Host-derived protease promotes aggregation of Staphylococcus aureus by cleaving the surface protein SasG.

Staphylococcus aureus is one of the leading causes of hospital-acquired infections, many of which begin following attachment and accumulation on indwelling medical devices or diseased tissue. These infections are often linked to the establishment of biofilms, but another often overlooked key characteristic allowing S. aureus to establish persistent infection is the formation of planktonic aggregates. Such aggregates are physiologically similar to biofilms and protect pathogens from innate immune clearance and increase antibiotic tolerance. The cell-wall-associated protein SasG has been implicated in biofilm formation via mechanisms of intercellular aggregation but the mechanism in the context of disease is largely unknown. We have previously shown that the expression of cell-wall-anchored proteins involved in biofilm formation is controlled by the ArlRS-MgrA regulatory cascade. In this work, we demonstrate that the ArlRS two-component system controls aggregation, by repressing the expression of sasG by activation of the global regulator MgrA. We also demonstrate that SasG must be proteolytically processed by a non-staphylococcal protease to induce aggregation and that strains expressing functional full-length sasG aggregate significantly upon proteolysis by a mucosal-derived host protease found in human saliva. We used fractionation and N-terminal sequencing to demonstrate that human trypsin within saliva cleaves within the A domain of SasG to expose the B domain and induce aggregation. Finally, we demonstrated that SasG is involved in virulence during mouse lung infection. Together, our data point to SasG, its processing by host proteases, and SasG-driven aggregation as important elements of S. aureus adaptation to the host environment.IMPORTANCEHere, we demonstrate that the Staphylococcus aureus surface protein SasG is important for cell-cell aggregation in the presence of host proteases. We show that the ArlRS two-component regulatory system controls SasG levels through the cytoplasmic regulator MgrA. We identified human trypsin as the dominant protease triggering SasG-dependent aggregation and demonstrated that SasG is important for S. aureus lung infection. The discovery that host proteases can induce S. aureus aggregation contributes to our understanding of how this pathogen establishes persistent infections. The observations in this study demonstrate the need to strengthen our knowledge of S. aureus surface adhesin function and processing, regulation of adhesin expression, and the mechanisms that promote biofilm formation to develop strategies for preventing chronic infections.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 µM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

A structure activity relationship study of 3,4'-dimethoxyflavone for ArlRS inhibition in Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat due to their resistance to many ß-lactam antibiotics, and their highly coordinated excretion of virulence factors. One way in which MRSA accomplishes this is by responding to environmental stimuli using two-component systems (TCS). The ArlRS TCS has been identified as having a key role in regulating virulence in both systemic and local infections caused by S. aureus. We recently disclosed 3,4'-dimethoxyflavone as a selective ArlRS inhibitor. In this study we explore the structure-activity relationship (SAR) of the flavone scaffold for ArlRS inhibition and identify several compounds with increased activity compared to the parent. Additionally, we identify a compound that suppresses oxacillin resistance in MRSA, and begin to probe the mechanism of action behind this activity.

The influenza-injured lung microenvironment promotes MRSA virulence, contributing to severe secondary bacterial pneumonia.

Influenza infection is substantially worsened by the onset of secondary pneumonia caused by bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). The bidirectional interaction between the influenza-injured lung microenvironment and MRSA is poorly understood. By conditioning MRSA ex vivo in bronchoalveolar lavage fluid collected from mice at various time points of influenza infection, we found that the influenza-injured lung microenvironment dynamically induces MRSA to increase cytotoxin expression while decreasing metabolic pathways. LukAB, a SaeRS two-component system-dependent cytotoxin, is particularly important to the severity of post-influenza MRSA pneumonia. LukAB's activity is likely shaped by the post-influenza lung microenvironment, as LukAB binds to (and is activated by) heparan sulfate (HS) oligosaccharide sequences shed from the epithelial glycocalyx after influenza. Our findings indicate that post-influenza MRSA pneumonia is shaped by bidirectional host-pathogen interactions: host injury triggers changes in bacterial expression of toxins, the activity of which may be shaped by host-derived HS fragments.

Staphylococcal Corneocyte Adhesion: Assay Optimization and Roles of Aap and SasG Adhesins in the Establishment of Healthy Skin Colonization.

Staphylococcus aureus is an opportunistic pathogen that causes the majority of wound and soft tissue infections. The accumulation-associated protein (Aap) from S. epidermidis and surface protein G (SasG) from S. aureus are cell wall-anchored (CWA) proteins known to be important in adhesion to healthy corneocytes from human skin. We investigated the mechanisms by which S. aureus colonizes healthy human skin by developing an optimized corneocyte adhesion assay. Trypan blue was used for enhanced red autofluorescent visualization of corneocytes with an overlay of green-fluorescent bacteria. The percent area of bacterial adhesion for images acquired by a fluorescence microscope was quantified using Fiji ImageJ. Using this optimized imaging procedure, differences in adhesion between various species and strains of staphylococci were measured. The ability of purified SasG to reduce Staphylococcus epidermidis adhesion was investigated in order to determine if these CWA proteins can compete for binding sites. To further test CWA-mediated adhesion, we engineered a nonadhering S. carnosus strain to express full-length SasG from two methicillin-resistant S. aureus (MRSA) strains. Finally, we demonstrated that the SasG A domain was a critical region of this surface protein for adherence to healthy human corneocytes. The developed imaging and expression methods are useful for studying staphylococcal adhesion to healthy human skin and have the potential to be used with a wide variety of fluorescently labeled organisms on both healthy and disease-state (such as atopic dermatitis) corneocytes. IMPORTANCE The skin is the largest organ of the human body and acts as a shield against hazards such as harmful bacteria like Staphylococcus aureus. A diverse skin microbiota and immune cross talk control S. aureus numbers. S. aureus can bind to healthy skin and subsequently proliferate when the skin barrier is compromised, such as in a wound or in patients with atopic dermatitis (AD). It is important to understand these mechanisms in an effort to prevent pathogenic bacteria from causing infection. We describe an augmented corneocyte adhesion assay using fluorescence microscopy to study binding of various staphylococcal species to healthy human skin cells. In addition, we tested the ability of homologous proteins from different staphylococcal species to reduce binding, and developed a new S. carnosus expression system to test individual protein binding properties. Our newly developed methods and findings will enhance the understanding of how staphylococci bind to healthy human skin.

Therapeutic Inhibition of Staphylococcus aureus ArlRS Two-Component Regulatory System Blocks Virulence.

Staphylococcus aureus is a common cause of severe infections, and its widespread antibiotic resistance necessitates search for alternative therapies, such as inhibition of virulence. As S. aureus produces multiple individual virulence factors, inhibition of an entire regulatory system might provide better effects than targeting each virulence factor separately. Herein, we describe two novel inhibitors of S. aureus two-component regulatory system ArlRS: 3,4'-dimethoxyflavone and homopterocarpin. Unlike other putative ArlRS inhibitors previously identified, these two compounds were effective and specific. In vitro kinase assays indicated that 3,4'-dimethoxyflavone directly inhibits ArlS autophosphorylation, while homopterocarpin did not exhibit such effect, suggesting that two inhibitors work through distinct mechanisms. Application of the inhibitors to methicillin-resistant S. aureus (MRSA) in vitro blocked ArlRS signaling, inducing an abnormal gene expression pattern that was reflected in changes at the protein level, enhanced sensitivity to oxacillin, and led to the loss of numerous cellular virulence traits, including the ability to clump, adhere to host ligands, and evade innate immunity. The pleiotropic antivirulence effect of inhibiting a single regulatory system resulted in a marked therapeutic potential, demonstrated by the ability of inhibitors to decrease severity of MRSA infection in mice. Altogether, this study demonstrated the feasibility of ArlRS inhibition as anti-S. aureus treatment, and identified new lead compounds for therapeutic development.

Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro and in vivo biofilm.

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo.

In Vitro Assay for Quantifying Clumping of Staphylococcus aureus.

Staphylococcus aureus interacts with fibrinogen in plasma to form macroscopic clumps of cells. A simple and rapid slide agglutination test using rabbit plasma has been employed in clinical labs to distinguish S. aureus from most coagulase-negative Staphylococci. The method described here is a quantitative clumping assay in which S. aureus cells are mixed with either plasma or purified fibrinogen, and clumps are allowed to sediment out of solution. Clearing of the overlying solution is monitored over time by measuring the optical density at 600 nm and comparing these values to the initial turbidity. This simple assay can be used to study regulation and expression of various cell wall-anchored adhesins.

Staphylococcus aureus uses the ArlRS and MgrA cascade to regulate immune evasion during skin infection.

Skin is one of the most common sites of host immune response against Staphylococcus aureus infection. Here, through a combination of in vitro assays, mouse models, and intravital imaging, we find that S. aureus immune evasion in skin is controlled by a cascade composed of the ArlRS two-component regulatory system and its downstream effector, MgrA. S. aureus lacking either ArlRS or MgrA is less virulent and unable to form correct abscess structure due to de-repression of a giant surface protein, Ebh. These S. aureus mutants also have decreased expression of immune evasion factors (leukocidins, chemotaxis-inhibitory protein of S. aureus [CHIPS], staphylococcal complement inhibitor [SCIN], and nuclease) and are unable to kill neutrophils, block their chemotaxis, degrade neutrophil extracellular traps, and survive direct neutrophil attack. The combination of disrupted abscess structure and reduced immune evasion factors makes S. aureus susceptible to host defenses. ArlRS and MgrA are therefore the main regulators of S. aureus immune evasion and promising treatment targets.

Novel Peptide from Commensal Staphylococcus simulans Blocks Methicillin-Resistant Staphylococcus aureus Quorum Sensing and Protects Host Skin from Damage.

Recent studies highlight the abundance of commensal coagulase-negative staphylococci (CoNS) on healthy skin. Evidence suggests that CoNS actively shape the skin immunological and microbial milieu to resist colonization or infection by opportunistic pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), in a variety of mechanisms collectively termed colonization resistance. One potential colonization resistance mechanism is the application of quorum sensing, also called the accessory gene regulator (agr) system, which is ubiquitous among staphylococci. Common and rare CoNS make autoinducing peptides (AIPs) that function as MRSA agr inhibitors, protecting the host from invasive infection. In a screen of CoNS spent media, we found that Staphylococcus simulans, a rare human skin colonizer and frequent livestock colonizer, released potent inhibitors of all classes of MRSA agr signaling. We identified three S. simulans agr classes and have shown intraspecies cross talk between noncognate S. simulans agr types for the first time. The S. simulans AIP-I structure was confirmed, and the novel AIP-II and AIP-III structures were solved via mass spectrometry. Synthetic S. simulans AIPs inhibited MRSA agr signaling with nanomolar potency. S. simulans in competition with MRSA reduced dermonecrotic and epicutaneous skin injury in murine models. The addition of synthetic AIP-I also effectively reduced MRSA dermonecrosis and epicutaneous skin injury in murine models. These results demonstrate potent anti-MRSA quorum sensing inhibition by a rare human skin commensal and suggest that cross talk between CoNS and MRSA may be important in maintaining healthy skin homeostasis and preventing MRSA skin damage during colonization or acute infection.

Staphylococcus aureus bloodstream infections: pathogenesis and regulatory mechanisms.

Staphylococcus aureus is an opportunistic pathogen that normally colonizes the human anterior nares. At the same time, this pathogen is one of the leading causes of life-threatening bloodstream infections, such as sepsis and endocarditis. In this review we will present the current understanding of the pathogenesis of these invasive infections, focusing on the mechanisms of S. aureus clearance from the bloodstream by the immune system, and how this pathogen hijacks the host defense and coagulation systems and further interacts with the blood vessel endothelium. Additionally, we will delve into the regulatory mechanisms S. aureus employs during an invasive infection. These new insights into host-pathogen interactions show promising avenues for the development of novel therapies for treating bloodstream infections.

Merit Ptah, "The First Woman Physician": Crafting of a Feminist History with an Ancient Egyptian Setting.

Merit Ptah is widely described as "the first woman physician and scientist" on the Internet and in popular history books. This essay explores the origins of this figure, showing that Merit Ptah came into being in the 1930s when Kate Campbell Hurd-Mead misinterpreted a report about an authentic ancient Egyptian healer. Merit Ptah gradually became a prominent figure in popular historical accounts during second-wave of feminism, and, in the twenty-first century she appeared in Wikipedia and subsequently spread throughout the Internet as a female (sometimes black African) founding figure. The history of Merit Ptah reveals powerful mechanisms of knowledge creation in the network of amateur historians, independently from the scholarly community. The case of Merit Ptah also pinpoints factors enabling the spread of erroneous historical accounts: the absence of professional audience, the development of echo chambers due to an obscured chain of knowledge transmission, the wide reach of the Internet, the coherence with existing preconceptions, the emotional charge of heritage, and even - in the case of ancient Egypt - the tendency to perceive certain pasts through a legendary lens. At the same time, the story of Merit Ptah reveals how important role models have been for women entering science and medicine.

Staphylococcus aureus toxin suppresses antigen-specific T cell responses.

Staphylococcus aureus remains a leading cause of human infection. These infections frequently recur when the skin is a primary site of infection, especially in infants and children. In contrast, invasive staphylococcal disease is less commonly associated with reinfection, suggesting that tissue-specific mechanisms govern the development of immunity. Knowledge of how S. aureus manipulates protective immunity has been hampered by a lack of antigen-specific models to interrogate the T cell response. Using a chicken egg OVA-expressing S. aureus strain to analyze OVA-specific T cell responses, we demonstrated that primary skin infection was associated with impaired development of T cell memory. Conversely, invasive infection induced antigen-specific memory and protected against reinfection. This defect in adaptive immunity following skin infection was associated with a loss of DCs, attributable to S. aureus α-toxin (Hla) expression. Gene- and immunization-based approaches to protect against Hla during skin infection restored the T cell response. Within the human population, exposure to α-toxin through skin infection may modulate the establishment of T cell-mediated immunity, adversely affecting long-term protection. These studies prompt consideration that vaccination targeting S. aureus may be most effective if delivered prior to initial contact with the organism.

The Staphylococcus aureus ArlRS two-component system regulates virulence factor expression through MgrA.

The Gram-positive bacterium, Staphylococcus aureus, is a versatile pathogen that can sense and adapt to a wide variety of environments within the human host, in part through its 16 two-component regulatory systems. The ArlRS two-component system has been shown to affect many cellular processes in S. aureus, including autolysis, biofilm formation, capsule synthesis and virulence. Yet the molecular details of this regulation remained largely unknown. We used RNA sequencing to identify the ArlRS regulon, and found 70% overlap with that of the global regulator MgrA. These genes included cell wall-anchored adhesins (ebh, sdrD), polysaccharide and capsule synthesis genes, cell wall remodeling genes (lytN, ddh), the urease operon, genes involved in metal transport (feoA, mntH, sirA), anaerobic metabolism genes (adhE, pflA, nrdDG) and a large number of virulence factors (lukSF, lukAB, nuc, gehB, norB, chs, scn and esxA). We show that ArlR directly activates expression of mgrA and identify a probable ArlR-binding site (TTTTCTCAT-N4 -TTTTAATAA). A highly similar sequence is also found in the spx P2 promoter, which was recently shown to be regulated by ArlRS. We also demonstrate that ArlS has kinase activity toward ArlR in vitro, although it has slower kinetics than other similar histidine kinases.

Identification of Key Determinants of Staphylococcus aureus Vaginal Colonization.

Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission.

Quorum Sensing, Virulence, and Antibiotic Resistance of USA100 Methicillin-Resistant Staphylococcus aureus Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) infections impact all patient populations both in the community and in health care settings. Despite advances in our knowledge of MRSA virulence, little is known about the regulatory mechanisms of USA100 health care-associated MRSA isolates, which are the second most frequently identified MRSA isolates found in all infections. This work focused on the contribution of the USA100 agr type II quorum-sensing system to virulence and antibiotic resistance. From a MRSA strain collection, we selected 16 representative USA100 isolates, constructed mutants with Δagr mutations, and characterized selected strain pairs for virulence factor expression, murine skin infection, and antibiotic resistance. For each strain pair, hemolysis and extracellular protease expression were significantly greater in the wild-type (WT) strains than in the Δagr mutants. Similarly, mice challenged with the WT strains had larger areas of dermonecrosis and greater weight loss than those challenged with the Δagr mutants, demonstrating that the USA100 agr system regulates virulence. Although USA100 isolates exhibit a high level of antibiotic resistance, the WT and Δagr strain pairs showed no difference in MICs by MIC testing. However, in the presence of a sub-MIC of vancomycin, most of the USA100 Δagr mutants exhibited slower growth than the WT isolates, and a couple of the Δagr mutants also grew more slowly in the presence of a sub-MIC of cefoxitin. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence, and it may have a contribution to the optimal survival of these MRSA strains in the presence of antibiotics.IMPORTANCE USA100 health care-associated MRSA isolates are highly antibiotic resistant and can cause invasive disease across all patient populations. Even though USA100 strains are some of the most frequently identified causes of infections, little is known about virulence regulation in these isolates. Our study demonstrates that the USA100 agr quorum-sensing system is important for the control of toxin and exoenzyme production and that the agr system has a key role in skin infection. In some USA100 isolates, the agr system is important for growth in the presence of low levels of antibiotics. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence and that it may make a contribution to the optimal survival of these MRSA strains in the presence of antibiotics.

Staphylococcus aureus adhesion in endovascular infections is controlled by the ArlRS-MgrA signaling cascade.

Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS-MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS-MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS-MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS-MgrA regulatory system.

Biofilm formation by Staphylococcus aureus clinical isolates correlates with the infection type.

BACKGROUND: Biofilms are involved in many Staphylococcus aureus infections, but relation of biofilm formation and the infection types or the clinical outcomes remain unclear. METHODS: We measured biofilm formation, with a microtiter plate assay, of a collection of methicillin-sensitive clinical isolates from 159 invasive S. aureus infections, encompassing all cases occurring within a hospital catchment area during two years, and from additional 49 non-invasive skin infections from the same region. Results were related to available clinical and microbiological documentation. RESULTS: Isolates from medical device infections (intravenous line-associated and prosthetic joint infections), as well as isolates from superficial skin infections, were particularly proficient in forming biofilms. No increased biofilm-forming capacity was seen in isolates from endocarditis, osteomyelitis, or other infections. There was also a correlation of biofilm formation with the agr type of the isolates. Thicker biofilms were more resistant to antibiotic treatment in vitro. No correlation between biofilm formation and clinical outcomes was noted. CONCLUSIONS: S. aureus isolates from 'classical' biofilm-related infections, but also from superficial skin infections, are especially proficient in forming biofilms. There is, however, no obvious relation of biofilm-forming capacity of isolates and the clinical outcome of the infection, and more studies on this issue are needed.