Inside Biofilms Research Articles

Magneto-Based Synergetic Therapy for Implant-Associated Infections via Biofilm Disruption and Innate Immunity Regulation.

Implant‐associated infections (IAIs) are a common cause of orthopedic surgery failure due to microbial biofilm‐induced antibiotic‐resistance and innate immune inactivation. Thus, the destruction of microbial biofilm plays a key role in reducing IAIs. Herein, first, a magneto‐based synergetic therapy (MST) is proposed and demonstrated against IAIs based on biofilm destruction. Under an alternating magnetic field (AMF), CoFe2O4@MnFe2O4 nanoparticles (MNPs), with a rather strong magnetic hyperthermal capacity, can generate sufficient thermal effect to cause dense biofilm dispersal. Loosened biofilms provide channels through which nitrosothiol‐coated MNPs (MNP‐SNOs) can penetrate. Subsequently, thermosensitive nitrosothiols rapidly release nitric oxide (NO) inside biofilms, thus efficiently killing sessile bacteria under the magnetothermal effect of MNPs. More importantly, MNP‐SNOs can trigger macrophage‐related immunity to prevent the relapse of IAIs by exposing the infected foci to a consistent innate immunomodulatory effect. The notable anti‐infection effect of this nanoplatform is also confirmed in a rat IAI model. This work presents the promising potential of combining magnetothermal therapy with immunotherapy, for the effective and durable control and elimination of IAIs.

Quantitative image analysis of microbial communities with BiofilmQ

Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ—a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.

Simultaneous inhibition of planktonic and biofilm bacteria by self-adapting semiconducting polymer dots.

Biofilm infections present an enormous challenge in today's healthcare settings. Currently, pH-switchable antibacterial agents are being developed to eradicate biofilms. However, most pH-switchable antibacterial agents are less lethal to planktonic bacteria under neutral conditions, and cannot prevent the dispersed bacteria from seeding acute infection again. Herein, this work reports the applications of semiconducting polymer dots (Pdots) with a double adhesion mechanism in imaging and inhibiting bacteria inside (weak acidic conditions) and outside (neutral conditions) biofilms. Clew-like Pdots were prepared by covalently linking phenylboronic acid (PBA) and pH-responsive naphthalimide (NA) ramification in semiconducting polymers. Under neutral conditions, the Pdots combined with bacteria through the formation of boronate esters between PBA and diols. Under weakly acidic conditions, the partial borate bond fractured, and the Pdots adhered onto the bacterial surface through the positively charged NA in Pdots. Furthermore, the Pdots display negligible toxicity to mammalian cells and tissues. More importantly, the Pdots can selectively damage the bacterial membrane and inhibit bacteria in vivo. This work highlights the feasibility of using semiconducting Pdots to image and inhibit bacteria inside and outside biofilms, which represents a highly effective strategy to cope with biofilm infections.

Multicellular and unicellular responses of microbial biofilms to stress.

Biofilms are a ubiquitous mode of microbial life and display an increased tolerance to different stresses. Inside biofilms, cells may experience both externally applied stresses and internal stresses that emerge as a result of growth in spatially structured communities. In this review, we discuss the spatial scales of different stresses in the context of biofilms, and if cells in biofilms respond to these stresses as a collection of individual cells, or if there are multicellular properties associated with the response. Understanding the organizational level of stress responses in microbial communities can help to clarify multicellular functions of biofilms.

Coordinated response of Au-NPs/rGO modified electroactive biofilms under phenolic compounds shock: Comprehensive analysis from architecture, composition, and activity

Electroactive biofilms (EABs) can be integrated with conductive nanomaterials to boost extracellular electron transfer (EET) for achieving efficient waste treatment and energy conversion in bioelectrochemical systems. However, the in situ nanomaterial-modified EABs of mixed-culture, and their response under environmental stress are rarely revealed. Here, two nanocatalyst-decorated EABs were established by self-assembled Au nanoparticles-reduced graphene oxide (Au-NPs/rGO) in mixed-biofilms with different maturities, then their multi-property were analyzed under long-term phenolic shock. Results showed that the power density of Au-NPs/rGO decorated EABs was significantly enhanced by 28.66–42.82% due to the intensified EET pathways inside biofilms. Meanwhile, the electrochemical and catalytic performance of EABs were controllably regulated by 0.3–3.0 g/L phenolic compounds, which, however, resulted in differential alterations in their architecture, composition, and viability. EABs originated with higher maturity displayed more compact structure, lower thickness (110 μm), higher biomass (8.67 mg/cm2) and viability (0.85–0.91), endowing it better antishock ability to phenolic compounds. Phenolic-shock also induced the heterogeneous distribution of extracellular polymeric substances in terms of both spatial and bonding degrees of the decorated EABs, which could be regarded as an active response to strike a balance between self-protection and EET under environmental pressure. Our findings provide a broader understanding of microbe-electrode interactions in the micro-ecology interface and improve their performance in the removal of complex contaminants for sustainable remediation and new-energy development.

Mechanisms and Control Measures of Mature Biofilm Resistance to Antimicrobial Agents in the Clinical Context.

Biofilms are the aggregation of micro-organisms, which are composed of extracellular polymeric substance (EPS) and many other biochemical components. Though they might be beneficial to some wastewater and soil treatment processes, they may expose chronic infection and risk to personal hygiene in the industrial as well as the clinical context. Despite having a well-established disinfection and hygiene monitoring program for the prevention of formation and growth, biofilm persistently remains in the medical settings because of its antibiotic resistance to antimicrobial agents and even the immune system. In this paper, the contributing factors of antibiotic resistance and the corresponding mechanisms, including heterogeneity inside biofilms, the roles of the EPS matrix, cell density, and quorum sensing, and cell mutability, are reviewed. Moreover, current clinical practice and strategic applications are also suggested to address the biofilm resistance issues.

Dissolvable Microneedles Coupled with Nanofiber Dressings Eradicate Biofilms via Effectively Delivering a Database-Designed Antimicrobial Peptide

Biofilms in chronic wounds, including diabetic foot ulcers, pressure ulcers, and venous leg ulcers, pose a major challenge to wound management. Herein, we report a Janus-type antimicrobial dressing for eradication of biofilms in chronic wounds. The dressing consists of electrospun nanofiber membranes coupled with dissolvable microneedle arrays to enable effective delivery of a database-designed antimicrobial peptide to both inside and outside biofilms. This antimicrobial dressing exhibited high efficacy against a broad spectrum of resistant pathogens in vitro. Importantly, such a dressing was able to eradicate methicillin-resistant Staphylococcus aureus (MRSA) biofilms in both an ex vivo human skin wound infection model and a type II diabetic mouse wound infection model after daily treatment without applying surgical debridement. Most importantly, the dressing can also completely remove the Pseudomonas aeruginosa and MRSA, dual-species biofilm in an ex vivo human skin infection model. In addition, our computational simulations also suggested that microneedles were more effective in the delivery of peptides to the biofilms than free drugs. Our results indicate that the Janus-type antimicrobial dressings may provide an effective treatment and management of chronic wound polymicrobial infections.

The microalga Haematococcus lacustris (Chlorophyceae) forms natural biofilms in supralittoral White Sea coastal rock ponds.

Haematococcus lacustris inhabits supralittoral rock ponds and forms, under natural conditions, biofilms including layered cyanobacterial and fermentative microbial mats. Dry mats, formed under extremely stressful conditions, contained only haematocysts. Under favorable growth conditions, modeled for dry biofilms in vitro, microalgal free-living stages were detected. Haematococcus lacustris is the microalga known for its high potential to survive under a wide range of unfavorable conditions, particularly in the supralittoral temporal rock ponds of the White Sea. Previously, we described microbial communities containing H. lacustris in this region. In many cases, they were organized into systems exhibiting complex three-dimensional structure similar to that of natural biofilms. In this study, for the first time, we clarify structural description and provide microscopic evidence that these communities of H. lacustris and bacteria are assembled into the true biofilms. There are (1) simple single layer biofilms on the surface of rocks and macrophytic algae, (2) floccules (or flocs) not attached to a surface, (3) as well as stratified (layered) biofilms, wet, and dehydrated in nature. Being involved into primary organic production, H. lacustris and cyanobacteria are located exclusively in the upper layers of stratified biofilms, where they are capable to absorb sufficient for photosynthesis amount of light. The presence of acidic polysaccharides in the extracellular matrix revealed by specific staining with ruthenium red in the H. lacustris-containing microbial communities is a biochemical evidence of biofilm formation. Meanwhile, the presence of bacterial L-form is an ultrastructural confirmation of that fact. Under favorable conditions, modeled in vitro, H. lacustris from the dry microbial mats moves to the free-living states represented by vegetative palmelloid cells and motile zoospores. Owing to the fact that inside biofilms cells of microorganisms exist under stable conditions, we consider the biofilm formation as an additional mechanism that contributes to the survival of H. lacustris in the supralittoral zone of the White Sea.

Lipid-polymer hybrid nanoparticles carrying linezolid improve treatment of methicillin-resistant Staphylococcus aureus (MRSA) harbored inside bone cells and biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent pathogen causing osteomyelitis. The tendency of MRSA to evade standard antibiotic treatment by hiding inside bone cells and biofilms is a major cause of frequent osteomyelitis recurrence. In this study, we developed a lipid-polymer hybrid nanoparticle loading the antibiotic linezolid (LIN-LPN), and focused on evaluating if this new nanoantibiotic can achieve significant in vitro activities against these intracellular and biofilm-embedded MRSA. The optimal LIN-LPN formulation demonstrated both high linezolid payload (12.0% by weight of nanoparticles) and controlled release characteristics (gradually released the entrapped antibiotic in 120 h). Although it achieved lower activities against bacteria including USA300-0114, CDC-587, RP-62A in planktonic form, it was substantially superior against the intracellular MRSA reservoir inside osteoblast cells. The differences of intracellular activities between LIN-LPN and linezolid were 87.0-fold, 12.3-fold, and 12.6-fold in CFU/ml (p < 0.05 or < 0.01) at 2 µg/ml, 4 µg/ml, and 8 µg/ml linezolid concentrations, respectively. LIN-LPN also suppressed the MRSA biofilm growth to 35–60% of the values achieved with free linezolid (p < 0.05). These enhanced intracellular and anti-biofilm activities of LIN-LPN were likely contributed by the extensive accumulation of LIN-LPN inside the MRSA-infected osteoblasts and biofilms as revealed in the confocal microscope images. The study thus validates the feasibility of exploiting the good nanoparticle-host cell and nanoparticle-biofilm interactions for improving the antibiotic drug activities against the poorly accessible bacteria, and supports LIN-LPN as a new alternative therapy for preventing the recurrence of MRSA-mediated bone infections.

Size and Charge Adaptive Clustered Nanoparticles Targeting the Biofilm Microenvironment for Chronic Lung Infection Management.

Chronic lung infection caused by bacterial biofilms is an extremely serious clinical problem, which can lead to the failure of antibiotic therapy. Although nanoparticles have shown great potential in the treatment of biofilms, the efficient penetration and retention of nanoparticles in biofilms is still a big challenge. To address this issue, we herein fabricate size and charge adaptive azithromycin (AZM)-conjugated clustered nanoparticles (denoted as AZM-DA NPs) as therapeutic agents for treating biofilms. The AZM-DA NPs are prepared by electrostatic complexation between AZM conjugated amino-ended poly(amidoamine) dendrimer (PAMAM) and 2,3-dimethyl maleic anhydride (DA) modified poly(ethylene glycol)-block-polylysine (PEG-b-PLys). It is noteworthy that the AZM-DA NPs can disassemble in an acidic biofilm microenvironment (pH 6.0), leading to the release of secondary AZM-conjugated PAMAM nanoparticles (PAMAM-AZM NPs). PAMAM-AZM NPs with small size and positive charge are beneficial for improved penetration and retention inside biofilms, enhanced permeabilization of the bacterial membrane, and increased internalization of AZM, thus exhibiting excellent antibiofilm activities. AZM-DA NPs are also favorable as long-term antibacterial agents due to the reduced occurrence of drug resistance. In vivo therapeutic performance is confirmed by the reduced bacterial burden and the alleviated inflammation in the chronic lung infection model. This research not only develops an innovative strategy for antibiotic delivery in vivo but also provides an effective way for the management of biofilm-associated infections, including chronic lung infection.

Metagenomic characterization of bacterial biofilm in four food processing plants in Colombia.

Bacteria inside biofilms are more persistent and resistant to stress conditions found in the production environment of food processing plants, thus representing a constant risk for product safety and quality. Therefore, the aim of this study was to characterize, using 16S rRNA sequencing, the bacterial communities from biofilms found in four food processing plants (P1, P2, P3, and P4). In total, 50 samples from these four processing plants were taken after cleaning and disinfection processes. Four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroides represented over 94% of the operational taxonomic units found across these four plants. A total of 102 families and 189 genera were identified. Two genera, Pseudomonas spp. and Acinetobacter spp., were the most frequently found (93.47%) across the four plants. In P1, Pseudomonas spp. and Lactobacillus spp. were the dominant genera, whereas Lactobacillus spp. and Streptococcus spp. were identified in P2. On the other hand, biofilms found in P3 and P4 mainly consisted of Pseudomonas spp. and Acinetobacter spp. Our results indicate that different bacterial genera of interest to the food industry due to their ability to form biofilm and affect food quality can coexist inside biofilms, and as such, persist in production environments, representing a constant risk for manufactured foods. In addition, the core microbiota identified across processing plants evaluated was probably influenced by type of food produced and cleaning and disinfection processes performed in each one of these.

Fast Biofilm Penetration and Anti-PAO1 Activity of Nebulized Azithromycin in Nanoarchaeosomes.

Azithromycin (AZ) is a broad-spectrum antibiotic with anti-inflammatory and antiquorum sensing activity against biofilm forming bacteria such as Pseudomonas aeruginosa. AZ administered by oral or parenteral routes, however, neither efficiently accesses nor remains in therapeutic doses inside pulmonary biofilm depths. Instead, inhaled nanocarriers loaded with AZ may revert the problem of low accessibility and permanence of AZ into biofilms, enhancing its antimicrobial activity. The first inhalable nanovesicle formulation of AZ, nanoarchaeosome-AZ (nanoARC-AZ), is here presented. NanoARC prepared with total polar archaeolipids (TPAs), rich in 2,3-di-O-phytanyl-sn-glycero-1-phospho-(3'-sn-glycerol-1'-methylphosphate) (PGP-Me) from Halorubrum tebenquichense archaebacteria, consisted of ∼180 nm-diameter nanovesicles, loaded with 0.28 w/w AZ/TPA. NanoARC-AZ displayed lower minimal inhibitory concentration and minimal bactericidal concentration, higher preformed biofilm disruptive, and anti-PAO1 activity in biofilms than AZ. NanoARC penetrated and disrupted the structure of the PAO1 biofilm within only 1 h. Two milliliters of 15 μg/mL AZ nanoARC-AZ nebulized for 5 min rendered AZ doses compatible with in vitro antibacterial activity. The strong association between AZ and the nanoARC bilayer, combined with electrostatic attraction and trapping into perpendicular methyl groups of archaeolipids, as determined by Laurdan fluorescence anisotropy, generalized polarization, and small-angle X-ray scattering, was critical to stabilize during storage and endure shear forces of nebulization. NanoARC-AZ was noncytotoxic on A549 cells and human THP-1-derived macrophages, deserving further preclinical exploration as enhancers of AZ anti-PAO1 activity.

Highly heterogeneous interior structure of biofilm wastewater for enhanced pollutant removals

Biofilm processes are widely used in wastewater treatment. The biofilm has highly heterogeneous interior structure, which can significantly affect the transport processes and the biological reactions over the biofilm. This study for the first time detailed the complicated velocity and concentration fields of substrate in a real biofilm structure. With a real biofilm interior being profiled and meshed to numerical solutions, the flow-through mode has significant distortion of inflow velocity fields and concentration distributions, which lead to enhanced biological reactions at regimes nearby major pores. Conversely, the crossflow mode depends weakly on the biofilm interior structure. The uniform biofilm model fails to describe the real biofilm processes. Future research needs based on real biofilm structures were discussed.

Accumulation of NH4 + and NO3 - inside Biofilms of Natural Microbial Consortia: Implication on Nutrients Seasonal Dynamic in Aquatic Ecosystems.

Microbial biofilms are ubiquitous in aquatic ecosystems. Inside the biofilm is the nutrient-rich microenvironment promoted by the accumulation of the nutrient ions such as NH4+ and NO3− from surrounding water. The present study investigated the characteristics of NH4+ and NO3− accumulation into the biofilm of natural microbial consortia collected from Lake Biwa, Japan. The results showed the following: (1) the concentrations of NH4+ and NO3− inside the biofilm were much higher than those in the surrounding water; (2) the nutrient ion concentration inside the biofilm changed in synchrony with those in the surrounding water; (3) biofilm polymers have both positively and negatively charged sites; (4) electrostatic attractive interactions between the charged sites on biofilm polymers and oppositely charged ions outside the biofilm seem to play important roles in the accumulation of nutrient ions into the biofilm from the surrounding water; (5) the bacterial community structure differs between the biofilm and surrounding water. The present study revealed that the accumulation of nutrient ions into the biofilm indicates the removal of these ions from water outside the biofilm. According to the result of this study, accumulation of ions such as NH4+ and NO3− into the biofilm of natural microbial consortia may have implications on nutrients seasonal dynamic in aquatic ecosystems.

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy.

The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

Predation creates unique void layer in membrane-aerated biofilms

The membrane-aerated biofilm reactor (MABR) is a novel wastewater treatment technology based on oxygen-supplying membranes. The counter diffusion of oxygen and electron donors in MABRs leads to unique behavior, and we hypothesized it also could impact predation. We used optical coherence tomography (OCT), microsensor analyses, and mathematical modeling to investigate predation in membrane-aerated biofilms (MABs). When protozoa were excluded from the inoculum, the MAB's OCT-observable void fraction was around 5%. When protozoa were included, the void fraction grew to nearly 50%, with large, continuous voids at the base of the biofilm. Real-time OCT imaging showed highly motile protozoa in the voids. MABs with protozoa and a high bulk COD (270 mg/L) only had 4% void fraction. DNA sequencing revealed a high relative abundance of amoeba in both high and low-COD MABs. Flagellates were only abundant in the low-COD MAB. Modeling also suggested a relationship between substrate concentrations, diffusion mode (co- or counter-diffusion), and biofilm void fraction. Results suggest that amoeba proliferate in the biofilm interior, especially in the aerobic zones. Voids form once COD limitation at the base of MABs allows predation rates to exceed microbial growth rates. Once formed, the voids provide a niche for motile protozoa, which expand the voids into a large, continuous gap. This increases the potential for biofilm sloughing, and may have detrimental effects on slow-growing, aerobic microorganisms such as nitrifying bacteria.

Sociological characteristics of biofilms

Biofilms are a kind of biological structure with high complexity and self-organization, composed of a variety of microorganisms (bacteria, archaea, algae, and micro eukaryotes) and biological substrates (polysaccharides, proteins, fatty acids, etc.). They own special structures and ecological functions, and play important roles in microbial colonization, niche construction and environmental adaptability. Many microorganisms’ behaviors, such as pathogenic infection, toxin production, drug resistance, biofouling and pipe blockage, are related with biofilms. The formation of these events is full of various microbial behaviors, including signal communication, cooperation/competition, division of labor, as well as “bacteria intelligence” under the adverse circumstances. These features offer further indication of the sociological attributes of biofilms, which also provide a new insight into them. Thus, based on the 3D structure of biofilms, we took the communication language, collaboration, and intelligent resistance (environmental stress, co-evolution) inside biofilms as the core to elaborate their social characteristics, in order to get a better understanding of their nature and sociomicrobiology.

Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus

Staphylococcus aureus is a major causative agent for biofilm-associated infections. Inside biofilms, S. aureus cells are embedded in an extracellular matrix (ECM) composed of polysaccharide-intercellular adhesins (PIA), proteins, and/or extracellular DNA (eDNA). However, the importance of each component and the relationship among them in biofilms of diverse strains are largely unclear. Here, we characterised biofilms formed by 47 S. aureus clinical isolates. In most (42/47) of the strains, biofilm formation was augmented by glucose supplementation. Sodium chloride (NaCl)-triggered biofilm formation was more prevalent in methicillin-sensitive S. aureus (15/24) than in methicillin-resistant strain (1/23). DNase I most effectively inhibited and disrupted massive biofilms, and Proteinase K was also effective. Anti-biofilm effects of Dispersin B, which cleaves PIA, were restricted to PIA-dependent biofilms formed by specific strains and showed significant negative correlations with those of Proteinase K, suggesting independent roles of PIA and proteins in each biofilm. ECM profiling demonstrated that eDNA was present in all strains, although its level differed among strains and culture conditions. These results indicate that eDNA is the most common component in S. aureus biofilms, whereas PIA is important for a small number of isolates. Therefore, eDNA can be a primary target for developing eradication strategies against S. aureus biofilms.

Probing Phenazine Electron Transfer and Retention in Pseudomonas Aeruginosa Biofilms

The opportunistic pathogen Pseudomonas aeruginosa forms metabolically stratified biofilms. Redox-active phenazine metabolites shuttle metabolic electrons from the oxygen depleted biofilm interior to the oxygen saturated exterior, facilitating anoxic survival. We are testing the hypothesis that phenazine retention and/or charge transfer through the biofilm matrix are mediated by extracellular DNA (eDNA), which is the most abundant polymer in the P. aeruginosa matrix and is known to provide structural integrity to the biofilm. Specifically, we are growing biofilms on electrode arrays and performing in vitro studies of phenazine interactions with DNA. Biofilm experiments show that conductivity through the matrix is mediated by sequential electron transfer between phenazine molecules. Transferring electrode grown biofilms to fresh liquid medium demonstrates that some phenazines are retained tightly in the biofilm matrix, while others rapidly diffuse away. The retention of different phenazines corresponds to their affinity for DNA in vitro, consistent with the idea that some phenazines might bind the eDNA in the biofilm. We are testing whether phenazines bound to DNA can also mediate charge transfer. Collectively, these experiments are helping us better understand how P. aeruginosa utilizes its self-produced extracellular electron shuttles to survive biofilm oxidant limitation, and are opening up new perspectives on the role(s) that eDNA may play in the biofilm matrix.

The effect of emodin on Staphylococcus aureus strains in planktonic form and biofilm formation in vitro.

Staphylococcus aureus (S. aureus) is a Gram-positive pathogen and forms biofilm easily. Bacteria inside biofilms display an increased resistance to antibiotics and disinfectants. The objective of the current study was to assess the antimicrobial activities of emodin, 1,2,8-trihydroxy-6-methylanthraquinone, an anthraquinone derivative isolated from Polygonum cuspidatum and Rheum palmatum, against S. aureus CMCC26003 grown in planktonic and biofilm cultures in vitro. In addition, a possible synergistic effect between emodin and berberine chloride was evaluated. As quantified by crystal violet method, emodin significantly decreased S. aureus biofilm growth in a dose-dependent manner. The above findings were further supported by scanning electron microscopy. Moreover, the present study demonstrated that sub-MICs emodin obviously intervened the release of extracellular DNA and inhibited expression of the biofilm-related genes (cidA, icaA, dltB, agrA, sortaseA and sarA) by real-time RT-PCR. These results revealed a promising application for emodin as a therapeutic agent and an effective strategy to prevent S. aureus biofilm-related infections.