Biological Characteristics of a Menadione-dependent Staphylococcus aureus Small Colony Variant (SCV) from Bovine Mastitis and Heme Promotion of Biofilm Formation

Biological Characteristics of a Menadione-dependent Staphylococcus aureus Small Colony Variant (SCV) from Bovine Mastitis and Heme Promotion of Biofilm Formation | 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 Research Article Biological Characteristics of a Menadione-dependent Staphylococcus aureus Small Colony Variant (SCV) from Bovine Mastitis and Heme Promotion of Biofilm Formation bin Yang, Guanghua Liao, Guofeng Xing, Zihao Wu, Jing Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7699109/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 While small colony variants (SCVs) of Staphylococcus aureus have been extensively documented, little is known about the properties of menadione-dependent SCVs from cow raw milk and how heme affects the ability to form biofilms. In this study, gentamicin-induced SCVs were generated from mastitis-derived Staphylococcus aureus , and the underlying mechanisms were investigated through nutrient dependency assays combined with genomic analysis. A comparative assessment of biological characteristics—including physiological and biochemical properties, antibiotic susceptibility, biofilm formation, and hemolytic activity—was conducted between the SCV and wild-type strains. Additionally, the effects of exogenous heme and menadione on biofilm formation were evaluated. The results revealed a frameshift deletion mutation due to a 95-nucleotide deletion in the menE gene. This mutation altered the corresponding amino acid sequence, which is likely the principal reason for the menadione-dependent SCV phenotype. The SCV strain presented not only typical characteristics, such as slow growth and increased resistance to aminoglycoside antibiotics, but also atypical ones, inculding significantly decreased biofilm formation capacity and increased hemolytic activity. Exogenous supplementation with menadione or heme markedly promoted biofilm formation in the SCV strain. These results suggesting that the SCV strain reduced biofilm-forming ability may stem from compromised metabolic activity. Moreover, the SCV strain potentially enabling the acquisition of exogenous heme via unidentified pathways, thereby restoring biofilm formation capacity. The regulatory mechanisms involved require further investigation. These findings provide new insights into the biofilm regulation and survival/pathogenicity mechanisms of Staphylococcus aureus SCVs. Staphylococcus aureus SCV biological characteristics biofilm hemolytic activity Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Mastitis is one of the most prevalent diseases in dairy cattle, leading to reduced milk yield and inferior milk quality, thereby causing substantial economic losses to the dairy industry. It can be categorized into clinical mastitis (CM) and subclinical mastitis (SCM). Although SCM does not present obvious clinical signs, it is often more challenging to diagnose and control in its early stages and is prone to recurrence. Consequently, SCM is generally considered more harmful than CM and is responsible for 70–80% of the total losses in milk production (Zaatout, 2022 ). SCM is commonly caused by multiple pathogens, among which Staphylococcus aureus ( Staphylococcus aureus ) is one of the primary etiological agents. Globally, the prevalence of SCM in dairy cows ranges from 5.6% to 70.0% (Shah et al., 2025 ). Staphylococcus aureus infections often result in chronic mastitis (Gonçalves et al., 2023 ), with some cases persisting for up to two years (Larsen et al., 2000 ; Ranasinghe et al., 2021 ). In addition to antimicrobial resistance mediated by resistance genes, Staphylococcus aureus can adapt to antibiotic pressure through additional mechanisms, such as biofilm formation, persister cells, and small colony variants (SCVs) (Rossi et al., 2019 ). SCVs of Staphylococcus aureus have been frequently reported in both human and veterinary clinical settings and are believed to play a key role in recurrent infections (Zhou et al., 2022 ). In the early 20th century, Jacobsen first identified SCVs in Salmonella . Since then, SCVs have been reported in numerous bacterial species, with Staphylococcus aureus being among the most frequently observed. SCVs are primarily categorized into two nutritionally deficient subtypes: those impaired in the electron transport chain and those deficient in thymidine deoxyribonucleoside synthesis (Alkasir et al., 2013 ). Staphylococcus aureus SCV typically exhibit traits such as small colony morphology, reduced hemolytic activity, attenuated virulence, enhanced biofilm-forming capacity, and increased antibiotic resistance (Zhou et al., 2022 ). Staphylococcus aureus is capable of surviving not only within phagocytic cells but also of invading nonphagocytic cells, including endothelial and epithelial cells (Kahl et al., 2016 ; Lee et al., 2020 ). Studies suggest that under host-induced stress, wild-type strains can convert intracellularly into SCVs, which persist in greater numbers and for longer durations inside host cells. This transition allows them to evade cellular immune responses, thereby facilitating long-term intracellular survival -a potential key factor in recurrent infections (Löffler et al., 2014 ). On the other hand, to survive in hostile environments, bacteria can secrete extracellular components—such as DNA, polysaccharides, and proteins—that promote aggregation and lead to biofilm formation, offering protection against adverse conditions. Staphylococcus aureus biofilms are typically composed of extracellular polymeric substances (EPS), proteins, and extracellular DNA (eDNA) (Schilcher & Horswill, 2020 ). Among these, EPS primarily consists of polysaccharide intercellular adhesin (PIA), which plays a critical role in bacterial colonization, biofilm formation, and multiple pathogenic processes—including biofilm-associated infections, immune evasion, antimicrobial resistance, and resistance to phagocytosis (Nguyen et al., 2020 ). Iron ions, which are essential trace elements for bacterial growth, are maintained at extremely low free concentrations within the host and are insufficient to support bacterial proliferation (Heinemann et al., 2008 ). In fact, more than 90% of the iron in the host is bound in the form of heme within heme-binding proteins (Tong & Guo, 2009 ). To overcome this limitation, many pathogenic bacteria have evolved specialized heme acquisition systems to utilize host heme as their primary iron source. Staphylococcus aureus , for example, secretes hemolysins to lyse red blood cells, releasing hemoglobin, from which heme is subsequently extracted. Additionally, research has indicated that heme may influence biofilm formation through multiple mechanisms. Aftab et al. ( 2025 ) and Kang & Kirienko ( 2018 ) reported that heme serves as a preferred iron source for nontuberculous mycobacteria and Pseudomonas aeruginosa , significantly increasing their biofilm formation capacity and stability. Furthermore, Ch'ng et al. ( 2022 ) demonstrated that Staphylococcus aureus can cross-feed heme to Enterococcus faecalis , thereby activating aerobic respiration in the latter and promoting biofilm development. Additionally, Lin et al. ( 2012 ) confirmed that iron ions can stimulate biofilm formation in Staphylococcus aureus . These findings collectively suggest that heme may play a regulatory role in the biofilm formation of Staphylococcus aureus , potentially contributing significantly to its pathogenicity and persistence in host tissues. In this study, a menadione-dependent SCV strain of Staphylococcus aureus was isolated following induction with gentamicin. Compared with the wild-type strain, this SCV strain presented a reduced biofilm-forming capacity but increased hemolytic activity. Accordingly, the present work compared the physiological and biochemical properties, as well as antibiotic susceptibility profiles, between the wild-type and SCV strains. Furthermore, we preliminarily investigated the effect of haem on the biofilm formation ability of the SCV strain. These findings provide foundational insights for further research on SCVs in Staphylococcus aureus. 2. Materials and methods 2.1 Induction of Small Colonies Variants of Staphylococcus aureus The experimental strain of Staphylococcus aureus , which was originally isolated from bovine mastitis, was obtained from the Key Laboratory of Tarim Animal Husbandry Science and Technology of the Corps. An activated culture of Staphylococcus aureus was inoculated into brain–heart infusion broth (BHI) (Hi-Media, Qingdao, China) and grown to an OD 600 of 1.0. The culture mixture was then diluted 1:1000 and introduced into BHI media containing gentamicin at concentrations of 0.25 µg/mL, 0.5 µg/mL, 1.0 µg/mL, 2 µg/mL, and 4 µg/mL. The inoculated broths were incubated at 37°C with shaking at 200 rpm for 24–48 hours. Subsequently, overnight cultures were plated onto Columbia blood agar (CBA) (Hibro, Qingdao, China) and incubated at 37°C for 24–72 hours to isolate slow-growing, pinpoint-sized small colonies. Putative SCVs were identified via 16S rRNA and nuc gene amplification. Following more than 10 successive subcultures on CBA to confirm phenotypic stability, the strains were cryopreserved at -80°C. 2.2 Staphylococcus aureus SCV nutrient deficiency test Staphylococcus aureus strain SCV was inoculated into BHI broth and incubated overnight at 37°C with shaking at 200 rpm. The bacterial suspension was then adjusted to a turbidity equivalent to a 0.5 McFarland standard and spread onto tryptic soy agar (TSA) (HaiBo, Qingdao, China). The TSA plates were supplemented with 10 µL of either 5 mg/mL menadione, hemin, or thymidine (Solaibao, Beijing, China). All plates were incubated aerobically at 37°C for 24–48 hours. In parallel, 3-1-S was streaked onto blood agar plates and incubated at 37°C under 5% CO 2 for 24–48 hours to assess potential phenotypic reversion. 2.3 Whole-Genome Sequencing Following revival, strains wild strain and SCV were subcultured in BHI broth for further propagation. Genomic DNA was extracted via the EasyPure Bacterial Genomic DNA Kit. The purity and concentration of the DNA samples were evaluated via 1% agarose gel electrophoresis. Qualified DNA samples were submitted to Shanghai Pisenno Biotechnology Co., Ltd. (Shanghai, China) for next-generation sequencing. A whole-genome shotgun (WGS) strategy was employed to construct libraries with insert sizes of 400 bp. These libraries were subjected to paired-end (PE) sequencing on an Illumina NovaSeq platform. The raw sequencing data were subjected to quality control assessment with FastQC, and the adapter sequences were trimmed via AdapterRemoval. Quality correction of the reads was performed via Kmer-based error correction implemented in SOAPec. De novo assembly of the Illumina data was carried out with SPAdes, followed by contig refinement and base correction via Pilon. Finally, genetic variants were annotated with ANNOVAR software, with a particular focus on mutations in genes associated with menaquinone biosynthesis. 2.4 Growth Curve Measurements and Biochemical Characteristics Strains wild strain and SCV were inoculated onto mannitol salt agar (MSA) (HaiBo, Qingdao, China) and incubated at 37°C for 18–24 hours. A single colony was selected from each strain and inoculated into tryptic soy broth (TSB) for cultivation. When the OD 600 reached 0.3–0.5, the cultures were subcultured at a 1:200 ratio in fresh TSB. Growth curves were generated by measuring the OD 590 at 1-hour intervals during incubation at 37°C with shaking at 180 rpm. Physiological and biochemical characteristics, including protease activity, coagulase production via rabbit plasma, and mannitol fermentation, were assessed for both wild strain and SCV. Staphylococcus aureus ATCC 25923 was used as a positive control, and sterile physiological saline served as the negative control. 2.5 Analysis of Mannitol metabolism-related gene expression levels The strains were cultured in liquid BHI medium at 37°C for 24 hours. Total RNA was extracted from strains wild strain and SCV via an RNA extraction kit (Quanshijin, Beijing, China). The extracted RNA was subsequently reverse transcribed into cDNA via an EasyScript kit (Quanshijin, Beijing, China). The synthesized cDNA was used as the template for real-time quantitative PCR (qPCR) amplification. qPCR was performed under the following three-step cycling conditions: initial denaturation at 94°C for 30 seconds, followed by 40 cycles of denaturation at 94°C for 5 seconds, annealing at 50°C for 15 seconds, and extension at 72°C for 10 seconds. The fluorescence data were collected and analyzed at the end of the extension step of each cycle. For detailed primer sequences, see Supplementary Material 1. 2.6 Scanning electron microscopy Strains wild strain and SCV were inoculated into TSB media and incubated at 37°C until they reached the plateau phase (26 hours). The cultures were centrifuged at 8,000 rpm for 2 minutes, and the resulting pellets were fixed with 2.5% glutaraldehyde for 24 hours. The fixed samples were subsequently submitted to the Instrumental Analysis Center of Tarim University for processing, which included supercritical drying and ion sputter coating. Morphological observations were carried out via a variable vacuum ultrahigh-resolution field emission scanning electron microscope equipped with an energy dispersive spectrometer (Thermo Fisher Scientific, USA). 2.7 Drug resistance analysis The minimum inhibitory concentrations (MICs) of gentamicin, enrofloxacin, kanamycin, vancomycin, streptomycin, paromomycin, and cefotaxime against strains 3 − 1 and 3-1-S were determined via the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The tested concentrations of antibiotics ranged from 0.125 µg/mL to 64 µg/mL. Mueller–Hinton (MH) medium without bacterial inoculum was used as the negative control. All experiments were performed in triplicate, with Staphylococcus aureus ATCC 25923 included as the quality control strain. 2.8 Biofilm formation capability testing The biofilm-forming capacity of strains wild strain and SCV was evaluated semiquantitatively via a 96-well plate model. Activated bacterial cultures were diluted 1:100 in fresh TSB broth, either plain or supplemented with one of the following: sheep erythrocytes (1%), heme (0.5, 1.0, 2.5, 5.0, 10.0, or 20.0 µg/mL), or menaquinone (0.5, 1.0, 2.5, or 5.0 µg/mL). A 200 µL aliquot of each diluted suspension was transferred into individual wells of a 96-well plate. Staphylococcus epidermidis ATCC 35984 was used as the positive control, and TSB medium alone served as the blank control. After 48 hours of incubation at 37°C, nonadherent cells were removed by washing with phosphate-buffered saline (PBS). The plates were air-dried, heat-fixed at 60°C for 30 minutes, and then stained with 1% crystal violet for 10 minutes. Following gentle rinsing and air drying, the bound dye was solubilized with an acetone/ethanol solution (1:4). The optical density at 570 nm (OD 570 ) was measured via a microplate reader. 2.9 Hemolytic activity assay The supernatant was collected from overnight bacterial cultures. A volume of 100 µL of the supernatant was mixed with 900 µL of PBS containing 3% sheep erythrocytes. The mixture was incubated at 37°C for 30 minutes and then centrifuged at 8000 rpm for 2 minutes. Two hundred microliters of the resulting supernatant was subsequently transferred to a 96-well plate. Hemolytic activity was assessed by measuring the optical density at 540 nm (OD 540 ) via a microplate reader. The positive control consisted of 1000 µL of a solution containing 3% sheep erythrocytes in double-distilled water (ddH 2 O), indicating complete hemolysis. The negative control contained 1000 µL of 3% sheep erythrocytes in PBS, indicating that there was no hemolysis. 2.10 Extracellular DNA, extracellular polysaccharide, and surface hydrophobicity detection eDNA was quantified according to the method described by Ivanov et al. ( 2022 ). Overnight cultures of strains 3 − 1 and 3-1-S were subcultured in TSB and grown to the mid-log phase. The cultures were diluted 1:100 in TSB supplemented with 2% glucose, aliquoted into a 96-well plate, and incubated at 37°C for 48 hours. For the treatment groups, heme (final concentration of 5 µg/mL) or menaquinone (final concentration of 2.5 µg/mL) was added to the culture medium of strain 3-1-S. After incubation, the culture broth was discarded, and the wells were washed twice with PBS. TE buffer was then added to each well, and the mixture was mixed vigorously. The mixtures were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 10,000 × g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in TE buffer by vortexing. After a second centrifugation step at 10,000 × g for 15 minutes, the absorbance of the resulting supernatant was measured at 260 nm. An overnight bacterial culture was inoculated at a 1:100 ratio into 50 mL of TSB medium. For the treatment group, either heme (5 µg/mL) or menaquinone (2.5 µg/mL) was added to the culture of strain SCV. The cultures were incubated at 37°C with shaking at 180 rpm for 24 hours. The cells were harvested via centrifugation at 6000 rpm for 15 minutes, and the supernatant was collected. Five volumes of 95% ethanol were added to the supernatant, and the mixture was precipitated at 4°C for 24 hours. The precipitate was collected by centrifugation at 10,000 rpm for 20 minutes. The resulting pellet was washed twice with 95% ethanol and air-dried at room temperature. The crude polysaccharide was dissolved in an appropriate volume of deionized water. A 1/10 volume of 20 mg/mL proteinase K was subsequently added, followed by incubation at 37°C for 2 hours. Then, a 1/4 volume of a chloroform:n-butanol mixture (5:1) was added, and the mixture was vortexed vigorously and centrifuged at 12,000 rpm and 4°C for 20 minutes. The aqueous layer was collected, transferred to a pretreated dialysis bag, and dialyzed overnight under running water. Finally, the sample was lyophilized under vacuum to obtain the extracellular polysaccharides. The overnight bacterial culture was inoculated into 50 mL of TSB medium at a 1:100 ratio. For the treatment groups, hemin or menadione was added to the SCV culture at final concentrations of 5 µg/mL and 2.5 µg/mL, respectively. The mixture was incubated at 37°C with shaking at 180 rpm for 24 hours. Bacterial cells were harvested by centrifugation at 6000 rpm for 15 minutes and washed twice with PUM buffer. Using PUM buffer as a blank, the absorbance of the bacterial suspension was measured at 600 nm (A 0 ). The suspension was adjusted to an optical density between 0.4 and 0.6. Hexadecane was added to the diluted bacterial suspension at a 4:1 ratio, followed by vigorous mixing and incubation for 15 minutes to allow phase separation. The lower aqueous phase was carefully collected, and its absorbance at 600 nm (A 1 ) was measured. The cell surface hydrophobicity was calculated as follows: surface hydrophobicity = (1 − A 1 /A 0 ) × 100%. 2.11 Statistical analysis Data analysis was performed via the t test via GraphPad Prism v.10.0 software. A p value of less than 0.05 was considered statistically significant (*: p < 0.05; **: p < 0.01). 3. Results 3.1 Gentamicin-induced acquisition of the menadione-dependent Staphylococcus aureus SCV In this study, screening with five different concentrations of gentamicin (0.25, 0.5, 1, 2, and 4 µg/mL) led to the successful isolation of an SCV strain, designated 3 − 1, which remained stable after more than 10 successive passages. Compared with the wild-type strain, the SCV isolate exhibited slow growth, formed transparent, pinpoint-sized colonies (Fig. 1 a) and was accordingly named 3-1-S. Nutritional deficiency assays revealed that phenotypic reversion occurred around filter paper discs impregnated with menadione, whereas no restoration of growth was observed around discs containing hemin chloride or thymidine or on blood agar plates. These results indicate that 3-1-S is a menadione-dependent SCV strain (Fig. 1 b). Sequence analysis of key genes involved in menaquinone biosynthesis ( menA , menB , menC , menD , menE , menF , menG , and menH ) revealed a total of 61 mutation sites. These mutations were distributed across all genes except menE . Spectrum analysis revealed that the majority of the mutations were synonymous (41 out of 61, 67.21%), which does not alter the encoded amino acid sequence. In addition, 20 nonsynonymous mutations were identified, leading to amino acid substitutions—including 9 conservative and 10 nonconservative changes (see supplementary material 2). Notably, a significant mutation was detected in the menE gene of strain 3-1-S: a 95-bp deletion starting at position 216. This deletion resulted in a frameshift mutation and the introduction of a premature stop codon (TAG) downstream of the deletion site (Fig. 1 c). It is predicted that this mutation leads to premature translational termination, likely yielding a truncated and dysfunctional MenE protein. 3.2 SCV exhibits slow growth and altered biochemical characteristics Biochemical characterization revealed that both strains, 3 − 1 and 3-1-S, tested positive in the protease activity assay (Fig. 2 a) and the rabbit plasma coagulase test (Fig. 2 b). The growth curve of strain 3 − 1 displayed typical bacterial kinetics, with a lag phase of approximately 4 hours followed by exponential growth, eventually reaching a stationary phase with an OD 600 between 2.0 and 2.5. In contrast, strain 3-1-S exhibited markedly impaired growth, lacked a distinct exponential phase and maintained an OD 600 below 0.5 throughout the incubation. Even after 30 h, its biomass remained significantly lower than that of strain 3 − 1 (Fig. 2 c). In the mannitol fermentation test, strain 3 − 1 utilized mannitol efficiently, whereas strain 3-1-S showed neither significant growth nor acid production (Fig. 2 d). qPCR analysis of genes within the mannitol operon revealed significant downregulation of mtlF and mtlD in the SCV strain compared with the wild-type strain, whereas the expression of mtlR was substantially upregulated (Fig. 2 d). These results suggest that mtlR may act as a negative regulator and that the loss of mannitol fermentation capability in the SCV is likely due to altered expression of key metabolic genes. Scanning electron microscopy (SEM) revealed no notable morphological differences between strains 3 − 1 and 3-1-S (Fig. 2 e). 3.3 SCV exhibits enhanced resistance to aminoglycoside antibiotics In this study, the broth microdilution method was used to evaluate the antimicrobial susceptibility of strains 3 − 1 and 3-1-S. The results revealed that the MIC values of enrofloxacin, ceftiofur, and vancomycin for strain 3-1-S were < 0.125, 1, and 0.25 µg/mL, respectively. In contrast, the MICs for several aminoglycoside antibiotics—including gentamicin, kanamycin, and streptomycin—reached 32 µg/mL or 64 µg/mL, indicating significantly elevated resistance compared with that of the wild-type strain 3 − 1. 3.4 Heme and menadione promote SCV biofilm formation Compared with that of strain 3 − 1, the biofilm-forming ability of 3-1-S was significantly reduced by 40.12% (p < 0.01) (Fig. 3 a), while its hemolytic activity increased markedly by 202.94% (p < 0.01) (Fig. 3 b). The addition of 1% sheep erythrocytes to TSB significantly enhanced the biofilm formation of 3-1-S by 78.72% (p < 0.01). Similarly, supplementation with different concentrations of haem also affected SCV biofilm formation. When cocultured with 5 µg/mL or 2.5 µg/mL haem, biofilm formation increased by 32.96% and 35.57%, respectively. However, heme concentrations below 2.5 µg/mL did not significantly promote biofilm formation, whereas concentrations exceeding 5 µg/mL resulted in inhibition (Fig. 3 c). Supplementing the culture medium with menadione at final concentrations of 1 µg/mL and 2.5 µg/mL increased the biofilm-forming capacity of 3-1-S by 41.95% and 54.08%, respectively. In contrast, no significant effect was observed at menadione concentrations of 0.5 µg/mL or 5 µg/mL (Fig. 3 d). 3.5 Heme and menaquinone regulate SCV biofilm formation by influencing extracellular polysaccharide levels and surface hydrophobicity Measurements of eDNA, EPS, and surface hydrophobicity were conducted for strains 3 − 1, 3-1-S, and 3-1-S supplemented with 5 µg/mL heme or 2.5 µg/mL menaquinone. Compared with the wild-type strain, the 3-1-S strain presented significantly lower eDNA content, lower EPS production, and lower surface hydrophobicity (Fig. 4 ). Following supplementation with heme or menaquinone, no significant change in eDNA content was observed in 3-1-S (Fig. 4 a). In contrast, both EPS production and surface hydrophobicity increased significantly (p < 0.01) (Figs. 4 b and 4 c). Notably, after the addition of menaquinone, the EPS content and surface hydrophobicity of 3-1-S were restored to levels comparable to those of the wild-type strain. These findings underscore the crucial role of menaquinone in regulating extracellular polysaccharide synthesis and modulating surface hydrophobicity in the SCV phenotype. 4. Discussion Staphylococcus aureus can develop SCVs in response to antibiotics and environmental stress. This phenotypic switch is closely associated with persistent, recurrent, and drug-resistant infections, posing considerable challenges for clinical diagnosis and treatment. In the present study, a menadione-dependent SCV strain was isolated from Staphylococcus aureus originating from a case of bovine mastitis through induction with gentamicin. Compared with the wild-type strain, the mutant strain displayed slow growth on blood agar plates, formed pinpoint-sized microcolonies, and exhibited a significantly reduced growth rate. These findings are consistent with previous reports by Alkasir and Atalla, who also isolated SCVs from cows with chronic mastitis, further confirming the ability of Staphylococcus aureus to form SCVs within the mastitis environment (Alkasir et al., 2013 ; Atalla et al., 2008 ). As a key component of the electron transport chain, menadione undergoes isoprenoid side-chain extension to form menaquinone derivatives. These act as electron acceptors for NADH/FADH₂ and participate in the respiratory electron transport process (Melter & Radojevič, 2010 ). Deficiency in menaquinone disrupts the electron transport chain, leading to a reduced proton motive force and consequently impairing ATP synthesis. Since bacterial cell wall biosynthesis is energy intensive, this energy deficit contributes to impaired growth and the formation of microcolonies. Furthermore, the electron transport chain is involved in the biosynthesis of carotenoid pigments. Its disruption can lead to the formation of nonpigmented colonies, reduced hemolytic activity, and a delayed coagulase reaction—a phenomenon that is partly attributable to diminished amino acid synthesis (Melter & Radojevič, 2010 ; Proctor et al., 1995 ). Supplementation with nutrients deficient in growth medium has been shown to restore the SCV to a normal colony phenotype. Gene expression analysis of the mannitol metabolism operon in the SCV revealed significant downregulation of mtlF and mtlD , along with marked upregulation of mtlR . These genes encode the mannitol-specific transporter mannitol-1-phosphate dehydrogenase and the transcriptional repressor of the mannitol operon (Anbalagan et al., 2022 ; Kumar et al., 2011 ). As a negative regulator, MtlR suppresses the expression of genes within the mannitol utilization operon (Byer et al., 2017 ), thereby inhibiting the transcription of mtlF and mtlD . The reduced expression of these genes impedes both the uptake and metabolic conversion of mannitol, limiting the ability of the bacterium to utilize mannitol as a carbon and energy source. This metabolic impairment likely exacerbates the energy deficiency already present in the SCV phenotype. The genes involved in menaquinone biosynthesis belong to the men operon, among which menD and menB play essential roles in this pathway (Kohler et al., 2008 ; Lannergård et al., 2008 ). Studies have shown that mutations in menD disrupt the electron transport chain in Staphylococcus aureus , impairing its ability to utilize oxygen or nitrate as terminal electron acceptors. These mutations also alter the expression of genes encoding enzymes involved in glycolysis, fermentation, and the tricarboxylic acid cycle (Kohler et al., 2008 ). Similarly, mutations in menB have been directly linked to a menaquinone-deficient phenotype, affecting the bacterial growth rate, susceptibility to gentamicin, and hemolytic activity (Lannergård et al., 2008 ). Mutation analysis of eight genes within the men operon revealed a notable 95-base pair deletion in menE , which induces a frameshift and introduces a premature stop codon. This mutation is predicted to cause premature termination of translation, likely resulting in a truncated and nonfunctional MenE protein. MenE functions as an O-succinylbenzoyl-CoA synthase, catalyzing the adenylation and thioesterification of O-succinylbenzoic acid (OSB), a key intermediate in menaquinone biosynthesis (Matarlo et al., 2015 ). This reaction converts OSB into OSB-CoA, which subsequently enters the menaquinone synthesis pathway (Lu et al., 2012 ). Therefore, severe mutations in menE disrupt menaquinone production, leading to dysfunction of the electron transport chain, reduced ATP synthesis, and cellular energy depletion. These metabolic deficiencies ultimately contribute to the development of the SCV phenotype. The pronounced antibiotic resistance of SCVs is a major factor underlying their role in chronic and recurrent infections that are challenging to eradicate. In this study, the SCV strain demonstrated significantly increased resistance to aminoglycoside antibiotics compared with the wild-type strain, whereas no marked differences were observed in its response to β-lactams, quinolones, or glycopeptide antibiotics. The uptake of aminoglycosides is an energy-dependent process that relies on an intact membrane potential and active electron transport (von Eiff et al., 2006 ). Menaquinone-deficient SCVs exhibit defects in their electron transport chain and respiratory activity, resulting in diminished ATP production and reduced membrane potential (Proctor et al., 2006 ). Consequently, impaired energy metabolism and depolarized membranes in SCVs limit the intracellular accumulation of aminoglycosides, thereby increasing bacterial resistance. Although SCVs do not present elevated MICs for β-lactam or glycopeptide antibiotics, their slow growth rate and reduced cell wall biosynthesis may diminish the bactericidal effectiveness of these drug classes—which target cell wall synthesis—in actual infections (von Eiff et al., 2006 ). Quinolones, on the other hand, enter bacterial cells primarily through passive diffusion or porin channels, a process that is not energy dependent (Hooper, 2001 ). Thus, even with the reduced metabolic activity and membrane potential of SCVs, the uptake and activity of fluoroquinolones remain largely unaffected, explaining the unchanged susceptibility profile. In summary, SCVs exhibit a well-established mechanism of high-level resistance to aminoglycoside antibiotics. Although no increase in MIC was detected for other drug classes under the tested conditions, the overall antibiotic tolerance and reduced rate of killing—attributable to the metabolic dormancy of SCVs—contribute substantially to phenotypic resistance, treatment failure, and infection recurrence. In most reported studies, SCVs have been shown to enhance biofilm formation but attenuate virulence(Lee et al., 2020 ). Interestingly, compared with the wild-type strain, the menadione-dependent SCV induced in this study presented significantly reduced biofilm-forming capacity but increased hemolytic activity. The synthesis of extracellular polysaccharides, release of eDNA, and maintenance of cell surface hydrophobicity—all key components of the biofilm matrix—are energy-demanding processes (Das & Manefield, 2012 ; Lin et al., 2022 ). The impaired biofilm formation observed in the SCV may thus stem from its severe energy deficiency, which is consistent with the restoration of biofilm capacity upon menadione supplementation. As noted earlier, iron availability regulates biofilm development, and in vivo, iron is predominantly complexed within heme-containing proteins (Tong & Guo, 2009 ). In this study, the SCV strain presented increased hemolytic activity, suggesting an increased capacity to acquire iron from host erythrocytes. Similarly, the addition of red blood cells or exogenous heme significantly increased SCV biofilm formation. Further experiments confirmed that heme supplementation notably augmented both extracellular polysaccharide production and surface hydrophobicity in the SCV, underscoring its role in promoting biofilm assembly. Swarupa et al. ( 2018 ) reported that iron supplementation substantially enhanced peptidyl deacetylase activity in Staphylococcus aureus , thereby facilitating biofilm formation. Under anaerobic conditions, iron increased the biofilm biomass by approximately 15-fold. Moreover, when hemoglobin serves as the iron source, the resulting biofilms are thicker and more structurally complex than those promoted by inorganic iron salts (Dauros-Singorenko et al., 2020 ). However, notably, high concentrations of heme can inhibit biofilm formation (Torres et al., 2007 ), a concentration-dependent effect that aligns with the trend observed in the present study. Therefore, we propose that, owing to its impaired biofilm formation and colonization capacity, the SCV may employ an alternative survival strategy by modulating the expression of virulence factors such as hemolysin. Enhanced hemolytic activity could facilitate the lysis of red blood cells, directly damaging host tissues and promoting bacterial dissemination and invasion. Concurrently, the nutrients released—including hemoglobin and iron—may serve as an energy source for the SCV, thereby partially compensating for its metabolic deficiencies. 5. Conclusion In this study, gentamicin-induced Staphylococcus aureus strains causing subclinical mastitis in dairy cows yielded one menE -mutant SCV strain exhibiting characteristics such as slow growth, reduced metabolic activity, enhanced drug resistance, weakened biofilm formation capacity, and increased hemolytic activity. Genomic analysis revealed that a mutation in the menE gene disrupted the menadione synthesis pathway, likely serving as a key molecular basis for its metabolic defects and phenotypic variation. Heme may regulate SCV biofilm formation, potentially representing an effective survival strategy by altering metabolic pathways; however, the underlying regulatory mechanisms require further investigation. Declarations Funding: This work was supported by the Engineering Laboratory of Tarim Animal Disease Diagnosis and Control, Xinjiang Production and Construction Corps (ELDC202002). Declaration of competing interests : The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions : Jing Wu ( [email protected] ), Bin Yang ( [email protected] ), and Guanghua Liao ( [email protected] ) conceived and designed this study and wrote the manuscript. Bin Yang, Guanghua Liao, Guofeng Xing ( [email protected] ), and Zihao Wu ( [email protected] ) performed the experiments and collected and analyzed the data. Ethics declaration: No animals were used in this study. Therefore, approval from an institutional animal ethics committee was not required. Data availability: The raw sequencing data are available in the Sequence Read Archive with the accession codes SAMN51698484 and SAMN51698485. References Aftab H, Samudio J, Wang G, Le L, Soni RK, Donegan RK (2025) Heme alters biofilm formation in Mycobacterium abscessus. Microbiol Spectr 13(2):e0241524. https://doi.org/10.1128/spectrum.02415-24 Alkasir R, Liu X, Zahra M, Ferreri M, Su J, Han B (2013) Characteristics of Staphylococcus aureus small colony variant and its parent strain isolated from chronic mastitis at a dairy farm in Beijing, China. 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Vet Microbiol 71(1–2):89–101. https://doi.org/10.1016/s0378-1135(99)00161-3 Lee J, Zilm PS, Kidd SP (2020) Novel Research Models for Staphylococcus aureus Small Colony Variants (SCV) Development: Co-pathogenesis and Growth Rate. Front Microbiol 11:321. https://doi.org/10.3389/fmicb.2020.00321 Lin MH, Shu JC, Huang HY, Cheng YC (2012) Involvement of iron in biofilm formation by Staphylococcus aureus . PLoS ONE 7(3):e34388. https://doi.org/10.1371/journal.pone.0034388 Lin Y, Zhou X, Li Y (2022) Strategies for Streptococcus mutans biofilm dispersal through extracellular polymeric substances disruption. Mol Oral Microbiol 37(1):1–8. https://doi.org/10.1111/omi.12355 Löffler B, Tuchscherr L, Niemann S, Peters G (2014) Staphylococcus aureus persistence in non-professional phagocytes. Int J Med Microbiol 304(2):170–176. https://doi.org/10.1016/j.ijmm.2013.11.011 Lu X, Zhou R, Sharma I, Li X, Kumar G, Swaminathan S, Tonge PJ, Tan DS (2012) Stable analogues of OSB-AMP: potent inhibitors of MenE , the o-succinylbenzoate-CoA synthetase from bacterial menaquinone biosynthesis. ChemBioChem 13(1):129–136. https://doi.org/10.1002/cbic.201100585 Matarlo JS, Evans CE, Sharma I, Lavaud LJ, Ngo SC, Shek R, Rajashankar KR, French JB, Tan DS, Tonge PJ (2015) Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents. Biochemistry 54(42):6514–6524. https://doi.org/10.1021/acs.biochem.5b00966 Melter O, Radojevič B (2010) Small colony variants of Staphylococcus aureus–r eview. Folia Microbiol (Praha) 55(6):548–558. https://doi.org/10.1007/s12223-010-0089-3 Nguyen HTT, Nguyen TH, Otto M (2020) The staphylococcal exopolysaccharide PIA - Biosynthesis and role in biofilm formation, colonization, and infection. Comput Struct Biotechnol J 18:3324–3334. https://doi.org/10.1016/j.csbj.2020.10.027 Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD (1995) Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus . Clin Infect Dis 20(1):95–102. https://doi.org/10.1093/clinids/20.1.95 Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M, Peters G (2006) Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4):295–305. https://doi.org/10.1038/nrmicro1384 Ranasinghe R, Deshapriya RMC, Abeygunawardana DI, Rahularaj R, Dematawewa CMB (2021) Subclinical mastitis in dairy cows in major milk-producing areas of Sri Lanka: Prevalence, associated risk factors, and effects on reproduction. J Dairy Sci 104(12):12900–12911. https://doi.org/10.3168/jds.2021-20223 Rossi BF, Bonsaglia ECR, Castilho IG, Dantas STA, Salina A, Langoni H, Pantoja JCF, Budri PE, Fitzgerald-Hughes D, Júnior AF, Rall VLM (2019) Genotyping of long term persistent Staphylococcus aureus in bovine subclinical mastitis. Microb Pathog 132:45–50. https://doi.org/10.1016/j.micpath.2019.04.031 Schilcher K, Horswill AR (2020) Staphylococcal Biofilm Development: Structure, Regulation, and Treatment Strategies. Microbiol Mol Biol Rev 84(3). https://doi.org/10.1128/mmbr.00026-19 Shah AU, Khan A, Avais J, Zaman M, Munir SH, Abbas Z, Tariq S, Rahman M, Tariq MU, Nawaz F, Fouad S, Anjum D, A. A., Ahmed QI (2025) Prevalence and chemotherapy of Staphylococcus aureus mastitis in dairy cattle. PLoS ONE 20(2):e0315480. https://doi.org/10.1371/journal.pone.0315480 Swarupa V, Chaudhury A, Sarma P (2018) Iron enhances the peptidyl deformylase activity and biofilm formation in Staphylococcus aureus . 3 Biotech 8(1):32. https://doi.org/10.1007/s13205-017-1050-9 Tong Y, Guo M (2009) Bacterial heme-transport proteins and their heme-coordination modes. Arch Biochem Biophys 481(1):1–15. https://doi.org/10.1016/j.abb.2008.10.013 Torres VJ, Stauff DL, Pishchany G, Bezbradica JS, Gordy LE, Iturregui J, Anderson KL, Dunman PM, Joyce S, Skaar EP (2007) A Staphylococcus aureus regulatory system that responds to host heme and modulates virulence. Cell Host Microbe 1(2):109–119. https://doi.org/10.1016/j.chom.2007.03.001 von Eiff C, McNamara P, Becker K, Bates D, Lei XH, Ziman M, Bochner BR, Peters G, Proctor RA (2006) Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype. J Bacteriol 188(2):687–693. https://doi.org/10.1128/jb.188.2.687-693.2006 Zaatout N (2022) An overview on mastitis-associated Escherichia coli : Pathogenicity, host immunity and the use of alternative therapies. Microbiol Res 256:126960. https://doi.org/10.1016/j.micres.2021.126960 Zhou S, Rao Y, Li J, Huang Q, Rao X (2022) Staphylococcus aureus small-colony variants: Formation, infection, and treatment. Microbiol Res 260:127040. https://doi.org/10.1016/j.micres.2022.127040 Tables Table 1 SCV minimum inhibitory concentration results Antibiotics MIC（μg/mL） 3-1 3-1-S Gentamicin 1 32 Streptomycin 1 32 Balongmycin 2 32 Ampicillin 8 >64 Kanamycin 1 >64 Enrofloxacin <0.125 <0.125 Cefotaxime 1 2 Vancomycin 0.25 0.25 Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":4059871,"visible":true,"origin":"","legend":"\u003cp\u003eColony morphology of SCV on CBA, the results of auxotrophy tests, and the deletion mutation pattern of the \u003cem\u003emenE \u003c/em\u003egene.\u003c/p\u003e\n\u003cp\u003eNote: a shows the colony morphology of 3-1 and 3-1-S on CBA medium; b shows the appearance of revertant strains in 3-1-S after the addition of menadione; c shows the deletion mutation pattern of the menE gene. The blue box indicates the normal base sequence; the red box indicates the deleted portion; the blue amino acid sequence represents the normal translation product; the red amino acid sequence represents the frameshift mutation translation result caused by the deletion; the red dashed line indicates the site of mutation deletion.\u003c/p\u003e","description":"","filename":"Onlinefloatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-7699109/v1/d200939b270e209e5839b809.png"},{"id":93513620,"identity":"942b650a-6d94-4fe4-990f-e86d23cfc493","added_by":"auto","created_at":"2025-10-14 15:55:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1443849,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of SCV biological characteristics.\u003c/p\u003e\n\u003cp\u003eNote: a represents catalase assay results; b represents plasma coagulase assay results; c represents growth curve results; d represents mannitol fermentation experiments and relative expression levels of the mannitol metabolism operon; e represents scanning electron microscopy observations; *: P \u0026lt; 0.05; **: P \u0026lt; 0.01; ns: no significant difference.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7699109/v1/e37427e8b7b54d7796b63745.png"},{"id":93513622,"identity":"0facb237-5bc4-4ab5-829c-bb173f4466bd","added_by":"auto","created_at":"2025-10-14 15:55:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95050,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of heme and menadione on SCV biofilm formation capacity.\u003c/p\u003e\n\u003cp\u003eNote: a represents biofilm formation capacity assays for 3-1 and 3-1-S; b: Hemolytic activity results for 3-1 and 3-1-S; c: Biofilm formation ability of SCV after adding red blood cells and heme; d: Biofilm formation ability of SCV after adding menadione; *: P \u0026lt; 0.05; **: P \u0026lt; 0.01; ns: No significant difference.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7699109/v1/08aaded97f324523d0a57450.png"},{"id":93514921,"identity":"64310dc7-485c-4f48-a25c-9150885159fc","added_by":"auto","created_at":"2025-10-14 16:19:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85008,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of heme and menadione on EDNA, extracellular polysaccharides, and surface hydrophobicity.\u003c/p\u003e\n\u003cp\u003eNote: a shows the eDNA content results for 3-1, 3-1-S, and 3-1-S after adding heme and menadione; b shows the extracellular polysaccharide content in 3-1, 3-1-S, and 3-1-S supplemented with heme and menadione; c shows the changes in surface hydrophobicity in 3-1, 3-1-S, and 3-1-S supplemented with heme and menadione; *: P \u0026lt; 0.05; **: P \u0026lt; 0.01; ns: no significant difference.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7699109/v1/f14b0c7a04c7337bfb5eb6fb.png"},{"id":94505682,"identity":"4b8a740c-34c7-4a8e-83b7-e108694dceec","added_by":"auto","created_at":"2025-10-28 16:20:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3423460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7699109/v1/a4c2e4e4-7350-4200-962d-3b5fe40c90a3.pdf"},{"id":93513988,"identity":"9dff8646-4471-4775-ad4e-73dc339df0ed","added_by":"auto","created_at":"2025-10-14 16:03:04","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15060,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7699109/v1/15af906e20af4e4a12f62854.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological Characteristics of a Menadione-dependent Staphylococcus aureus Small Colony Variant (SCV) from Bovine Mastitis and Heme Promotion of Biofilm Formation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMastitis is one of the most prevalent diseases in dairy cattle, leading to reduced milk yield and inferior milk quality, thereby causing substantial economic losses to the dairy industry. It can be categorized into clinical mastitis (CM) and subclinical mastitis (SCM). Although SCM does not present obvious clinical signs, it is often more challenging to diagnose and control in its early stages and is prone to recurrence. Consequently, SCM is generally considered more harmful than CM and is responsible for 70\u0026ndash;80% of the total losses in milk production (Zaatout, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). SCM is commonly caused by multiple pathogens, among which \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e) is one of the primary etiological agents. Globally, the prevalence of SCM in dairy cows ranges from 5.6% to 70.0% (Shah et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infections often result in chronic mastitis (Gon\u0026ccedil;alves et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with some cases persisting for up to two years (Larsen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Ranasinghe et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to antimicrobial resistance mediated by resistance genes, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e can adapt to antibiotic pressure through additional mechanisms, such as biofilm formation, persister cells, and small colony variants (SCVs) (Rossi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). SCVs of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e have been frequently reported in both human and veterinary clinical settings and are believed to play a key role in recurrent infections (Zhou et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the early 20th century, Jacobsen first identified SCVs in \u003cem\u003eSalmonella\u003c/em\u003e. Since then, SCVs have been reported in numerous bacterial species, with \u003cem\u003eStaphylococcus aureus\u003c/em\u003e being among the most frequently observed. SCVs are primarily categorized into two nutritionally deficient subtypes: those impaired in the electron transport chain and those deficient in thymidine deoxyribonucleoside synthesis (Alkasir et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SCV typically exhibit traits such as small colony morphology, reduced hemolytic activity, attenuated virulence, enhanced biofilm-forming capacity, and increased antibiotic resistance (Zhou et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is capable of surviving not only within phagocytic cells but also of invading nonphagocytic cells, including endothelial and epithelial cells (Kahl et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies suggest that under host-induced stress, wild-type strains can convert intracellularly into SCVs, which persist in greater numbers and for longer durations inside host cells. This transition allows them to evade cellular immune responses, thereby facilitating long-term intracellular survival -a potential key factor in recurrent infections (L\u0026ouml;ffler et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). On the other hand, to survive in hostile environments, bacteria can secrete extracellular components\u0026mdash;such as DNA, polysaccharides, and proteins\u0026mdash;that promote aggregation and lead to biofilm formation, offering protection against adverse conditions. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e biofilms are typically composed of extracellular polymeric substances (EPS), proteins, and extracellular DNA (eDNA) (Schilcher \u0026amp; Horswill, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among these, EPS primarily consists of polysaccharide intercellular adhesin (PIA), which plays a critical role in bacterial colonization, biofilm formation, and multiple pathogenic processes\u0026mdash;including biofilm-associated infections, immune evasion, antimicrobial resistance, and resistance to phagocytosis (Nguyen et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIron ions, which are essential trace elements for bacterial growth, are maintained at extremely low free concentrations within the host and are insufficient to support bacterial proliferation (Heinemann et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In fact, more than 90% of the iron in the host is bound in the form of heme within heme-binding proteins (Tong \u0026amp; Guo, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). To overcome this limitation, many pathogenic bacteria have evolved specialized heme acquisition systems to utilize host heme as their primary iron source. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, for example, secretes hemolysins to lyse red blood cells, releasing hemoglobin, from which heme is subsequently extracted. Additionally, research has indicated that heme may influence biofilm formation through multiple mechanisms. Aftab et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and Kang \u0026amp; Kirienko (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that heme serves as a preferred iron source for nontuberculous mycobacteria and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, significantly increasing their biofilm formation capacity and stability. Furthermore, Ch'ng et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that \u003cem\u003eStaphylococcus aureus\u003c/em\u003e can cross-feed heme to \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, thereby activating aerobic respiration in the latter and promoting biofilm development. Additionally, Lin et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) confirmed that iron ions can stimulate biofilm formation in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. These findings collectively suggest that heme may play a regulatory role in the biofilm formation of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, potentially contributing significantly to its pathogenicity and persistence in host tissues.\u003c/p\u003e\u003cp\u003eIn this study, a menadione-dependent SCV strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was isolated following induction with gentamicin. Compared with the wild-type strain, this SCV strain presented a reduced biofilm-forming capacity but increased hemolytic activity. Accordingly, the present work compared the physiological and biochemical properties, as well as antibiotic susceptibility profiles, between the wild-type and SCV strains. Furthermore, we preliminarily investigated the effect of haem on the biofilm formation ability of the SCV strain. These findings provide foundational insights for further research on SCVs in \u003cem\u003eStaphylococcus aureus.\u003c/em\u003e\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Induction of Small Colonies Variants of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe experimental strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, which was originally isolated from bovine mastitis, was obtained from the Key Laboratory of Tarim Animal Husbandry Science and Technology of the Corps. An activated culture of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was inoculated into brain\u0026ndash;heart infusion broth (BHI) (Hi-Media, Qingdao, China) and grown to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0. The culture mixture was then diluted 1:1000 and introduced into BHI media containing gentamicin at concentrations of 0.25 \u0026micro;g/mL, 0.5 \u0026micro;g/mL, 1.0 \u0026micro;g/mL, 2 \u0026micro;g/mL, and 4 \u0026micro;g/mL. The inoculated broths were incubated at 37\u0026deg;C with shaking at 200 rpm for 24\u0026ndash;48 hours. Subsequently, overnight cultures were plated onto Columbia blood agar (CBA) (Hibro, Qingdao, China) and incubated at 37\u0026deg;C for 24\u0026ndash;72 hours to isolate slow-growing, pinpoint-sized small colonies. Putative SCVs were identified via 16S rRNA and nuc gene amplification. Following more than 10 successive subcultures on CBA to confirm phenotypic stability, the strains were cryopreserved at -80\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SCV nutrient deficiency test\u003c/h2\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e strain SCV was inoculated into BHI broth and incubated overnight at 37\u0026deg;C with shaking at 200 rpm. The bacterial suspension was then adjusted to a turbidity equivalent to a 0.5 McFarland standard and spread onto tryptic soy agar (TSA) (HaiBo, Qingdao, China). The TSA plates were supplemented with 10 \u0026micro;L of either 5 mg/mL menadione, hemin, or thymidine (Solaibao, Beijing, China). All plates were incubated aerobically at 37\u0026deg;C for 24\u0026ndash;48 hours. In parallel, 3-1-S was streaked onto blood agar plates and incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e for 24\u0026ndash;48 hours to assess potential phenotypic reversion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Whole-Genome Sequencing\u003c/h2\u003e\u003cp\u003eFollowing revival, strains wild strain and SCV were subcultured in BHI broth for further propagation. Genomic DNA was extracted via the EasyPure Bacterial Genomic DNA Kit. The purity and concentration of the DNA samples were evaluated via 1% agarose gel electrophoresis. Qualified DNA samples were submitted to Shanghai Pisenno Biotechnology Co., Ltd. (Shanghai, China) for next-generation sequencing. A whole-genome shotgun (WGS) strategy was employed to construct libraries with insert sizes of 400 bp. These libraries were subjected to paired-end (PE) sequencing on an Illumina NovaSeq platform. The raw sequencing data were subjected to quality control assessment with FastQC, and the adapter sequences were trimmed via AdapterRemoval. Quality correction of the reads was performed via Kmer-based error correction implemented in SOAPec. De novo assembly of the Illumina data was carried out with SPAdes, followed by contig refinement and base correction via Pilon. Finally, genetic variants were annotated with ANNOVAR software, with a particular focus on mutations in genes associated with menaquinone biosynthesis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Growth Curve Measurements and Biochemical Characteristics\u003c/h2\u003e\u003cp\u003eStrains wild strain and SCV were inoculated onto mannitol salt agar (MSA) (HaiBo, Qingdao, China) and incubated at 37\u0026deg;C for 18\u0026ndash;24 hours. A single colony was selected from each strain and inoculated into tryptic soy broth (TSB) for cultivation. When the OD\u003csub\u003e600\u003c/sub\u003e reached 0.3\u0026ndash;0.5, the cultures were subcultured at a 1:200 ratio in fresh TSB. Growth curves were generated by measuring the OD\u003csub\u003e590\u003c/sub\u003e at 1-hour intervals during incubation at 37\u0026deg;C with shaking at 180 rpm.\u003c/p\u003e\u003cp\u003ePhysiological and biochemical characteristics, including protease activity, coagulase production via rabbit plasma, and mannitol fermentation, were assessed for both wild strain and SCV. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 was used as a positive control, and sterile physiological saline served as the negative control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Analysis of Mannitol metabolism-related gene expression levels\u003c/h2\u003e\u003cp\u003eThe strains were cultured in liquid BHI medium at 37\u0026deg;C for 24 hours. Total RNA was extracted from strains wild strain and SCV via an RNA extraction kit (Quanshijin, Beijing, China). The extracted RNA was subsequently reverse transcribed into cDNA via an EasyScript kit (Quanshijin, Beijing, China). The synthesized cDNA was used as the template for real-time quantitative PCR (qPCR) amplification. qPCR was performed under the following three-step cycling conditions: initial denaturation at 94\u0026deg;C for 30 seconds, followed by 40 cycles of denaturation at 94\u0026deg;C for 5 seconds, annealing at 50\u0026deg;C for 15 seconds, and extension at 72\u0026deg;C for 10 seconds. The fluorescence data were collected and analyzed at the end of the extension step of each cycle. For detailed primer sequences, see Supplementary Material 1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Scanning electron microscopy\u003c/h2\u003e\u003cp\u003eStrains wild strain and SCV were inoculated into TSB media and incubated at 37\u0026deg;C until they reached the plateau phase (26 hours). The cultures were centrifuged at 8,000 rpm for 2 minutes, and the resulting pellets were fixed with 2.5% glutaraldehyde for 24 hours. The fixed samples were subsequently submitted to the Instrumental Analysis Center of Tarim University for processing, which included supercritical drying and ion sputter coating. Morphological observations were carried out via a variable vacuum ultrahigh-resolution field emission scanning electron microscope equipped with an energy dispersive spectrometer (Thermo Fisher Scientific, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Drug resistance analysis\u003c/h2\u003e\u003cp\u003eThe minimum inhibitory concentrations (MICs) of gentamicin, enrofloxacin, kanamycin, vancomycin, streptomycin, paromomycin, and cefotaxime against strains 3\u0026thinsp;\u0026minus;\u0026thinsp;1 and 3-1-S were determined via the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The tested concentrations of antibiotics ranged from 0.125 \u0026micro;g/mL to 64 \u0026micro;g/mL. Mueller\u0026ndash;Hinton (MH) medium without bacterial inoculum was used as the negative control. All experiments were performed in triplicate, with \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 included as the quality control strain.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Biofilm formation capability testing\u003c/h2\u003e\u003cp\u003eThe biofilm-forming capacity of strains wild strain and SCV was evaluated semiquantitatively via a 96-well plate model. Activated bacterial cultures were diluted 1:100 in fresh TSB broth, either plain or supplemented with one of the following: sheep erythrocytes (1%), heme (0.5, 1.0, 2.5, 5.0, 10.0, or 20.0 \u0026micro;g/mL), or menaquinone (0.5, 1.0, 2.5, or 5.0 \u0026micro;g/mL). A 200 \u0026micro;L aliquot of each diluted suspension was transferred into individual wells of a 96-well plate. \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e ATCC 35984 was used as the positive control, and TSB medium alone served as the blank control. After 48 hours of incubation at 37\u0026deg;C, nonadherent cells were removed by washing with phosphate-buffered saline (PBS). The plates were air-dried, heat-fixed at 60\u0026deg;C for 30 minutes, and then stained with 1% crystal violet for 10 minutes. Following gentle rinsing and air drying, the bound dye was solubilized with an acetone/ethanol solution (1:4). The optical density at 570 nm (OD\u003csub\u003e570\u003c/sub\u003e) was measured via a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Hemolytic activity assay\u003c/h2\u003e\u003cp\u003eThe supernatant was collected from overnight bacterial cultures. A volume of 100 \u0026micro;L of the supernatant was mixed with 900 \u0026micro;L of PBS containing 3% sheep erythrocytes. The mixture was incubated at 37\u0026deg;C for 30 minutes and then centrifuged at 8000 rpm for 2 minutes. Two hundred microliters of the resulting supernatant was subsequently transferred to a 96-well plate. Hemolytic activity was assessed by measuring the optical density at 540 nm (OD\u003csub\u003e540\u003c/sub\u003e) via a microplate reader. The positive control consisted of 1000 \u0026micro;L of a solution containing 3% sheep erythrocytes in double-distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO), indicating complete hemolysis. The negative control contained 1000 \u0026micro;L of 3% sheep erythrocytes in PBS, indicating that there was no hemolysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Extracellular DNA, extracellular polysaccharide, and surface hydrophobicity detection\u003c/h2\u003e\u003cp\u003eeDNA was quantified according to the method described by Ivanov et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overnight cultures of strains 3\u0026thinsp;\u0026minus;\u0026thinsp;1 and 3-1-S were subcultured in TSB and grown to the mid-log phase. The cultures were diluted 1:100 in TSB supplemented with 2% glucose, aliquoted into a 96-well plate, and incubated at 37\u0026deg;C for 48 hours. For the treatment groups, heme (final concentration of 5 \u0026micro;g/mL) or menaquinone (final concentration of 2.5 \u0026micro;g/mL) was added to the culture medium of strain 3-1-S. After incubation, the culture broth was discarded, and the wells were washed twice with PBS. TE buffer was then added to each well, and the mixture was mixed vigorously. The mixtures were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 10,000 \u0026times; g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in TE buffer by vortexing. After a second centrifugation step at 10,000 \u0026times; g for 15 minutes, the absorbance of the resulting supernatant was measured at 260 nm.\u003c/p\u003e\u003cp\u003eAn overnight bacterial culture was inoculated at a 1:100 ratio into 50 mL of TSB medium. For the treatment group, either heme (5 \u0026micro;g/mL) or menaquinone (2.5 \u0026micro;g/mL) was added to the culture of strain SCV. The cultures were incubated at 37\u0026deg;C with shaking at 180 rpm for 24 hours. The cells were harvested via centrifugation at 6000 rpm for 15 minutes, and the supernatant was collected. Five volumes of 95% ethanol were added to the supernatant, and the mixture was precipitated at 4\u0026deg;C for 24 hours. The precipitate was collected by centrifugation at 10,000 rpm for 20 minutes. The resulting pellet was washed twice with 95% ethanol and air-dried at room temperature. The crude polysaccharide was dissolved in an appropriate volume of deionized water. A 1/10 volume of 20 mg/mL proteinase K was subsequently added, followed by incubation at 37\u0026deg;C for 2 hours. Then, a 1/4 volume of a chloroform:n-butanol mixture (5:1) was added, and the mixture was vortexed vigorously and centrifuged at 12,000 rpm and 4\u0026deg;C for 20 minutes. The aqueous layer was collected, transferred to a pretreated dialysis bag, and dialyzed overnight under running water. Finally, the sample was lyophilized under vacuum to obtain the extracellular polysaccharides.\u003c/p\u003e\u003cp\u003eThe overnight bacterial culture was inoculated into 50 mL of TSB medium at a 1:100 ratio. For the treatment groups, hemin or menadione was added to the SCV culture at final concentrations of 5 \u0026micro;g/mL and 2.5 \u0026micro;g/mL, respectively. The mixture was incubated at 37\u0026deg;C with shaking at 180 rpm for 24 hours. Bacterial cells were harvested by centrifugation at 6000 rpm for 15 minutes and washed twice with PUM buffer. Using PUM buffer as a blank, the absorbance of the bacterial suspension was measured at 600 nm (A\u003csub\u003e0\u003c/sub\u003e). The suspension was adjusted to an optical density between 0.4 and 0.6. Hexadecane was added to the diluted bacterial suspension at a 4:1 ratio, followed by vigorous mixing and incubation for 15 minutes to allow phase separation. The lower aqueous phase was carefully collected, and its absorbance at 600 nm (A\u003csub\u003e1\u003c/sub\u003e) was measured. The cell surface hydrophobicity was calculated as follows: surface hydrophobicity = (1\u0026thinsp;\u0026minus;\u0026thinsp;A\u003csub\u003e1\u003c/sub\u003e/A\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eData analysis was performed via the t test via GraphPad Prism v.10.0 software. A p value of less than 0.05 was considered statistically significant (*: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Gentamicin-induced acquisition of the menadione-dependent \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SCV\u003c/h2\u003e\u003cp\u003eIn this study, screening with five different concentrations of gentamicin (0.25, 0.5, 1, 2, and 4 \u0026micro;g/mL) led to the successful isolation of an SCV strain, designated 3\u0026thinsp;\u0026minus;\u0026thinsp;1, which remained stable after more than 10 successive passages. Compared with the wild-type strain, the SCV isolate exhibited slow growth, formed transparent, pinpoint-sized colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and was accordingly named 3-1-S. Nutritional deficiency assays revealed that phenotypic reversion occurred around filter paper discs impregnated with menadione, whereas no restoration of growth was observed around discs containing hemin chloride or thymidine or on blood agar plates. These results indicate that 3-1-S is a menadione-dependent SCV strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSequence analysis of key genes involved in menaquinone biosynthesis (\u003cem\u003emenA\u003c/em\u003e, \u003cem\u003emenB\u003c/em\u003e, \u003cem\u003emenC\u003c/em\u003e, \u003cem\u003emenD\u003c/em\u003e, \u003cem\u003emenE\u003c/em\u003e, \u003cem\u003emenF\u003c/em\u003e, \u003cem\u003emenG\u003c/em\u003e, and \u003cem\u003emenH\u003c/em\u003e) revealed a total of 61 mutation sites. These mutations were distributed across all genes except \u003cem\u003emenE\u003c/em\u003e. Spectrum analysis revealed that the majority of the mutations were synonymous (41 out of 61, 67.21%), which does not alter the encoded amino acid sequence. In addition, 20 nonsynonymous mutations were identified, leading to amino acid substitutions\u0026mdash;including 9 conservative and 10 nonconservative changes (see supplementary material 2). Notably, a significant mutation was detected in the \u003cem\u003emenE\u003c/em\u003e gene of strain 3-1-S: a 95-bp deletion starting at position 216. This deletion resulted in a frameshift mutation and the introduction of a premature stop codon (TAG) downstream of the deletion site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). It is predicted that this mutation leads to premature translational termination, likely yielding a truncated and dysfunctional MenE protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 SCV exhibits slow growth and altered biochemical characteristics\u003c/h2\u003e\u003cp\u003eBiochemical characterization revealed that both strains, 3\u0026thinsp;\u0026minus;\u0026thinsp;1 and 3-1-S, tested positive in the protease activity assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and the rabbit plasma coagulase test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The growth curve of strain 3\u0026thinsp;\u0026minus;\u0026thinsp;1 displayed typical bacterial kinetics, with a lag phase of approximately 4 hours followed by exponential growth, eventually reaching a stationary phase with an OD\u003csub\u003e600\u003c/sub\u003e between 2.0 and 2.5. In contrast, strain 3-1-S exhibited markedly impaired growth, lacked a distinct exponential phase and maintained an OD\u003csub\u003e600\u003c/sub\u003e below 0.5 throughout the incubation. Even after 30 h, its biomass remained significantly lower than that of strain 3\u0026thinsp;\u0026minus;\u0026thinsp;1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In the mannitol fermentation test, strain 3\u0026thinsp;\u0026minus;\u0026thinsp;1 utilized mannitol efficiently, whereas strain 3-1-S showed neither significant growth nor acid production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). qPCR analysis of genes within the mannitol operon revealed significant downregulation of mtlF and mtlD in the SCV strain compared with the wild-type strain, whereas the expression of \u003cem\u003emtlR\u003c/em\u003e was substantially upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results suggest that mtlR may act as a negative regulator and that the loss of mannitol fermentation capability in the SCV is likely due to altered expression of key metabolic genes. Scanning electron microscopy (SEM) revealed no notable morphological differences between strains 3\u0026thinsp;\u0026minus;\u0026thinsp;1 and 3-1-S (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 SCV exhibits enhanced resistance to aminoglycoside antibiotics\u003c/h2\u003e\u003cp\u003eIn this study, the broth microdilution method was used to evaluate the antimicrobial susceptibility of strains 3\u0026thinsp;\u0026minus;\u0026thinsp;1 and 3-1-S. The results revealed that the MIC values of enrofloxacin, ceftiofur, and vancomycin for strain 3-1-S were \u0026lt;\u0026thinsp;0.125, 1, and 0.25 \u0026micro;g/mL, respectively. In contrast, the MICs for several aminoglycoside antibiotics\u0026mdash;including gentamicin, kanamycin, and streptomycin\u0026mdash;reached 32 \u0026micro;g/mL or 64 \u0026micro;g/mL, indicating significantly elevated resistance compared with that of the wild-type strain 3\u0026thinsp;\u0026minus;\u0026thinsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Heme and menadione promote SCV biofilm formation\u003c/h2\u003e\u003cp\u003eCompared with that of strain 3\u0026thinsp;\u0026minus;\u0026thinsp;1, the biofilm-forming ability of 3-1-S was significantly reduced by 40.12% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), while its hemolytic activity increased markedly by 202.94% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe addition of 1% sheep erythrocytes to TSB significantly enhanced the biofilm formation of 3-1-S by 78.72% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, supplementation with different concentrations of haem also affected SCV biofilm formation. When cocultured with 5 \u0026micro;g/mL or 2.5 \u0026micro;g/mL haem, biofilm formation increased by 32.96% and 35.57%, respectively. However, heme concentrations below 2.5 \u0026micro;g/mL did not significantly promote biofilm formation, whereas concentrations exceeding 5 \u0026micro;g/mL resulted in inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eSupplementing the culture medium with menadione at final concentrations of 1 \u0026micro;g/mL and 2.5 \u0026micro;g/mL increased the biofilm-forming capacity of 3-1-S by 41.95% and 54.08%, respectively. In contrast, no significant effect was observed at menadione concentrations of 0.5 \u0026micro;g/mL or 5 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Heme and menaquinone regulate SCV biofilm formation by influencing extracellular polysaccharide levels and surface hydrophobicity\u003c/h2\u003e\u003cp\u003eMeasurements of eDNA, EPS, and surface hydrophobicity were conducted for strains 3\u0026thinsp;\u0026minus;\u0026thinsp;1, 3-1-S, and 3-1-S supplemented with 5 \u0026micro;g/mL heme or 2.5 \u0026micro;g/mL menaquinone. Compared with the wild-type strain, the 3-1-S strain presented significantly lower eDNA content, lower EPS production, and lower surface hydrophobicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Following supplementation with heme or menaquinone, no significant change in eDNA content was observed in 3-1-S (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, both EPS production and surface hydrophobicity increased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Notably, after the addition of menaquinone, the EPS content and surface hydrophobicity of 3-1-S were restored to levels comparable to those of the wild-type strain. These findings underscore the crucial role of menaquinone in regulating extracellular polysaccharide synthesis and modulating surface hydrophobicity in the SCV phenotype.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e can develop SCVs in response to antibiotics and environmental stress. This phenotypic switch is closely associated with persistent, recurrent, and drug-resistant infections, posing considerable challenges for clinical diagnosis and treatment. In the present study, a menadione-dependent SCV strain was isolated from \u003cem\u003eStaphylococcus aureus\u003c/em\u003e originating from a case of bovine mastitis through induction with gentamicin. Compared with the wild-type strain, the mutant strain displayed slow growth on blood agar plates, formed pinpoint-sized microcolonies, and exhibited a significantly reduced growth rate. These findings are consistent with previous reports by Alkasir and Atalla, who also isolated SCVs from cows with chronic mastitis, further confirming the ability of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e to form SCVs within the mastitis environment (Alkasir et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Atalla et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). As a key component of the electron transport chain, menadione undergoes isoprenoid side-chain extension to form menaquinone derivatives. These act as electron acceptors for NADH/FADH₂ and participate in the respiratory electron transport process (Melter \u0026amp; Radojevič, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Deficiency in menaquinone disrupts the electron transport chain, leading to a reduced proton motive force and consequently impairing ATP synthesis. Since bacterial cell wall biosynthesis is energy intensive, this energy deficit contributes to impaired growth and the formation of microcolonies. Furthermore, the electron transport chain is involved in the biosynthesis of carotenoid pigments. Its disruption can lead to the formation of nonpigmented colonies, reduced hemolytic activity, and a delayed coagulase reaction\u0026mdash;a phenomenon that is partly attributable to diminished amino acid synthesis (Melter \u0026amp; Radojevič, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Proctor et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Supplementation with nutrients deficient in growth medium has been shown to restore the SCV to a normal colony phenotype. Gene expression analysis of the mannitol metabolism operon in the SCV revealed significant downregulation of \u003cem\u003emtlF\u003c/em\u003e and \u003cem\u003emtlD\u003c/em\u003e, along with marked upregulation of \u003cem\u003emtlR\u003c/em\u003e. These genes encode the mannitol-specific transporter mannitol-1-phosphate dehydrogenase and the transcriptional repressor of the mannitol operon (Anbalagan et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As a negative regulator, MtlR suppresses the expression of genes within the mannitol utilization operon (Byer et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), thereby inhibiting the transcription of \u003cem\u003emtlF\u003c/em\u003e and \u003cem\u003emtlD\u003c/em\u003e. The reduced expression of these genes impedes both the uptake and metabolic conversion of mannitol, limiting the ability of the bacterium to utilize mannitol as a carbon and energy source. This metabolic impairment likely exacerbates the energy deficiency already present in the SCV phenotype.\u003c/p\u003e\u003cp\u003eThe genes involved in menaquinone biosynthesis belong to the \u003cem\u003emen\u003c/em\u003e operon, among which \u003cem\u003emenD\u003c/em\u003e and \u003cem\u003emenB\u003c/em\u003e play essential roles in this pathway (Kohler et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lannerg\u0026aring;rd et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Studies have shown that mutations in \u003cem\u003emenD\u003c/em\u003e disrupt the electron transport chain in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, impairing its ability to utilize oxygen or nitrate as terminal electron acceptors. These mutations also alter the expression of genes encoding enzymes involved in glycolysis, fermentation, and the tricarboxylic acid cycle (Kohler et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similarly, mutations in \u003cem\u003emenB\u003c/em\u003e have been directly linked to a menaquinone-deficient phenotype, affecting the bacterial growth rate, susceptibility to gentamicin, and hemolytic activity (Lannerg\u0026aring;rd et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Mutation analysis of eight genes within the \u003cem\u003emen\u003c/em\u003e operon revealed a notable 95-base pair deletion in \u003cem\u003emenE\u003c/em\u003e, which induces a frameshift and introduces a premature stop codon. This mutation is predicted to cause premature termination of translation, likely resulting in a truncated and nonfunctional MenE protein. MenE functions as an O-succinylbenzoyl-CoA synthase, catalyzing the adenylation and thioesterification of O-succinylbenzoic acid (OSB), a key intermediate in menaquinone biosynthesis (Matarlo et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This reaction converts OSB into OSB-CoA, which subsequently enters the menaquinone synthesis pathway (Lu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, severe mutations in \u003cem\u003emenE\u003c/em\u003e disrupt menaquinone production, leading to dysfunction of the electron transport chain, reduced ATP synthesis, and cellular energy depletion. These metabolic deficiencies ultimately contribute to the development of the SCV phenotype.\u003c/p\u003e\u003cp\u003eThe pronounced antibiotic resistance of SCVs is a major factor underlying their role in chronic and recurrent infections that are challenging to eradicate. In this study, the SCV strain demonstrated significantly increased resistance to aminoglycoside antibiotics compared with the wild-type strain, whereas no marked differences were observed in its response to β-lactams, quinolones, or glycopeptide antibiotics. The uptake of aminoglycosides is an energy-dependent process that relies on an intact membrane potential and active electron transport (von Eiff et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Menaquinone-deficient SCVs exhibit defects in their electron transport chain and respiratory activity, resulting in diminished ATP production and reduced membrane potential (Proctor et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Consequently, impaired energy metabolism and depolarized membranes in SCVs limit the intracellular accumulation of aminoglycosides, thereby increasing bacterial resistance. Although SCVs do not present elevated MICs for β-lactam or glycopeptide antibiotics, their slow growth rate and reduced cell wall biosynthesis may diminish the bactericidal effectiveness of these drug classes\u0026mdash;which target cell wall synthesis\u0026mdash;in actual infections (von Eiff et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Quinolones, on the other hand, enter bacterial cells primarily through passive diffusion or porin channels, a process that is not energy dependent (Hooper, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Thus, even with the reduced metabolic activity and membrane potential of SCVs, the uptake and activity of fluoroquinolones remain largely unaffected, explaining the unchanged susceptibility profile. In summary, SCVs exhibit a well-established mechanism of high-level resistance to aminoglycoside antibiotics. Although no increase in MIC was detected for other drug classes under the tested conditions, the overall antibiotic tolerance and reduced rate of killing\u0026mdash;attributable to the metabolic dormancy of SCVs\u0026mdash;contribute substantially to phenotypic resistance, treatment failure, and infection recurrence.\u003c/p\u003e\u003cp\u003eIn most reported studies, SCVs have been shown to enhance biofilm formation but attenuate virulence(Lee et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Interestingly, compared with the wild-type strain, the menadione-dependent SCV induced in this study presented significantly reduced biofilm-forming capacity but increased hemolytic activity. The synthesis of extracellular polysaccharides, release of eDNA, and maintenance of cell surface hydrophobicity\u0026mdash;all key components of the biofilm matrix\u0026mdash;are energy-demanding processes (Das \u0026amp; Manefield, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The impaired biofilm formation observed in the SCV may thus stem from its severe energy deficiency, which is consistent with the restoration of biofilm capacity upon menadione supplementation. As noted earlier, iron availability regulates biofilm development, and in vivo, iron is predominantly complexed within heme-containing proteins (Tong \u0026amp; Guo, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this study, the SCV strain presented increased hemolytic activity, suggesting an increased capacity to acquire iron from host erythrocytes. Similarly, the addition of red blood cells or exogenous heme significantly increased SCV biofilm formation. Further experiments confirmed that heme supplementation notably augmented both extracellular polysaccharide production and surface hydrophobicity in the SCV, underscoring its role in promoting biofilm assembly. Swarupa et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that iron supplementation substantially enhanced peptidyl deacetylase activity in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, thereby facilitating biofilm formation. Under anaerobic conditions, iron increased the biofilm biomass by approximately 15-fold. Moreover, when hemoglobin serves as the iron source, the resulting biofilms are thicker and more structurally complex than those promoted by inorganic iron salts (Dauros-Singorenko et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, notably, high concentrations of heme can inhibit biofilm formation (Torres et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), a concentration-dependent effect that aligns with the trend observed in the present study.\u003c/p\u003e\u003cp\u003eTherefore, we propose that, owing to its impaired biofilm formation and colonization capacity, the SCV may employ an alternative survival strategy by modulating the expression of virulence factors such as hemolysin. Enhanced hemolytic activity could facilitate the lysis of red blood cells, directly damaging host tissues and promoting bacterial dissemination and invasion. Concurrently, the nutrients released\u0026mdash;including hemoglobin and iron\u0026mdash;may serve as an energy source for the SCV, thereby partially compensating for its metabolic deficiencies.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, gentamicin-induced \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains causing subclinical mastitis in dairy cows yielded one \u003cem\u003emenE\u003c/em\u003e-mutant SCV strain exhibiting characteristics such as slow growth, reduced metabolic activity, enhanced drug resistance, weakened biofilm formation capacity, and increased hemolytic activity. Genomic analysis revealed that a mutation in the \u003cem\u003emenE\u003c/em\u003e gene disrupted the menadione synthesis pathway, likely serving as a key molecular basis for its metabolic defects and phenotypic variation. Heme may regulate SCV biofilm formation, potentially representing an effective survival strategy by altering metabolic pathways; however, the underlying regulatory mechanisms require further investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Engineering Laboratory of Tarim Animal Disease Diagnosis and Control, Xinjiang Production and Construction Corps (ELDC202002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecompeting interests\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Wu ([email protected]), Bin Yang ([email protected]), and Guanghua Liao ([email protected]) conceived and designed this study and wrote the manuscript. Bin Yang, Guanghua Liao, Guofeng Xing ([email protected]), and Zihao Wu ([email protected]) performed the experiments and collected and analyzed the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo animals were used in this study. Therefore, approval from an institutional animal ethics committee was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data are available in the Sequence Read Archive with the accession codes SAMN51698484 and SAMN51698485.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAftab H, Samudio J, Wang G, Le L, Soni RK, Donegan RK (2025) Heme alters biofilm formation in Mycobacterium abscessus. 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Microbiol Res 260:127040. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micres.2022.127040\u003c/span\u003e\u003cspan address=\"10.1016/j.micres.2022.127040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 SCV minimum inhibitory concentration results\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 33px;\"\u003e\n \u003cp\u003eAntibiotics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003eMIC（\u0026mu;g/mL）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e3-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e3-1-S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eStreptomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eBalongmycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026gt;64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eKanamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026gt;64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eEnrofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026lt;0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026lt;0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eCefotaxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003eVancomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"Staphylococcus aureus, SCV, biological characteristics, biofilm, hemolytic activity","lastPublishedDoi":"10.21203/rs.3.rs-7699109/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7699109/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile small colony variants (SCVs) of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e have been extensively documented, little is known about the properties of menadione-dependent SCVs from cow raw milk and how heme affects the ability to form biofilms. In this study, gentamicin-induced SCVs were generated from mastitis-derived \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and the underlying mechanisms were investigated through nutrient dependency assays combined with genomic analysis. A comparative assessment of biological characteristics\u0026mdash;including physiological and biochemical properties, antibiotic susceptibility, biofilm formation, and hemolytic activity\u0026mdash;was conducted between the SCV and wild-type strains. Additionally, the effects of exogenous heme and menadione on biofilm formation were evaluated. The results revealed a frameshift deletion mutation due to a 95-nucleotide deletion in the \u003cem\u003emenE\u003c/em\u003e gene. This mutation altered the corresponding amino acid sequence, which is likely the principal reason for the menadione-dependent SCV phenotype. The SCV strain presented not only typical characteristics, such as slow growth and increased resistance to aminoglycoside antibiotics, but also atypical ones, inculding significantly decreased biofilm formation capacity and increased hemolytic activity. Exogenous supplementation with menadione or heme markedly promoted biofilm formation in the SCV strain. These results suggesting that the SCV strain reduced biofilm-forming ability may stem from compromised metabolic activity. Moreover, the SCV strain potentially enabling the acquisition of exogenous heme via unidentified pathways, thereby restoring biofilm formation capacity. The regulatory mechanisms involved require further investigation. These findings provide new insights into the biofilm regulation and survival/pathogenicity mechanisms of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SCVs.\u003c/p\u003e","manuscriptTitle":"Biological Characteristics of a Menadione-dependent Staphylococcus aureus Small Colony Variant (SCV) from Bovine Mastitis and Heme Promotion of Biofilm Formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 15:55:00","doi":"10.21203/rs.3.rs-7699109/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":"82968b55-d0c8-4ef5-bada-92be6049605e","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:01:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-14 15:55:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7699109","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7699109","identity":"rs-7699109","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}