Biofilm

Polymicrobial infection with Candida albicans and Staphylococcus aureus may result in a concomitant increase in virulence and resistance to antimicrobial drugs. This enhanced pathogenicity phenotype is mediated by numerous factors including metabolic processes and direct interaction of S. aureus with C. albicans hyphae. The overall structure of biofilms is known to contribute to their recalcitrance to treatment, however the dynamics of direct interaction between species and how it contributes to pathogenicity is poorly understood. To address this, a novel time-lapse mesoscopic optical imaging method was developed to enable the formation of C. albicans/S. aureus whole dual-species biofilms to be followed. It was found that yeast-form or hyphal-form C. albicans in the biofilm founder-population profoundly affects the structure of the biofilm as it matures. Different sub-populations of C. albicans and S. aureus arise within each biofilm as a result of the different C. albicans morphotypes, resulting in distinct sub-regions. These data reveal that C. albicans cell morphology is pivotal in the development of global biofilm architecture and the emergence of colony macrostructures and may temporally influence synergy in infection.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances (EPS) and other components such as proteins. Matrix components can be produced by the microorganisms themselves but can also originate from the environment and then be incorporated into the biofilm. For example, we and our collaborators have recently shown that collagen, a host-produced protein that is abundant in many different infection sites, can be taken up into the matrices of Pseudomonas aeruginosa biofilms, altering biofilm mechanics. In an infection, the biofilm matrix protects bacteria from clearance by the immune system, and some of that protection likely arises from the mechanical properties of the biofilm. P. aeruginosa, Staphylococcus aureus, and Burkholderia pseudomallei are human pathogens notable for forming biofilms in vitro and in vivo in tissues rich in collagen such as lung and skin. Here, we show that the incorporation of Type I collagen into P. aeruginosa, S. aureus, and B. pseudomallei biofilms significantly enhances biofilm elasticity and hinders phagocytosis of biofilm bacteria by human neutrophils. Additionally, enzymatic degradation of collagen using collagenase reverses these effects, increasing biofilm susceptibility to neutrophils. Our findings suggest that host materials play significant roles in stabilizing biofilms and may present promising targets for therapeutic interventions.

Biofilms are a cause of chronic, non-healing infections. Staphylococcus aureus is a proficient biofilm forming pathogen commonly isolated from prosthetic joint infections that develop following primary arthroplasty. Extracellular adhesion protein (Eap), previously characterized in planktonic or non-biofilm populations as being an adhesin and immune evasion factor, was recently identified in the exoproteome of S. aureus biofilms. This work demonstrates that Eap and its two functionally orphaned homologs EapH1 and EapH2, contribute to biofilm structure and prevent macrophage invasion and phagocytosis into these communities. Biofilms unable to express Eap proteins demonstrated increased porosity and reduced biomass. We describe a role for Eap proteins in vivo using a mouse model of S. aureus prosthetic joint infection. Results suggest that the protection conferred to biofilms by Eap proteins is a function of biofilm structural stability that interferes with the leukocyte response to biofilm-associated bacteria.

Healthcare-associated infections (HAIs) significantly contribute to the burden of antimicrobial resistance (AMR). A major factor in HAIs is the colonisation of indwelling medical devices by biofilm-forming opportunistic pathogens such as Candida albicans and Staphylococcus aureus. These organisms frequently co-infect, resulting in synergistic interactions with enhanced virulence and resistance to treatment. C. albicans and S. aureus readily form dual-species biofilms on silicone elastomers, a commonly used medical device material, yet the colonisation phenotypes of these organisms on such surfaces remains poorly understood. We developed a simple, optically tractable model to mimic the colonisation of indwelling medical devices to investigate C. albicans and S. aureus biofilm formation. The system utilises discs of a silicone elastomer embedded in agar, reflecting device-associated conditions and enabling high-resolution imaging of biofilms formed by C. albicans and S. aureus co-culture. Initial results using the silicone elastomer colonisation model reveal robust biofilm formation. These biofilms exhibited morphological differences between dual species biofilms formed by S. aureus co-cultures with either yeast- or hyphal-form C. albicans, indicating the impact of differing C. albicans cell morphotypes in biofilm-associated medical device colonization on silicone elastomers. Quantification of biofilm formation by crystal violet staining provided further validation of the system. These findings underscore the importance of developing tools for biofilm study which more closely resemble the infectious microenvironment, with our work detailing such a system which can be employed in further study to improve strategies against device-related HAIs.

Recent mesoscopic characterisation of nutrient-transporting channels in E. coli has allowed the identification and measurement of individual channels in whole mature biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of biofilms formed by cell shape mutants of E. coli shows that channels have a high fractal complexity, regardless of cell phenotype or growth medium. In particular, biofilms formed by the mutant strain {Delta}ompR, which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentration in the medium. Osmotic stress leads to a dramatic reduction in {Delta}ompR cell size, but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli biofilms.

Defined as a pneumonia occurring after more than 48 hours of mechanical ventilation via an endotracheal tube, ventilator-associated pneumonia results from biofilm formation on the indwelling tube, seeding the patients lower airways with pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Currently there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates biofilm formation of these pathogens in a host-mimicking environment, and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilator airway mucus (SVAM) medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-SEM were employed to characterise the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication, or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved biofilm eradication of all pathogens. Our in vitro endotracheal tube (IVETT) model informs on fundamental microbiology in the ventilator-associated pneumonia context, and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants. ImportanceThe incidence of ventilator-associated pneumonia in mechanically ventilated patients is between 5-40%, increasing to 50-80% in patients suffering from coronavirus disease 2019 (COVID-19). The mortality rate of ventilator-associated pneumonia patients can reach 45%. Treatment of the endotracheal tube biofilms that cause ventilator-associated pneumonia is extremely challenging, with causative organisms able to persist in endotracheal tube biofilm despite appropriate antimicrobial treatment in 56% of ventilator-associated pneumonia patients. Flawed antimicrobial susceptibility testing often means that ventilator-associated pneumonia pathogens are insufficiently treated, resulting in patients experiencing ventilator-associated pneumonia recurrence. Here we present an in vitro endotracheal tube biofilm model that recapitulates key aspects of endotracheal tube biofilms, including dense biofilm growth and elevated antimicrobial tolerance. Thus our biofilm model can be used as a ventilated airway simulating environment, aiding the development of anti-ventilator-associated pneumonia therapies and antimicrobial endotracheal tubes that can one day improve the clinical outcomes of mechanically ventilated patients.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in non-trivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/563605v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@126dc6aorg.highwire.dtl.DTLVardef@50dab8org.highwire.dtl.DTLVardef@477fd6org.highwire.dtl.DTLVardef@19e8e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Implant-associated biofilm infections, particularly those caused by Staphylococcus aureus and Enterococcus faecalis, present significant challenges in clinical settings, often necessitating surgical removal. This study investigates the potential of the invertebrate model Galleria mellonella for evaluating biofilm formation of S. aureus and E. faecalis clinical isolates, the leading causes of implant-associated infections. Utilizing expanded polytetrafluoroethylene (ePTFE) sutures as a surrogate for cardiac implants, we employed two biofilm formation methodologies reflecting the two main routes of implant infections: in vivo biofilm development within larvae mimicking hematogenous spread and pre-formed biofilm transplantation mimicking contamination during surgery. Scanning electron microscopy revealed complex biofilm structures for the biofilm formed inside of the larvae on implant, closely mimicking clinical conditions. Antibiotic treatments with vancomycin and rifampicin demonstrated significant reductions in bacterial biofilms, proving highly effective. The study highlights the G. mellonella models potential for preclinical biofilm research, offering a cost-effective and ethical alternative to vertebrate models while providing valuable insights into biofilm-related infections and their treatment.

Catheter-associated urinary tract infection (CAUTI) is one of the most common healthcare-associated infections and biofilm formation plays a key role in its pathogenesis. Indwelling medical devices introduce ideal pathways inside the body for invading pathogens and feature surfaces conducive to biofilm development. These devices are the root cause of severe clinical infections often recalcitrant to antimicrobials. When bacteria and fungi switch to biofilm mode of growth, they produce a matrix in the form of extracellular polymeric substances (EPS). This creates a unique environment for growing virulent colonizers and persisting cells while also forming a shielding barrier against immune system attacks, antimicrobial agents and mechanical removal by fluid shear forces. To address this challenge, LubriShield TM - a novel permanent coating was invented, and evenly applied to both the internal and external surfaces of indwelling urinary Foley catheters. Without releasing active substances, it effectively prevented pathogens from producing biofilm. The coating was superhydrophilic and incorporated a proprietary anti-fouling ligand, which created a surface that significantly inhibited up to 99% of colonizing uropathogens from forming biofilm for the duration of use without any microbial killing (p< 0.001). The predominant uropathogens, including Gram-positive and Gram-negative as well as Candida albicans, were inhibited from forming biofilm on the LubriShieldTM coated surfaces for up to 14 days in artificial urine medium. After challenging the adhering bacteria in a glass bladder flow model, the coating still significantly reduced biofilm formation by 83% (p<0.001). The growth mode and emergent properties of adhering bacteria on uncoated silicone catheter surfaces were compared with those on LubriShieldTM coated surfaces. RNA-seq analysis revealed that gene expression associated with microbial EPS formation was significantly downregulated on the coated surfaces. Additionally, microorganisms adhering to LubriShieldTM coated catheters were 46% more susceptible to antibiotics compared to those on uncoated silicone catheters (p<0.01).

Pseudomonas aeruginosa is a pathogen notorious for its antimicrobial resistance and is currently classified as a high-priority pathogen for which new drugs are needed. Tetrasodium EDTA (tEDTA) is one of the new antimicrobial compounds that have been shown to have good antibacterial and antibiofilm efficacy against P. aeruginosa. Due to the diversity and highly drug-tolerant nature of P. aeruginosa biofilms in different infection environments, it is important to carry out pre-clinical testing of new antibiofilm agents against this pathogen in media and models that accurately mimic diverse infection environments. In this study, we used different high validity media and biofilm models that mimic chronic wounds, endotracheal tubes, and cystic fibrosis lung infections to assess the efficacy of tEDTA against P. aeruginosa biofilms. We report that different infection environments influence the susceptibility of both planktonic and biofilm forms of P. aeruginosa to tEDTA. The highest tolerance to tEDTA was observed in the media and biofilm model that mimics the endotracheal tube environment. In conclusion, we show that although different infection environments influence the efficacy of tEDTA against P. aeruginosa biofilms, it has good potential for use as an alternative antimicrobial in treating P. aeruginosa-associated biofilm infections.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA. MRSA cells also secrete micrococcal nuclease (Nuc1) which degrades double-stranded DNA. In this study we used a nuc1 mutant strain to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. Biofilm was quantitated in microplates using a crystal violet binding assay. Extracellular DNA (eDNA) was isolated from colony biofilms and analyzed by agarose gel electrophoresis. In some experiments, broth or agar was supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods, transferring the rods to fresh broth, and enumerating CFUs that detached from the rods. Biofilm sloughing was investigated by culturing biofilms in glass tubes perfused with broth and measuring the sizes of the detached cell aggregates. We found that a nuc1 mutant strain produced significantly more biofilm and more eDNA than a wild-type strain in both the absence and presence of sub-MIC amoxicillin. nuc1 mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or a wild-type nuc1 gene on a plasmid. In the sloughing assay, nuc1 mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.

Biofilms pose a significant challenge in medical settings, leading to persistent infections. Phage therapy has shown promise in biofilm eradication, but its effectiveness under dynamic flow conditions remains unclear. Here we use two novel phages isolated on Klebsiella, Llofrudd and Samara, and characterized their genomes, host range and virulence. In this study, we built a simple catheterised bladder model with flow to investigate the impact of phage treatment on biofilm viability in a flow-based catheter model. Our analyses demonstrate that phages Llofrudd and Samara are the same species and infect a limited number of strains (3/222), but across three species: Klebsiella aerogenes, Klebsiella pneumoniae and E. coli. Phage treatment significantly reduced E. coli biofilm viability in catheters both in static conditions and under flow, highlighting the potential of phage therapy as an intervention strategy for catheter associated urinary tract infections (CAUTI).

Optotracers have a wide-range of applications in the detection, visualization, and characterization of biofilms. More recently, optotracers have been used in antibiotic susceptibility assays for biofilms and for the detection of biomarkers in clinical biofilm infections. This is particularly important, given that the field is increasingly focused on the evaluation of novel anti-biofilm agents and the study of biofilms in host-relevant systems. Given this, the possibility of using optotracers for an expanded set of biofilm focus areas is important to explore. In this study, we examine the application of the optotracer EbbaBiolight 680 to study biofilms in a host-relevant system and for the evaluation of a non-conventional anti-biofilm remedy. Using Staphylococcus aureus wound biofilms as a case study, we leverage a previously built in vitro 4-D wound microenvironment platform and a plant-based wound remedy. We find that EbbaBiolight 680 can be used to visualize S. aureus biofilms, likely detecting both bacterial cells and bacterial EPS components. Further, the optotracer can be used to evaluate and quantify the effects of the plant-based wound remedy on S. aureus biomass formation. However, in the 4-D wound microenvironment, EbbaBiolight 680 detected host cellular and matrix elements, which confounded the detection of biofilms. Taken together, this study opens the possibility of using optotracers as screening tools for the identification of novel anti-biofilm treatments and underscores the need for modifications for their use in host-relevant systems.

The use of enzymes represents an approach to combat bacterial infections by degrading extracellular biomolecules to disperse Staphylococcus aureus biofilms. Commercial enzyme preparations, including cellulase, amylase, pectinase, zymolyase, and pepsin, exhibit concentration-dependent dispersion of S. aureus biofilms. Here, we report that low concentrations of these enzymes generally lack synergy when combined or added together sequentially to biofilms. Only the addition of a protease (pepsin) followed by a commercial mixture of degradative enzymes from Arthrobacter luteus (zymolyase 20T), demonstrated synergy and was effective at dispersing S. aureus biofilms. A more purified mixture of Arthrobacter luteus enzymes (zymolyase 100T) showed improved dispersal of S. aureus biofilms compared to zymolyase 20T but lacked synergy with pepsin. This study emphasizes the complexity of enzymatic biofilm dispersal and the need for tailored approaches based on the properties of degradative enzymes and biofilm composition.

Class 1 integrons play a major role in antibiotic resistance dissemination among Gram-negative bacteria. They are genetic platforms able to capture, exchange and express antibiotic resistance gene cassettes. The integron integrase, whose expression is regulated by the bacterial SOS response, is the key element of the integron catalyzing insertion/excision/shuffling of gene cassettes. We previously demonstrated that the basal level of integrase expression and in consequence, its activity, is increased via the starvation-induced stringent response in the biofilm population. However, biofilms are heterogeneous environments where bacteria are under various physiological states. Here we thus analyzed at the bacterial level, the SOS response and integrase expression within the biofilm, using confocal microscopy and flow cytometry. We showed that in the absence of exogenous stress, only a small number of bacteria (~ 1%) located in the depth of the biofilm induce the SOS-response leading to a high level of integrase expression, through both a stringent response-dependent and -independent manner. Our results thus indicate that few bacteria located in microniches of the biofilm depth undergo sufficient endogenous stress to promote the acquisition of antibiotic resistance, forming a reservoir of bacteria ready to rapidly resist antibiotic treatments.

Urinary catheterization facilitates Escherichia coli colonization of the urinary tract and increases infection risk. While specific pathotypes are well-recognized for some E. coli infections, it is unclear whether strain-specific characteristics among E. coli are associated with infection risk in catheterized patients. Here we used comparative genomics and a simulated catheter biofilm model to compare strains associated with catheter-associated urinary tract infection (CAUTI) and catheter-associated asymptomatic bacteriuria (CAASB). CAUTI was associated with a phylotype B2 sub-clade dominated by the multidrug resistant ST131 lineage, while CAASB isolates were genetically more diverse. Catheter-associated biofilm formation was widespread but quantitatively variable among isolates. Network community analysis resolved distinct groups of genes associated with infection or biofilm formation, with iron acquisition-associated genes prominent throughout. Using a reporter construct and targeted mutagenesis, we detected a biofilm phenotype for the ferric citrate transport (Fec) system, the most prominent correlate of high catheter biofilm formation in these patients. In mixed cultures, catheter biofilms formed by some CAASB strains suppressed catheter colonization by ST131 CAUTI isolates. These results are consistent with a paradigm in which catheter biofilm-associated genes increase infection risk in strains with a high pathogenic potential and decrease infection risk through niche exclusion in strains with low pathogenic potential.

ObjectivesBacteriophage (phage) therapy is being explored as a strategy for clinical control of periprosthetic joint infections, addressing the challenges of antibiotic-resistant bacteria and limitations around the complete eradication of methicillin-resistant Staphylococcus aureus biofilms by conventional antibiotic treatment. Additionally, phage-antibiotic combinations may enhance treatment success through synergistic interactions. This study aimed to compare the efficacy of phage Silviavirus remus (Remus) and vancomycin, independently and together, against S. aureus biofilms on Kirschner-wire (K-wire) implants using Galleria mellonella as an in vivo model, and to assess the biocompatibility of K-wire implantation. ResultsSurvival of G. mellonella larvae implanted with S. aureus biofilms and treated with Remus ([~]3 x 104 PFU/worm) and/or vancomycin (5 {micro}g/worm) after 72 hours was assessed using Kaplan-Meier curves. Statistical analyses among treatment groups were performed using log-rank tests at p-value < 0.10. Implantation of uncolonized K-wires with treatment administration did not affect survival (100%). Compared with untreated biofilm-infected larvae (77%), vancomycin alone (97%; p = 0.023) or in combination with Remus (93%; p = 0.087) improved survival, whereas Remus alone did not increase survival (67%, p = 0.33). Scanning electron microscopy confirmed the presence of biofilm-associated attachment of S. aureus on K-wires, although adherence was non-uniform.

Bacterial cells detached from Staphylococcus epidermidis biofilms are found to release predominantly as small oblate clusters ([~]1.9 {micro}m) in both untreated biofilms and biofilms treated with matrix-targeted disruptors. Quantitative image analysis common to colloidal science was applied to quantitatively evaluate the physical properties of 9,147 bacterial clusters detached from S. epidermidis biofilms with and without targeted disruption of individual matrix components (polysaccharides, proteins, extracellular DNA) or solubilization of the extracellular polymeric substances (EPS). Concentrations of S. epidermidis biofilm-detached cells are highest after matrix-targeted disruption of polysaccharides. K-means clustering, an unsupervised machine learning technique, was used to reveal that S. epidermidis biofilm-detached cells are released in five distinct phenotypes: small oblate, mid-sized oblate, large oblate, small spherical, and mid-sized prolate clusters. S. epidermidis biofilm detached cell clusters are predominantly oblate across three size groups (79.5%), with the small oblate phenotype representing 60.1% of cell clusters that have 3.1 {+/-} 1.2 cells per cluster, Euclidean diameters of 1.9 {+/-} 0.4 {micro}m, anisotropy indices of 0.98 {+/-} 0.05, and asphericities of -1.75 {+/-} 0.31 on average. The proportion of S. epidermidis cell clusters within each biofilm-detached cell phenotype differs between matrix-targeted disruptors. There are also variations in the abundance of S. epidermidis biofilm detached cells after matrix-targeted disruption between growth conditions and strains. Evaluating the physical properties of biofilm-detached cells after matrix-targeted disruption is critical to understanding their translocation in fluid flow and susceptibility to the host immune response as well as in evaluating matrix-targeted disruption for biofilm control.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilm infections in the lungs of people with the genetic condition cystic fibrosis (CF) that can persist for decades. There are numerous P. aeruginosa lifestyle changes associated with chronic biofilm infection cued by the CF lung environment. These include a loss of virulence, metabolic changes and increased antimicrobial tolerance. We have investigated P. aeruginosa PA14 biofilm infection over 7 d in an ex vivo pig lung (EVPL) model for CF, previously shown to facilitate formation of a clinically relevant P. aeruginosa biofilm structure with expression of key genes comparable to human infection. We have compared P. aeruginosa gene expression between sequential time points: 24 h, 48 h and 7 d post infection, and investigated tolerance to polymyxins. Our results demonstrate that the EVPL model can maintain a P. aeruginosa biofilm population, which exhibits increased antibiotic tolerance, for at least 7 d. Differential expression of antimicrobial resistance-associated genes was not observed, however there was significant upregulation of sulfur metabolism and maintenance of a structured biofilm. Our findings provide further insight into the increased P. aeruginosa antibiotic tolerance during chronic infection of the CF lung, and suggest we can cue aspects of chronic infection in 7 d under the right lab conditions.

Staphylococcus epidermidis and Cutibacterium acnes are among the most abundant members of the human skin microbiome. Both species are associated with skin health and disease. Although skin microbes typically grow in surface-associated biofilms, few studies on the interaction between S. epidermidis and C. acnes in biofilms have been reported. In the present study we measured the ability of S. epidermidis and C. acnes, either individually or jointly, to form biofilms in glass culture tubes. Since S. epidermidis is a facultative anaerobe and C. acnes is an aerotolerant anaerobe, tubes were incubated both aerobically and anaerobically to assess the role of atmosphere in biofilm forma4on. When cultured individually, we found that S. epidermidis formed biofilms under both aerobic and anaerobic conditions, whereas C. acnes formed biofilms only under aerobic conditions. When co-cultured, the presence of C. acnes had no effect on S. epidermidis biofilm formation under either aerobic or anaerobic conditions. However, the presence of S. epidermidis significantly enhanced the growth of C. acnes biofilms under anaerobic conditions and enabled C. acnes to form biofilms under aerobic condi2ons. This finding may be clinically relevant to the interaction between S. epidermidis and C. acnes on human skin.