Biofilm最新文献

{"title":"Comparative transcriptomics analysis of the Oleidesulfovibrio alaskensis G20 biofilms grown on copper and polycarbonate surfaces","authors":"Priya Saxena ,&nbsp;Dipayan Samanta ,&nbsp;Payal Thakur ,&nbsp;Vinoj Gopalakrishnan ,&nbsp;Rajesh K. Sani","doi":"10.1016/j.bioflm.2025.100309","DOIUrl":"10.1016/j.bioflm.2025.100309","url":null,"abstract":"<div><div>Sulfate-reducing bacterial (SRB) biofilms are prevalent across diverse environments, playing key roles in biogeochemical sulfur cycling while also contributing to industrial challenges such as biofouling and biocorrosion. Understanding the genetic and physiological adaptations of SRB biofilms to different surfaces is crucial for developing mitigation strategies. This study presents a comparative transcriptomic analysis of <em>Oleidesulfovibrio alaskensis</em> G20 biofilms grown on copper and polycarbonate surfaces, aimed at elucidating their differential responses at the molecular level. RNA sequencing revealed 1255 differentially expressed genes, with copper-grown biofilms exhibiting upregulation of Dde_1570 (flagellin; log2FC 2.31) and Dde_0831 (polysaccharide chain length determinant; log2FC 1.15), highlighting enhanced motility and extracellular polymeric substance production. Conversely, downregulated genes on copper included Dde_0132 (Cu/Zn efflux transporter; log2FC −3.37) and Dde_0369 (methyl-accepting chemotaxis protein; log2FC −1.19), indicating a metabolic shift and stress adaptation to metal exposure. Morphological analysis via SEM revealed denser biofilm clusters with precipitates on copper, whereas biofilms on polycarbonate were more dispersed. AFM analysis showed a 4.6-fold increase in roughness on copper (44.3 ± 3.1 to 205.89 ± 8.7 nm) and a 3.8-fold increase on polycarbonate (521.12 ± 15.2 to 1975.64 ± 52.6 nm), indicating surface erosion and structural modifications. Protein-protein interaction analysis identified tightly regulated clusters associated with ribosomal synthesis, folate metabolism, and quorum sensing, underscoring their role in biofilm resilience. Additionally, functional annotations of uncharacterized genes revealed potential biofilm regulators, such as Dde_4025 (cytochrome-like protein; log2FC 4.18) and Dde_3288 (DMT superfamily permease; log2FC 3.55). These findings provide mechanistic insights into surface-dependent biofilm formation, with implications for designing antifouling materials and controlling microbial-induced corrosion.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100309"},"PeriodicalIF":4.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144831276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Linoleic acid addition prevents Staphylococcus aureus biofilm formation on PMMA bone cement","authors":"Linglu Hong ,&nbsp;Karin Hjort ,&nbsp;Dan I. Andersson ,&nbsp;Cecilia Persson","doi":"10.1016/j.bioflm.2025.100311","DOIUrl":"10.1016/j.bioflm.2025.100311","url":null,"abstract":"<div><div>Acrylic bone cement is widely used in vertebroplasty to treat osteoporosis-induced vertebral compression fractures. However, infection after vertebroplasty is problematic and previous work has suggested loading the bone cement with an antibiotic for prophylaxis. Linoleic acid (LA) has been investigated as a promising additive to improve the mechanical properties of bone cement for vertebroplasty, but LA could potentially also have an antibacterial effect. In this study, we evaluated the antibacterial properties of LA-loaded bone cement by comparing its antibiofilm properties with that of original bone cement through quantification of bacterial growth using viable cell count and scanning electron microscopy. The released monomer (MMA) concentration and the monomer minimum inhibitory concentration were determined to clarify the monomer's potential role in inhibiting bacterial growth. The LA release profile was measured, and a checkerboard assay was done to determine any synergistic effects of LA and the commonly used antibiotic gentamicin. Results show that LA-loaded bone cement could significantly inhibit <em>Staphylococcus aureus</em> biofilm formation, including gentamicin-resistant strains, but with limited effect on <em>Escherichia coli</em>. Furthermore, the released MMA did not have a significant influence on bacterial growth. The checkerboard assay results show that the LA and gentamicin combination could broaden the antibacterial spectrum and increase gentamicin efficacy. In conclusion, LA merits further investigation as an antibacterial agent in bone cement, alone or in combination with antibiotics.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100311"},"PeriodicalIF":4.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144809573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"S. aureus biofilm disruption using ultrasound and microbubbles: Influence of radiation force, bubble dynamics and biofilm growth conditions","authors":"Damien V.B. Batchelor ,&nbsp;Anjali Lad ,&nbsp;Kathryn L. Burr ,&nbsp;Kristian Hollie ,&nbsp;James R. McLaughlan ,&nbsp;W. Bruce Turnbull ,&nbsp;Jonathan A.T. Sandoe ,&nbsp;Stephen D. Evans","doi":"10.1016/j.bioflm.2025.100327","DOIUrl":"10.1016/j.bioflm.2025.100327","url":null,"abstract":"<div><div><em>Staphylococcus aureus</em> is a human pathogen and a major cause of bloodstream infections, which can readily form biofilms on implanted medical devices. Here, we utilise a combination of lipid-shelled microbubbles (MBs) and ultrasound (US) to physically disperse the biofilm from the growth surface. The effects of two peak negative pressures (PNPs) and the direction of the acoustic radiation force (ARF) were evaluated. At 1.1 MHz, a clinically relevant frequency, and low PNP of 360 kPa, no significant biofilm dispersal occurred regardless of ultrasound (US) orientation. In contrast, at a high PNP of 2500 kPa, directing the ultrasound beam upward (US↑) pushed microbubbles (MBs) toward the biofilm, resulting in near-complete dispersal of the biofilm (94 ± 2 %) within the focal zone. Reversing direction to US↓, which pushes MBs away from the biofilm, reduced biofilm dispersal to 81 ± 3 %. Pre-treatment of the biofilm growth surface with fibrinogen or human plasma significantly altered the biofilm morphology and thickness, but did not affect the efficiency of ultrasound and microbubbles (US + MB)-mediated dispersal. Furthermore, multiple consecutive US + MB treatments could be applied to treat larger areas of biofilm without requiring MB replenishment between treatments. High-speed imaging was used to observe MB behaviour (e.g. translation and destruction) during US exposure. We revealed that the near instantaneous destruction of smaller MBs (∼1 μm) at high pressure did not induce significant biofilm dispersal and hypothesise that the translational motion of larger MBs (&gt;10 μm) across the surface of the biofilm was the dominant mechanism behind biofilm dispersal.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100327"},"PeriodicalIF":4.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145519283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of Staphylococcus lugdunensis biofilm reveals key differences according to clonal lineage and iron availability","authors":"Laurie Destruel ,&nbsp;Sandrine Dahyot ,&nbsp;Laurent Coquet ,&nbsp;Magalie Barreau ,&nbsp;Stéphanie Legris ,&nbsp;Marie Leoz ,&nbsp;Maxime Grand ,&nbsp;Xavier Argemi ,&nbsp;Gilles Prevost ,&nbsp;Nicolas Nalpas ,&nbsp;Emmanuelle Dé ,&nbsp;Sylvie Chevalier ,&nbsp;Martine Pestel-Caron","doi":"10.1016/j.bioflm.2025.100329","DOIUrl":"10.1016/j.bioflm.2025.100329","url":null,"abstract":"<div><div>To understand the mechanisms involved in the evolutionary success of <em>Staphyloccocus lugdunensis</em> clones, we compared the biofilm-forming ability of representative strains of the seven clonal complexes (CCs) in rich and iron-restricted conditions, and characterized the extracellular matrix (ECM) of two highly biofilm-forming strains under each condition.</div><div>Over 90 % of the 49 <em>S. lugdunensis</em> strains produced biofilm in both conditions, with a level of production depending on the iron availability and clonal lineage. Two behaviors were observed: a significantly higher production in rich medium than in iron-restricted medium for CC1, CC2, and some CC3 strains, and the opposite phenomenon for CC6 ones. Analysis of the ECM of two representative strains using confocal microscopy showed that biofilm of the CC3 strain in rich medium contained similar amounts of proteins, eDNA and polysaccharides while that of CC6 strain was predominantly proteinaceous. Under iron-restricted conditions, biofilm structure and composition of both strains completely differed from those obtained in rich conditions. The proteomic analysis of their biofilm ECM by liquid chromatography coupled to tandem mass spectrometry identified 321 proteins common to both strains, mainly intracellular and in particular ribosomal. Of note, 202 proteins differed between the strains in terms of abundance, with a higher proportion of membrane proteins in the CC3 strain.</div><div>This study performed on a large cohort of strains shows that <em>S. lugdunensis</em> biofilm-forming capacity is strongly associated with CC and iron availability. This analysis of biofilm-associated proteins in <em>S. lugdunensis</em> opens the way to propose new molecular targets for anti-biofilm strategies.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100329"},"PeriodicalIF":4.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145464880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unfolding the collective functional potential of a synergistic multispecies community through genotypic and phenotypic analyses","authors":"Dana Ronin ,&nbsp;Mads Frederik Hansen ,&nbsp;Maximilian Lukas Flaig ,&nbsp;Morten Kam Dahl Dueholm ,&nbsp;Anders Ogechi Hostrup Daugberg ,&nbsp;Joseph Nesme ,&nbsp;Witold Kot ,&nbsp;Mette Burmølle","doi":"10.1016/j.bioflm.2025.100290","DOIUrl":"10.1016/j.bioflm.2025.100290","url":null,"abstract":"<div><div>By studying model multispecies biofilm systems, we can further our knowledge regarding why some properties emerge solely in a multispecies setting. In this study, the model system under investigation is composed of four bacterial species: <em>Paenibacillus amylolyticus</em>, <em>Microbacterium oxydans</em>, <em>Stenotrophomonas rhizophila</em> and <em>Stenotrophomonas maltophilia</em>. This community was isolated from soil and has previously shown synergistic biofilm formation capabilities in vitro, along with other intrinsic properties, some of which could lead to potential industrial and agricultural applications. In this study, we conducted the first complete genome assemblies for these four strains and performed a manually curated annotation of the genomes to identify genomic features that could guide the selection of relevant phenotypic assays. In all four strains, we identified genes linked to interspecies communication, biofilm formation, secondary metabolite production, antibiotic resistance, enzymatic activity and metabolism of toxic xenobiotics. With metabolism being the largest gene function category identified, we then conducted growth assays on various carbon sources and relevant polysaccharides. This revealed interesting emergent behaviors - regarding growth and enzymatic activity - in the four-species community which were not seen in the monocultures. Overall, analysis of the complete genomes of this model community uncovered gene functions which could play a role in the previously observed community intrinsic properties, as well as provided insight to the positive social interactions observed in vitro.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100290"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144480342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum to “Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle” [Biofilm 7 100196]","authors":"Ana Barbosa ,&nbsp;Nuno F. Azevedo ,&nbsp;Darla M. Goeres ,&nbsp;Laura Cerqueira","doi":"10.1016/j.bioflm.2025.100291","DOIUrl":"10.1016/j.bioflm.2025.100291","url":null,"abstract":"","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100291"},"PeriodicalIF":4.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145736089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacillus-derived lipopeptides inhibit and eradicate Candida tropicalis Biofilms: An in vitro exploration","authors":"Madhuri Madduri ,&nbsp;Shivaprakash M Rudramurthy ,&nbsp;Utpal Roy","doi":"10.1016/j.bioflm.2025.100294","DOIUrl":"10.1016/j.bioflm.2025.100294","url":null,"abstract":"<div><div><em>Candida tropicalis</em> is a WHO-prioritised opportunistic fungal pathogen capable of causing superficial-to-systemic fungal infections and forming biofilms on both biotic and abiotic surfaces, such as tissues and medical implants. The rising incidence of fungal infections and the emergence of drug resistance underscore the urgent need for alternative, natural therapeutic agents such as antifungal macromolecules. AF₄ and AF₅, natural antifungal lipopeptide homologs derived from the <em>Bacillus subtilis</em> RLID strain, were purified to homogeneity from the cell free supernatant using semi-preparative scale HPLC with a C18 column at the final step. Using these lipopeptide fractions AF₄ and AF₅, the <em>anti</em>-biofilm efficacies were evaluated against <em>C. tropicalis</em> ATCC 750 biofilms and their synergistic potential with the standard antifungal fluconazole. Two compounds with amphiphilic nature were highly effective in preventing adhesion and inhibiting the formation of biofilm within 6 h. Additionally, they eradicated 24 h mature biofilms, as were demonstrated through various biofilm quantification techniques, including the crystal violet assay, XTT reduction assay, quantification of EPS, and ATR-FTIR analysis of biochemical composition of treated biofilms. <em>In vitro</em> studies revealed that AF₄ and AF₅ significantly reduced biofilm biomass and metabolic activity of cells embedded within the biofilm. Furthermore, morphological analysis of biofilms stained with Con A and FUN-1 using CLSM and COMSTAT analyses demonstrated a strong antibiofilm effect, including damaged cell surfaces and reduced hyphae structures, indicating effective biofilm disruption. Combinations of fluconazole with AF₅ exhibited improved synergistic <em>anti</em>-biofilm effects. However, AF₄ alone demonstrated strong potential to combat all phases of <em>C. tropicalis</em> biofilms.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100294"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144502209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spatial structure of yeast biofilms and the role of cell adhesion across different media","authors":"Vichi Sicha Irianto ,&nbsp;Vítězslav Plocek ,&nbsp;Rashim Bharti ,&nbsp;Jana Maršíková ,&nbsp;Libuše Váchová ,&nbsp;Zdena Palková","doi":"10.1016/j.bioflm.2025.100306","DOIUrl":"10.1016/j.bioflm.2025.100306","url":null,"abstract":"<div><div>The ability of yeast cells to adhere to solid surfaces or even penetrate semi-solid surfaces and form multicellular biofilms are critical factors in infection. This study examines the relationship between cell adhesion capability and the ability to create spatially organized biofilms in selected <em>Saccharomyces cerevisiae</em> strains, including clinical isolates, and five <em>Candida</em> species (<em>C. albicans</em>, <em>C. glabrata</em>, <em>C. krusei</em>, <em>C. parapsilosis</em>, and <em>C. tropicalis</em>). We assessed cell adhesion to polystyrene surface in four media varying in source of carbon and other nutrients. Using microscopy of vertical cell arrangement profiles within yeast populations grown at the solid-liquid interface, we evaluated their internal organization to determine whether the populations exhibit typical biofilm characteristics, such as the spatial organization of distinct cell types. Results indicate that well adherent <em>S. cerevisiae</em> strains form spatial biofilms with typical internal organization, highlighting strain-specific responses to media composition and supporting the use of natural <em>S. cerevisiae</em> strains for biofilm research. Among <em>Candida</em> species, biofilm formation did not consistently align with adhesion efficiency to plastic; while <em>C. albicans</em> and <em>C. krusei</em> formed spatially structured biofilms on media where they adhered well, <em>C. tropicalis</em> and <em>C. glabrata</em> exhibited efficient adhesion without biofilm structuring. Interestingly, <em>C. parapsilosis</em> formed a structured biofilm despite minimal adhesion. These findings emphasize the role of media composition, particularly components of yeast extract and defined medium for mammalian cell growth RPMI, which differentially impacted adhesion and biofilm formation in <em>S. cerevisiae</em> and <em>C. albicans</em>.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100306"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144663356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accurate quantitation of antibiotic penetration through staphylococcal biofilms","authors":"Lou Bin ,&nbsp;David McGiffin ,&nbsp;Thuy Nguyen ,&nbsp;Lv Wang ,&nbsp;Yao Sun ,&nbsp;Lumei Ye ,&nbsp;Meiling Han ,&nbsp;Chengju Sheng ,&nbsp;Tzong-Hsien Lee ,&nbsp;Marie-Isabel Aguilar ,&nbsp;Anton Y. Peleg ,&nbsp;Yue Qu","doi":"10.1016/j.bioflm.2025.100316","DOIUrl":"10.1016/j.bioflm.2025.100316","url":null,"abstract":"<div><h3>Objectives</h3><div>Limited antimicrobial penetration is an important mechanism underlying antimicrobial resistance of biofilms and has often been incorrectly quantitated. We adopted a rationalized antibiotic agar-diffusion model to accurately interpret experimental results of a widely-accepted biofilm penetration assay, and to determine drug-related physiochemical properties impacting antimicrobial biofilm penetration.</div></div><div><h3>Methods</h3><div>Staphylococcal reference strains and eight conventional antibiotics were selected for this study. A well-established biofilm penetration assay based on disk diffusion and colony biofilms was used. Sizes of the zone of inhibition (ZOI) were converted to concentrations of antibiotics, using linear regressions of squared radii of the ZOI on the natural logarithm of antibiotic concentrations. Biofilm penetration ratios were calculated by comparing concentrations of antibiotics reaching the agar surface with or without biofilm barriers. Multiple regression analysis was performed to assess the impact of antibiotic physicochemical properties, such as surface charge, on their biofilm penetration.</div></div><div><h3>Results</h3><div>Ciprofloxacin and oxacillin showed great capacities in penetrating staphylococcal biofilms. Rifampicin penetrated biofilms at low rates of ∼20 %. Aminoglycosides showed strain- and agent-specific penetration ratios, with tobramycin showing the least penetration (17.8 % for <em>Staphylococcus aureus</em> and 35.6 % for <em>Staphylococcus epidermidis</em>) and kanamycin presenting good penetration (∼82.3 %) against <em>S. aureus</em> biofilms. Surface charges of antibiotics at neutral and acidic conditions were important for their biofilm penetration.</div></div><div><h3>Conclusions</h3><div>Accurate quantitation of antibiotic biofilm penetration can be achieved using the transformed linear regression between the ZOI and antibiotic concentrations. Mathematical evidence was provided to support the importance of surface charge of antimicrobials on their biofilm penetration.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100316"},"PeriodicalIF":4.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145048575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correlative Imaging and super resolution microscopy studies reveal complexities in determining live-dead state of bacteria","authors":"Jiaqi Luo ,&nbsp;Rasmita Raval","doi":"10.1016/j.bioflm.2025.100302","DOIUrl":"10.1016/j.bioflm.2025.100302","url":null,"abstract":"<div><div>Imaging techniques are widely used to determine the physiological state of bacterial cells and provide an important platform for antibacterial evaluation in biofilms research. The commercial counter-staining SYTO 9 – propidium iodine kit is a popular choice for viability studies, with cell membrane damage due to antimicrobial action leading to replacement of the SYTO 9 dye with propidium iodine. This study investigates the live-dead state of cells in early-stage <em>Staphylococcus aureus</em> biofilms using correlative Fluorescence Microscopy (FM), Scanning Electron Microscopy (SEM) and super resolution Structural Illumination Microscopy (SIM). Correlative imaging data obtained at the single-cell level show that the physiological states of individual bacterial cells indicated by the SYTO 9 – propidium iodine counterstain in FM does not correlate directly with the detailed cell morphology observed by SEM. In addition, SIM was used to map sub-cellular distributions of SYTO 9 – propidium iodine dyes within single cells and revealed greater complexity than hitherto assumed, with 4 different cell-states identified, including double-stained ones and those where SYTO-9 is bound to substances at the cell perimeter. With this knowledge, we present ternary plots to illustrate the significant impacts of this complex staining behaviour on underestimation of cell-membrane damage due to antimicrobial actions and, thus, overestimation of bacterial survival rate in biofilms research.</div></div>","PeriodicalId":55844,"journal":{"name":"Biofilm","volume":"10 ","pages":"Article 100302"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144654377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}