CTT CAG TGA TTC GTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Sambanthamoorthy, Smeltzer, and Elasri 2006)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u0026thinsp;s rRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTGT CGT GAG ATG TTG GG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCGA TTC CAG CTT CAT GT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Lee et al. 2013)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRelative gene expression was calculated using the 2^(-ΔΔCt) method, normalizing target gene expression to 16 s rRNA (Livak and Schmittgen 2001).\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5. Statistical Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll experiments were performed in quadruplicate. Data was analyzed using GraphPad Prism 5 (GraphPad Software, USA). One-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used to compare experimental conditions. Results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Hydrogen Peroxide on Biofilm Formation\u003c/h2\u003e\u003cp\u003eExposure to increasing concentrations of hydrogen peroxide (H₂O₂) resulted in a statistically significant, dose-dependent reduction in biofilm biomass (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The mean OD₅₇₀ values decreased from 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 in the control to 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 at 1%, 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05at 2%, and 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 3% H₂O₂, representing 81%, 84%, and 85% reductions, respectively [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA]. Additionally, the genotypic analysis revealed a dose-dependent downregulation of \u003cem\u003eicaA, icaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e under hydrogen peroxide (H₂O₂) stress compared to the untreated control. At 1% H₂O₂, \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaD\u003c/em\u003e showed moderate repression (56.76% and 50.24%, respectively), while \u003cem\u003esarA\u003c/em\u003e expression was relatively less affected (63.03%). Increasing concentration to 2% further reduced \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaD\u003c/em\u003e to 37.19% and 30.35%, with \u003cem\u003esarA\u003c/em\u003e maintaining slightly higher expression (41.75%). At 3% H₂O₂, all three genes were markedly repressed, with \u003cem\u003eicaA\u003c/em\u003e (19.38%) and \u003cem\u003eicaD\u003c/em\u003e (16.01%) showing the greatest decline, and \u003cem\u003esarA\u003c/em\u003e retaining relatively higher expression (22.8%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEffect of pH on Biofilm Formation\u003c/h2\u003e\u003cp\u003eBiofilm formation was significantly affected by the pH of the growth medium. The highest biofilm biomass was observed at neutral pH 7 (OD₅₇₀ = 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00). In contrast, both acidic (pH 5) and alkaline (pH 9) conditions led to a significant reduction in biomass by 33.1% and 42.5%, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA]. In accordance with phenotypic results of the effect of pH on biofilm formation, the gene expression analysis under different pH conditions demonstrated that both acidic (pH 5) and alkaline (pH 9) environments significantly reduced the transcription of \u003cem\u003eicaA, icaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e compared to the neutral pH 7 control (100%). At pH 5, \u003cem\u003eicaA\u003c/em\u003e (51.76%) and \u003cem\u003eicaD\u003c/em\u003e (46.65%) showed moderate repression, while \u003cem\u003esarA\u003c/em\u003e decreased to 40.61% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Exposure to alkaline stress (pH 9) resulted in a stronger downregulation, with \u003cem\u003eicaA\u003c/em\u003e (39.23%), \u003cem\u003eicaD\u003c/em\u003e (34.15%), and \u003cem\u003esarA\u003c/em\u003e (30.78%) showing marked reductions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Saline Concentration on Biofilm Formation\u003c/h2\u003e\u003cp\u003eBiofilm formation was significantly influenced by sodium chloride (NaCl) concentration. The maximum biomass was recorded at 1.25% NaCl, reaching 171.06% \u0026plusmn; 0.03% relative to the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A moderate increase was observed at 5% NaCl (133% \u0026plusmn; 0.03), while biofilm biomass returned to near-control levels at 2.5% and 10% NaCl (~\u0026thinsp;97%) as shown in [Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA]. The expressions of \u003cem\u003eicaA, icaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e were differentially modulated by increasing NaCl concentrations compared to the 0% control (100%). At 1.25% NaCl, all three genes were markedly upregulated, with \u003cem\u003eicaA\u003c/em\u003e (160.55%), \u003cem\u003eicaD\u003c/em\u003e (168.18%), and \u003cem\u003esarA\u003c/em\u003e (149.8%) showing significant induction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that mild osmotic stress stimulates biofilm-associated gene expression. At 2.5% NaCl, expression levels stabilized near control values (\u003cem\u003eicaA\u003c/em\u003e 103.53%, \u003cem\u003eicaD\u003c/em\u003e 100%, \u003cem\u003esarA\u003c/em\u003e 100%), indicating an adaptive balance. Exposure to 5% NaCl again enhanced transcription, with \u003cem\u003eicaA\u003c/em\u003e (123.11%), \u003cem\u003eicaD\u003c/em\u003e (123.11%), and \u003cem\u003esarA\u003c/em\u003e (114.87%) showing moderate but significant upregulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At the highest concentration tested (10% NaCl), expression returned close to baseline, with \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaD\u003c/em\u003e at 103.53% and \u003cem\u003esarA\u003c/em\u003e at 107.18% [Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB]. Collectively, these findings indicate that NaCl induces a biphasic transcriptional response, where mild to moderate osmotic stress promotes \u003cem\u003eica\u003c/em\u003e and \u003cem\u003esarA\u003c/em\u003e expression, potentially enhancing biofilm formation, while extreme salinity drives expression back to near basal levels, reflecting stress adaptation and regulatory control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Serum Concentration on Biofilm Formation\u003c/h2\u003e\u003cp\u003eBiofilm biomass exhibited a concentration-dependent response to human serum. The highest formation was recorded at 5% serum (100.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007%) and 10% serum (99.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005%), both statistically comparable to the control. A moderate reduction was observed at 20% serum (92.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006%) as shown in [Figure 4A], while a significant inhibition was noted at 50% serum, where biofilm biomass dropped to 4.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The expressions of \u003cem\u003eicaA, icaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e was influenced in a concentration-dependent manner by serum exposure compared with the 0% control (100%). At low serum levels (5\u0026ndash;10%), a slight upregulation was observed, with \u003cem\u003eicaA\u003c/em\u003e (110.96% at 5%, 108.45% at 10%), \u003cem\u003eicaD\u003c/em\u003e (110.96% at both 5% and 10%), and \u003cem\u003esarA\u003c/em\u003e (107.18% at 5%, 107.7% at 10%) showing modest but consistent increases (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, at 20% serum, expression declined significantly, with \u003cem\u003eicaA\u003c/em\u003e (81.23%), \u003cem\u003eicaD\u003c/em\u003e (73.2%), and \u003cem\u003esarA\u003c/em\u003e (84.09%) all showing suppression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). At the highest tested concentration (50%), a marked downregulation was observed, with \u003cem\u003eicaA\u003c/em\u003e (12.94%), \u003cem\u003eicaD\u003c/em\u003e (10.88%), and \u003cem\u003esarA\u003c/em\u003e (12.79%) exhibiting profound repression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) [Figure 4B]. These results suggest that while low serum concentrations may transiently enhance biofilm-associated gene expression, higher serum levels strongly inhibit transcription, indicating a threshold-dependent regulatory effect of host factors on \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm genotypic response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBiofilm formation by \u003cem\u003eStaph. aureus\u003c/em\u003e is a key factor contributing to its pathogenicity and antibiotic resistance, allowing bacterial survival in hostile environments, including medical devices and host tissues (Stewart and Franklin 2008). This study investigated the effects of oxidative stress (hydrogen peroxide), pH variation, saline concentration, and serum levels on \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm formation, using both phenotypic (crystal violet assay) and genotypic (qRT-PCR) approaches. The results demonstrate that these environmental factors significantly influence biofilm development and the expression of key biofilm-associated genes (\u003cem\u003eicaA\u003c/em\u003e, \u003cem\u003eicaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e), providing deeper insights into potential strategies for biofilm control.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e Newman strain has its well-characterized genome, clinical relevance, and robust biofilm machinery including \u003cem\u003eicaA\u003c/em\u003e, \u003cem\u003eicaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e. Previous research has indicated that Newman exhibits unique responses to stress, particularly under iron-limited conditions, where biofilm formation is regulated by Emp and Eap proteins (Yin et al. 2017a). Moreover, genetic studies have shown that the Newman strain has a point mutation in the \u003cem\u003esaeS\u003c/em\u003e gene affecting its biofilm phenotype, further supporting the need to study its environmental adaptability (Cue et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnlike prior studies that focused on single stressors or solely on phenotypic biofilm quantification, our research utilized a dual approach combining crystal violet staining with qRT-PCR analysis of core biofilm genes. This approach allowed for a deeper understanding of the biofilm regulatory mechanisms in response to multiple clinically relevant environmental challenges. Such an integrated analysis has not been systematically applied to the Newman strain, thus filling a critical gap in the literature and providing a comprehensive model for environmental modulation of biofilm formation.\u003c/p\u003e\u003cp\u003eThe results showed that hydrogen peroxide significantly reduced biofilm biomass in a dose-dependent manner, with 3% H₂O₂ leading to the highest inhibition (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This was accompanied by a significant downregulation of \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaD\u003c/em\u003e expression, which are essential for polysaccharide intercellular adhesion (PIA) production, a major component of \u003cem\u003eStaph. aureus\u003c/em\u003e biofilms (Marques et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hydrogen peroxide acts as an oxidative stressor, damaging bacterial DNA, proteins, and membranes, thereby impairing biofilm integrity (Baldeck and Marquis \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). These findings are consistent with previous studies that demonstrated H₂O₂-mediated inhibition of \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation and suppression of the \u003cem\u003eica\u003c/em\u003e operon (Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given the potent anti-biofilm effects of hydrogen peroxide, its use in wound care, disinfectants, and medical device sterilization may be an effective strategy for controlling \u003cem\u003eStaph. aureus\u003c/em\u003e infections (Lineback et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBiofilm formation was highest at neutral pH and significantly reduced under acidic (pH 5) and alkaline (pH 9) conditions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Acidic environments disrupt bacterial cell wall stability and metabolic activity, leading to decreased biofilm production (O'Leary et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Conversely, alkaline conditions may interfere with protein stability and enzyme function, reducing bacterial adhesion (Nostro et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These findings align with previous reports indicating that \u003cem\u003eStaph. aureus\u003c/em\u003e biofilms are highly sensitive to pH fluctuations (Tango et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The ability to manipulate pH could serve as an adjunct strategy for biofilm control in medical and industrial settings (Nostro et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). pH-responsive polymeric nanomaterials can enhance antibacterial activity against biofilm cells, potentially addressing biofilm-related microbial infections (Jeong et al. 2023).\u003c/p\u003e\u003cp\u003eThe observed variations in \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm formation under different NaCl concentrations align with existing research highlighting the influence of environmental factors on biofilm development. Studies have demonstrated that NaCl can significantly impact biofilm formation in \u003cem\u003eStaph. aureus\u003c/em\u003e. For instance, research indicates that biofilm production increases with NaCl concentrations up to a certain threshold, beyond which it plateaus or decreases. This multiphasic response suggests the involvement of specific regulatory pathways that mediate biofilm formation in response to varying NaCl levels (Lim et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).​\u003c/p\u003e\u003cp\u003eThe production of polysaccharide intercellular adhesin (PIA), an essential component of the biofilm matrix, depends on the \u003cem\u003eica\u003c/em\u003e operon. The expression of these genes can be influenced by environmental factors such as NaCl. Studies have shown that NaCl can activate the ica operon, leading to increased PIA production and enhanced biofilm formation (Agarwal and Jain \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).​\u003c/p\u003e\u003cp\u003eThe concurrent upregulation of \u003cem\u003esarA\u003c/em\u003e, a global regulator of biofilm-associated genes, further supports its role in biofilm adaptation under saline stress. However, at 10% NaCl, gene expression and biofilm formation decline, indicating an inhibitory threshold beyond which salt disrupts biofilm regulatory networks. These findings align with previous research highlighting the influence of NaCl on \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm development, emphasizing its role in modulating virulence and persistence in saline environments (Islam, Ross, and Marten \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn our study, biofilm biomass was highest at 5% serum, comparable to the control, but significantly inhibited at 50% serum, suggesting the presence of inhibitory components at higher concentrations. The serum component affects the transcription of biofilm-related genes, such as the intercellular adhesin gene \u003cem\u003eicaA\u003c/em\u003e in \u003cem\u003eStaph. aureus\u003c/em\u003e, suggesting a regulatory role in biofilm formation. Additionally, serum proteases like plasma kallikrein and plasmin have been found to cleave bacterial adhesins, preventing biofilm formation (Abraham and Jefferson \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Arenas et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is found that Serum provides a dual effect on \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm formation depending on its concentration. Low serum concentrations (5\u0026ndash;10%) is associated with upregulation of adhesion-related genes and biofilm inducers like \u003cem\u003esarA\u003c/em\u003e and \u003cem\u003eicaA\u003c/em\u003e, which promotes biofilm formation by increasing bacterial aggregation and enhancing adherence to host proteins such as fibronectin and fibrinogen (Yin et al. 2017b). However, at higher concentrations (around 50%), serum introduces inhibitory factors, which are potentially low molecular weight components or immune-related molecules that suppress biofilm formation although it supports planktonic growth (Abraham and Jefferson \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).This dual actions reflects clinical scenarios as in case of bloodstream infections, initial adhesion with immune evasion may occur in areas with low serum content such as tissues., while higher serum levels in circulation could inhibit biofilm maturation, possibly selecting for strains or conditions that overcome these inhibitory effects (Abraham and Jefferson \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yin et al. 2017b). These findings focus on the complex interplay between host environment and \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm dynamics, with important effects for infection persistence and treatment strategies.\u003c/p\u003e\u003cp\u003eOur study emphasizes the importance of biofilm-associated genes in the alternation of the biomass-related transcription profiles of \u003cem\u003eStaph. aureus\u003c/em\u003e. In addition to planktonic growth of the bacteria, several genes involved in cellular adhesion, production of extracellular matrix and adaptation to metabolic states exhibit altered expression, mostly upregulation, causing significant transcriptional reprogramming in biofilm-embedded cells. For instance, a global gene regulator such as \u003cem\u003esarA\u003c/em\u003e in addition to some additional gene regulators such as \u003cem\u003eagr\u003c/em\u003e system are responsible for expression of various downstream effects, including extracellular protease production and intracellular adhesion. Thus, it affects both biomass accumulation and biofilm production (Beenken et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Humeniuk et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Syed et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, the expression of \u003cem\u003eica\u003c/em\u003e operon, which is responsible for synthesis of polysaccharide intracellular adhesin (PIA) and expression of genes capable of autolysis such as atlA, are linked closely to stability and maturation of biofilm resulting in firms biomass structure (Beenken et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Boles et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEnvironmental factors such as oxidative stress, pH fluctuations, osmotic changes, and serum-derived factors are induced by \u003cem\u003eStaph. aureus\u003c/em\u003e and transduced into genetic regulation through complex networks involving two-component systems, global regulators, and small RNAs (Liu, Zhang, and Ji \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These environmental cues can affect the activity of \u003cem\u003esarA\u003c/em\u003e, a global regulator that influences biofilm formation by modulating the expression of \u003cem\u003eica\u003c/em\u003e operon, which encodes vital enzymes for polysaccharide intercellular adhesin (PIA) production (Peng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Adjustment of PIA synthesis and biofilm architecture in response to different stressors can be done by either upregulation or downregulation of \u003cem\u003eicaA\u003c/em\u003e and \u003cem\u003eicaD\u003c/em\u003e, which are mainly influenced by \u003cem\u003esarA\u003c/em\u003e as a global regulator for biofilm formation. The complex dynamicity of biofilm formation of \u003cem\u003eStaph. aureus\u003c/em\u003e can enhance its survival and persistence, especially in hostile environments such as those encountered during infection or on medical devices (Wu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu, Zhang, and Ji \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe findings of the research have significant therapeutic and industrial applications as they shed light on \u003cem\u003eStaph. aureus\u003c/em\u003e biofilm development is modulated. In clinical settings, biofilm-associated infections pose challenges in wound care, bloodstream infections, and medical device-related complications due to their resistance to antibiotics and host immune responses. The observed inhibition of biofilm formation at higher hydrogen peroxide concentrations (\u0026ge;\u0026thinsp;3%) suggests potential applications in antimicrobial therapies and hospital disinfection protocols and this align with (Lineback et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eControlling the formation of biofilms is essential from an industrial aspect in the manufacturing of biomedical devices, water treatment, and food processing. Understanding the effects of oxidative stress, salinity, and pH on biofilm formation could assist in developing antibiofilm coatings, improve industrial cleaning techniques, and design improved sanitation protocols for preventing microbiological contamination (Shineh et al. 2023).\u003c/p\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eLimitation and future directions:\u003c/h2\u003e\u003cp\u003eThis study is limited using a single laboratory strain under in-vitro conditions, which may not reflect the genetic diversity of clinical or environmental isolates or the complexity of in-vivo biofilms. Future work should include multiple clinical strains, explore combined environmental stresses (e.g., immune factors, nutrient limitation), and test anti-biofilm strategies in in-vivo models to bridge laboratory findings with clinical applications.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eStaphylococcus aureus\u003c/em\u003e biofilm formation is highly sensitive to environmental and host-related stresses. Hydrogen peroxide and non-neutral pH conditions significantly reduced biomass and downregulated \u003cem\u003eicaA\u003c/em\u003e, \u003cem\u003eicaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e, with polysaccharide-associated genes most affected. Sodium chloride induced a biphasic response, where mild osmotic stress enhanced biofilm formation, but extreme salinity normalized expression to basal levels. Serum exerted a threshold effect, with low concentrations slightly stimulating gene expression and high concentrations strongly suppressing biofilm development. Together, these results indicate to different but related stress-mediated processes that control the regulation of biofilms and offer possible directions for anti-biofilm therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eORCID\u003c/h2\u003e\u003cp\u003e[0000-0002-5193-9662]\u003csup\u003e1\u003c/sup\u003e, [0000-0003-2745-3811] \u003csup\u003e2\u003c/sup\u003e, [0000-0002-1147-336X] \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\u003ch2\u003eEthics Approval\u003c/h2\u003e\u003cp\u003eNot applicable. The study did not involve human participants or animal experiments; only a standard laboratory strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Newman (ATCC 25904) was used\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Ahmed R. Ragab; Methodology: Ahmed R. Ragab, Ahmed R. El-Sheakh; Formal analysis and investigation: Ahmed R. Ragab; Writing \u0026ndash; original draft preparation: Ahmed R. Ragab; Writing \u0026ndash; review and editing: Ahmed R. El-Sheakh, Shokri M. Shafik; Funding acquisition: Not applicable; Resources: Shokri M. Shafik; Supervision: Shokri M. Shafik. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article. Additional raw datasets are available from the corresponding author upon reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbraham N, Jefferson K (2010) A low molecular weight component of serum inhibits biofilm formation in Staphylococcus aureus. Microb Pathog 49 6:388\u0026ndash;391\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgarwal A, Jain A (2013) Glucose \u0026amp; sodium chloride induced biofilm production \u0026amp; ica operon in clinical isolates of staphylococci. 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Front Microbiol, 8: 1191\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin S, Jiang B, Huang G, Gong Y, You B, Yang Z, Chen Y, Chen J, Yuan Z, Li M, Hu F, Zhao Y, and Yizhi Peng. 2017b. 'Burn Serum Increases \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Biofilm Formation via Oxidative Stress'. Front Microbiol, 8\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":true,"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":"Staphylococcus aureus Newman, Environmental factors, Oxidative stress, ica operon, Gene expression","lastPublishedDoi":"10.21203/rs.3.rs-7714339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7714339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e biofilm formation enhances survival on host tissues and medical devices. This study tested how oxidative stress (H₂O₂), pH (5\u0026ndash;9), NaCl (0\u0026ndash;10%), and human serum (5\u0026ndash;50%) affect Newman strain biofilm and key genes (\u003cem\u003eicaA\u003c/em\u003e, \u003cem\u003eicaD\u003c/em\u003e, \u003cem\u003esarA\u003c/em\u003e). Biofilm was quantified by crystal violet assays, and gene expression measured by quantitative real-time PCR. Biofilm biomass was quantified using crystal violet staining assays under various environmental conditions. Statistical significance was determined using ANOVA with post-hoc analysis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Hydrogen peroxide induced a dose-dependent reduction in biomass, with significant repression of \u003cem\u003eicaA, icaD\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e expression at 3% H₂O₂ (\u0026le;\u0026thinsp;22.8%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, deviations from neutral pH markedly impaired biofilm formation, with acidic (pH 5) and alkaline (pH 9) conditions reducing biomass by 34.6% and 41.7%, respectively, accompanied by strong downregulation of biofilm-associated genes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, NaCl exerted a biphasic effect: mild osmotic stress (1.25% and 5%) enhanced biofilm biomass (up to 154.2%) and gene expression (\u003cem\u003eicaA\u003c/em\u003e 160.55%, \u003cem\u003eicaD\u003c/em\u003e 168.18%, \u003cem\u003esarA\u003c/em\u003e 149.8%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas higher concentrations (\u0026ge;\u0026thinsp;10%) restored expression to near-control levels. Serum exposure produced a threshold-dependent response, with low concentrations (5\u0026ndash;10%) slightly enhancing gene expression (~\u0026thinsp;110%), while higher concentrations (20\u0026ndash;50%) significantly repressed both biomass and transcription, with profound inhibition observed at 50% (\u003cem\u003eicaA\u003c/em\u003e 12.94%, \u003cem\u003eicaD\u003c/em\u003e 10.88%, \u003cem\u003esarA\u003c/em\u003e 12.79%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Environmental stressors modulate \u003cem\u003eStaphylococcus aureus\u003c/em\u003e biofilm formation in a dose-dependent manner via regulation of the \u003cem\u003eica\u003c/em\u003e operon and \u003cem\u003esarA\u003c/em\u003e, offering molecular insights that may guide strategies for biofilm control.\u003c/p\u003e","manuscriptTitle":"Environmental and Chemical Modulation of Staphylococcus aureus Newman Biofilm Formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 19:03:23","doi":"10.21203/rs.3.rs-7714339/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":"41d3da08-1f22-4dc9-b7e3-6789ac34802d","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:18:53+00:00","versionOfRecord":{"articleIdentity":"rs-7714339","link":"https://doi.org/10.1007/s00253-026-13781-6","journal":{"identity":"applied-microbiology-and-biotechnology","isVorOnly":false,"title":"Applied Microbiology and Biotechnology"},"publishedOn":"2026-03-24 16:10:56","publishedOnDateReadable":"March 24th, 2026"},"versionCreatedAt":"2025-10-29 19:03:23","video":"","vorDoi":"10.1007/s00253-026-13781-6","vorDoiUrl":"https://doi.org/10.1007/s00253-026-13781-6","workflowStages":[]},"version":"v1","identity":"rs-7714339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7714339","identity":"rs-7714339","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}