Staphylococcus aureus membrane vesicles: an evolving story.

Staphylococcus aureus is an important bacterial pathogen that causes a wide variety of human diseases in community and hospital settings. S. aureus employs a diverse array of virulence factors, both surface-associated and secreted, to promote colonization, infection, and immune evasion. Over the past decade, a growing body of research has shown that S. aureus generates extracellular membrane vesicles (MVs) that package a variety of bacterial components, many of which are virulence factors. In this review, we summarize recent advances in our understanding of S. aureus MVs and highlight their biogenesis, cargo, and potential role in the pathogenesis of staphylococcal infections. Lastly, we present some emerging questions in the field.

Staphylococcus aureus delta toxin modulates both extracellular membrane vesicle biogenesis and amyloid formation.

IMPORTANCE: Extracellular membrane vesicles (MVs) produced by Staphylococcus aureus in planktonic cultures encapsulate a diverse cargo of bacterial proteins, nucleic acids, and glycopolymers that are protected from destruction by external factors. δ-toxin, a member of the phenol soluble modulin family, was shown to be critical for MV biogenesis. Amyloid fibrils co-purified with MVs generated by virulent, community-acquired S. aureus strains, and fibril formation was dependent on expression of the S. aureus δ-toxin gene (hld). Mass spectrometry data confirmed that the amyloid fibrils were comprised of δ-toxin. Although S. aureus MVs were produced in vivo in a localized murine infection model, amyloid fibrils were not observed in the in vivo setting. Our findings provide critical insights into staphylococcal factors involved in MV biogenesis and amyloid formation.

On-person adaptive evolution of Staphylococcus aureus during treatment for atopic dermatitis.

The opportunistic pathogen Staphylococcus aureus frequently colonizes the inflamed skin of people with atopic dermatitis (AD) and worsens disease severity by promoting skin damage. Here, we show, by longitudinally tracking 23 children treated for AD, that S. aureus adapts via de novo mutations during colonization. Each patient's S. aureus population is dominated by a single lineage, with infrequent invasion by distant lineages. Mutations emerge within each lineage at rates similar to those of S. aureus in other contexts. Some variants spread across the body within months, with signatures of adaptive evolution. Most strikingly, mutations in capsule synthesis gene capD underwent parallel evolution in one patient and across-body sweeps in two patients. We confirm that capD negativity is more common in AD than in other contexts, via reanalysis of S. aureus genomes from 276 people. Together, these findings highlight the importance of the mutation level when dissecting the role of microbes in complex disease.

Staphylococcus aureus Delta Toxin Modulates both Extracellular Membrane Vesicle Biogenesis and Amyloid Formation.

Staphylococcus aureus secretes phenol-soluble modulins (PSMs), a family of small, amphipathic, secreted peptides with multiple biologic activities. Community-acquired S. aureus strains produce high levels of PSMs in planktonic cultures, and PSM alpha peptides have been shown to augment the release of extracellular membrane vesicles (MVs). We observed that amyloids, aggregates of proteins characterized by a fibrillar morphology and stained with specific dyes, co-purified with MVs harvested from cell-free culture supernatants of community-acquired S. aureus strains. δ-toxin was a major component of amyloid fibrils that co-purified with strain LAC MVs, and δ-toxin promoted the production of MVs and amyloid fibrils in a dose-dependent manner. To determine whether MVs and amyloid fibrils were generated under in vivo conditions, we inoculated mice with S. aureus harvested from planktonic cultures. Bacterial MVs could be isolated and purified from lavage fluids recovered from infected animals. Although δ-toxin was the most abundant PSM in lavage fluids, amyloid fibrils could not be detected in these samples. Our findings expand our understanding of amyloid fibril formation in S. aureus cultures, reveal important roles of δ-toxin in amyloid fibril formation and MV biogenesis, and demonstrate that MVs are generated in vivo in a staphylococcal infection model. Importance: Extracellular membrane vesicles (MVs) produced by Staphylococcus aureus in planktonic cultures encapsulate a diverse cargo of bacterial proteins, nucleic acids, and glycopolymers that are protected from destruction by external factors. δ-toxin, a member of the phenol soluble modulin family, was shown to be critical for MV biogenesis. Amyloid fibrils co-purified with MVs generated by virulent, community-acquired S. aureus strains, and fibril formation was dependent on expression of the S. aureus δ-toxin gene ( hld ). Mass spectrometry data confirmed that the amyloid fibrils were comprised of δ-toxin. Although S. aureus MVs were produced in vivo in a localized murine infection model, amyloid fibrils were not observed in the in vivo setting. Our findings provide critical insights into staphylococcal factors involved in MV biogenesis and amyloid formation.

Contribution of Extracellular Membrane Vesicles To the Secretome of Staphylococcus aureus.

The microbial secretome modulates how the organism interacts with its environment. Included in the Staphylococcus aureus secretome are extracellular membrane vesicles (MVs) that consist of cytoplasmic and membrane proteins, as well as exoproteins, some cell wall-associated proteins, and glycopolymers. The extent to which MVs contribute to the diverse composition of the secretome is not understood. We performed a proteomic analysis of MVs purified from the S. aureus strain MRSA252 along with a similar analysis of the whole secretome (culture supernatant) before and after depletion of MVs. The MRSA252 secretome was comprised of 1,001 proteins, of which 667 were also present in MVs. Cell membrane-associated proteins and lipoteichoic acid in the culture supernatant were highly associated with MVs, followed by cytoplasmic and extracellular proteins. Few cell wall-associated proteins were contained in MVs, and capsular polysaccharides were found both in the secretome and MVs. When MVs were removed from the culture supernatant by ultracentrifugation, 54 of the secretome proteins were significantly depleted in abundance. Proteins packaged in MVs were characterized by an isoelectric point that was significantly higher than that of proteins excluded from MVs. Our data indicate that the generation of S. aureus MVs is a mechanism by which lipoteichoic acid, cytoplasmic, and cell membrane-associated proteins are released into the secretome. IMPORTANCE The secretome of Staphylococcus aureus includes soluble molecules and nano-sized extracellular membrane vesicles (MVs). The protein composition of both the secretome and MVs includes cytoplasmic and membrane proteins, as well as exoproteins, some cell wall-associated proteins, and glycopolymers. How the MV cargo differs from the protein composition of the secretome has not yet been addressed. Although the compositions of the secretome and MVs were strikingly similar, we identified 54 proteins that were specifically packaged in MVs. Proteins highly associated with MVs were characterized by their abundance in the secretome, an association with the bacterial membrane, and a basic isoelectric point. This study deepens our limited understanding about the contribution of MVs to the secretome of S. aureus.

GltS regulates biofilm formation in methicillin-resistant Staphylococcus aureus.

Biofilm-based infection is a major healthcare burden. Methicillin-resistant Staphylococcus aureus (MRSA) is one of major organisms responsible for biofilm infection. Although biofilm is induced by a number of environmental signals, the molecule responsible for environmental sensing is not well delineated. Here we examined the role of ion transporters in biofilm formation and found that the sodium-glutamate transporter gltS played an important role in biofilm formation in MRSA. This was shown by gltS transposon mutant as well as its complementation. The lack of exogenous glutamate also enhanced biofilm formation in JE2 strain. The deficiency of exogenous glutamate intake accelerated endogenous glutamate/glutamine production, which led to the activation of the urea cycle. We also showed that urea cycle activation was critical for biofilm formation. In conclusion, we showed that gltS was a critical regulator of biofilm formation by controlling the intake of exogenous glutamate. An intervention to target glutamate intake may be a potential useful approach against biofilm.

Pilot comparison of the ease of swallowing of single tablet antiretroviral regimens.

Daily adherence to lifelong antiretroviral therapy (ART) is required to achieve long term treatment success. However, patient preferences for ART tablet size have not been well studied. Our study assessed factors associated with the ease of swallowing (EoS) and tolerability of two placebo tablets representing and matching B/F/TAF (BPT) and DTG/ABC/3TC (DPT). Fifty ART-naïve patients were randomized into a two-period cross-over study. Likert scale (1-5) questionnaires were administered to assess patient factors influencing the ease of swallowing, adherence, home medications, medication preferences and perceptions. Comparisons were done using Student t-tests and ordinal regression. Participants were 64% female, 61% white, mean age 43 years, and taking a mean (median) of 4(1) pills/day. BPT was reported to be easier than DPT with ease of swallowability 1.76 vs. 2.42 (p < 0.001) (1 = very easy). DPT tablet was correctly perceived as larger than BPT (p < 0.001); with both tablets perceived as smaller than actual size (p < 0.001). EoS of either tablet was positively associated with the EoS of the largest home tablet medication (p = 0.021, p = 0.03). Patient's perceptions of EoS can affect their medication adherence, especially in HIV, and should be considered in treatment regimens.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

IBT-V02: A Multicomponent Toxoid Vaccine Protects Against Primary and Secondary Skin Infections Caused by Staphylococcus aureus.

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.

Staphylococcus aureus Extracellular Vesicles: A Story of Toxicity and the Stress of 2020.

Staphylococcus aureus generates and releases extracellular vesicles (EVs) that package cytosolic, cell-wall associated, and membrane proteins, as well as glycopolymers and exoproteins, including alpha hemolysin, leukocidins, phenol-soluble modulins, superantigens, and enzymes. S. aureus EVs, but not EVs from pore-forming toxin-deficient strains, were cytolytic for a variety of mammalian cell types, but EV internalization was not essential for cytotoxicity. Because S. aureus is subject to various environmental stresses during its encounters with the host during infection, we assessed how these exposures affected EV production in vitro. Staphylococci grown at 37 °C or 40 °C did not differ in EV production, but cultures incubated at 30 °C yielded more EVs when grown to the same optical density.S. aureus cultivated in the presence of oxidative stress, in iron-limited media, or with subinhibitory concentrations of ethanol, showed greater EV production as determined by protein yield and quantitative immunoblots. In contrast, hyperosmotic stress or subinhibitory concentrations of erythromycin reduced S. aureus EV yield. EVs represent a novel S. aureus secretory system that is affected by a variety of stress responses and allows the delivery of biologically active pore-forming toxins and other virulence determinants to host cells.

Orchestration of human macrophage NLRP3 inflammasome activation by Staphylococcus aureus extracellular vesicles.

Release of extracellular vesicles (EVs) is a common feature among eukaryotes, archaea, and bacteria. However, the biogenesis and downstream biological effects of EVs released from gram-positive bacteria remain poorly characterized. Here, we report that EVs purified from a community-associated methicillin-resistant Staphylococcus aureus strain were internalized into human macrophages in vitro and that this process was blocked by inhibition of the dynamin-dependent endocytic pathway. Human macrophages responded to S. aureus EVs by TLR2 signaling and activation of NLRP3 inflammasomes through K+ efflux, leading to the recruitment of ASC and activation of caspase-1. Cleavage of pro-interleukin (IL)-1ß, pro-IL-18, and gasdermin-D by activated caspase-1 resulted in the cellular release of the mature cytokines IL-1ß and IL-18 and induction of pyroptosis. Consistent with this result, a dose-dependent cytokine response was detected in the extracellular fluids of mice challenged intraperitoneally with S. aureus EVs. Pore-forming toxins associated with S. aureus EVs were critical for NLRP3-dependent caspase-1 activation of human macrophages, but not for TLR2 signaling. In contrast, EV-associated lipoproteins not only mediated TLR2 signaling to initiate the priming step of NLRP3 activation but also modulated EV biogenesis and the toxin content of EVs, resulting in alterations in IL-1ß, IL-18, and caspase-1 activity. Collectively, our study describes mechanisms by which S. aureus EVs induce inflammasome activation and reveals an unexpected role of staphylococcal lipoproteins in EV biogenesis. EVs may serve as a novel secretory pathway for S. aureus to transport protected cargo in a concentrated form to host cells during infections to modulate cellular functions.

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus.

Capsular polysaccharide (CP) biosynthesis in Staphylococcus aureus is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in nongrowing cells. Expression of the corresponding biosynthesis gene cluster is driven by one promoter element (Pcap ). Here, we demonstrate that Pcap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat (IR) that is crucial for cap expression. The essentiality of the IR is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, Pcap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for cap repressors such as SaeR, CodY and Rot. Heterogeneous CP synthesis is determined by SigB activity and repressor binding to the upstream region. SigB dependency and regulation by the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when transcription is rendered constitutive, suggesting additional posttranscriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.

Release of Staphylococcus aureus extracellular vesicles and their application as a vaccine platform.

Secretion of extracellular vesicles (EVs), a process common to eukaryotes, archae, and bacteria, represents a secretory pathway that allows cell-free intercellular communication. Microbial EVs package diverse proteins and influence the host-pathogen interaction, but the mechanisms underlying EV production in Gram-positive bacteria are poorly understood. Here we show that EVs purified from community-associated methicillin-resistant Staphylococcus aureus package cytosolic, surface, and secreted proteins, including cytolysins. Staphylococcal alpha-type phenol-soluble modulins promote EV biogenesis by disrupting the cytoplasmic membrane; whereas, peptidoglycan cross-linking and autolysin activity modulate EV production by altering the permeability of the cell wall. We demonstrate that EVs purified from a S. aureus mutant that is genetically engineered to express detoxified cytolysins are immunogenic in mice, elicit cytolysin-neutralizing antibodies, and protect the animals in a lethal sepsis model. Our study reveals mechanisms underlying S. aureus EV production and highlights the usefulness of EVs as a S. aureus vaccine platform.

Optimization of Stress-Based Microfluidic Testing for Methicillin Resistance in Staphylococcusaureus Strains.

The rapid evolution of antibiotic resistance in bacterial pathogens is driving the development of innovative, rapid antibiotic susceptibility testing (AST) tools as a way to provide more targeted and timely antibiotic treatment. We have previously presented a stress-based microfluidic method for the rapid determination of antibiotic susceptibility in methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). In this method, stress is used to potentiate the action of antibiotics, and cell death is measured as a proxy for susceptibility. The method allows antibiotic susceptibility to be determined within an hour from the start of the antibiotic introduction. However, the relatively low dynamic range of the signal (2–10% cell response) even with high antibiotic concentrations (10–50 µg/mL) left room for the method’s optimization. We have conducted studies in which we varied the flow patterns, the media composition, and the antibiotic concentration to increase the cell death response and concordantly decrease the required antibiotic concentration down to 1–3 µg/mL, in accordance with the Clinical and Laboratory Standards Institute’s (CLSI) guidelines for AST breakpoint concentrations.

Rapid phenotypic stress-based microfluidic antibiotic susceptibility testing of Gram-negative clinical isolates.

Bacteremia is a life-threatening condition for which antibiotics must be prescribed within hours of clinical diagnosis. Since the current gold standard for bacteremia diagnosis is based on conventional methods developed in the mid-1800s-growth on agar or in broth-identification and susceptibility profiling for both Gram-positive and Gram-negative bacterial species requires at least 48-72 h. Recent advancements in accelerated phenotypic antibiotic susceptibility testing have centered on the microscopic growth analysis of small bacterial populations. These approaches are still inherently limited by the bacterial growth rate. Our approach is fundamentally different. By applying environmental stress to bacteria in a microfluidic platform, we can correctly assign antibiotic susceptibility profiles of clinically relevant Gram-negative bacteria within two hours of antibiotic introduction rather than 8-24 h. The substantial expansion to include a number of clinical isolates of important Gram-negative species-Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa-reported here underscores the broad utility of our approach, complementing the method's proven utility for Gram-positive bacteria. We also demonstrate that the platform is compatible with antibiotics that have varying mechanisms of action-meropenem, gentamicin, and ceftazidime-highlighting the versatility of this platform.

Evaluation of serotypes 5 and 8 capsular polysaccharides in protection against Staphylococcus aureus in murine models of infection.

Staphylococcus aureus is the leading cause of nosocomial and community-acquired infections, including soft tissue and skin infections and bacteremia. However, efforts to develop an effective vaccine against S. aureus infections have not been successful. We evaluated serotypes 5 and 8 capsule polysaccharides (CP) CRM197 conjugates as vaccine candidates in murine models of bacteremia, lethal sepsis, and skin infection. The conjugate vaccines elicited a good antibody response, and active immunization of CP5-CRM or CP8-CRM conjugates protected against staphylococcal bacteremia. In the skin infection model, CP8-CRM but not CP5-CRM protected against dermonecrosis, and CP8-CRM immunization significantly decreased the bacterial burden in the lesion. However, neither CP5-CRM nor CP8-CRM protected against mortality in the lethal sepsis model. The results indicate the capsular vaccines elicit protection against some, but not all, aspects of staphylococcal infection.

Antibodies to Staphylococcus aureus capsular polysaccharides 5 and 8 perform similarly in vitro but are functionally distinct in vivo.

The capsular polysaccharide (CP) produced by Staphylococcus aureus is a virulence factor that allows the organism to evade uptake and killing by host neutrophils. Polyclonal antibodies to the serotype 5 (CP5) and type 8 (CP8) capsular polysaccharides are opsonic and protect mice against experimental bacteremia provoked by encapsulated staphylococci. Thus, passive immunotherapy using CP antibodies has been considered for the prevention or treatment of invasive antibiotic-resistant S. aureus infections. In this report, we generated monoclonal antibodies (mAbs) against S. aureus CP5 or CP8. Backbone specific mAbs reacted with native and O-deacetylated CPs, whereas O-acetyl specific mAbs reacted only with native CPs. Reference strains of S. aureus and a selection of clinical isolates reacted by colony immunoblot with the CP5 and CP8 mAbs in a serotype-specific manner. The mAbs mediated in vitro CP type-specific opsonophagocytic killing of S. aureus strains, and mice passively immunized with CP5 mAbs were protected against S. aureus bacteremia. Neither CP8-specific mAbs or polyclonal antibodies protected mice against bacteremia provoked by serotype 8 S. aureus clinical isolates, although these same antibodies did protect against a serotype 5 S. aureus strain genetically engineered to produce CP8. We detected soluble CP8 in culture supernatants of serotype 8 clinical isolates and in the plasma of infected animals. Serotype 5 S. aureus released significantly less soluble CP5 in vitro and in vivo. The release of soluble CP8 by S. aureus may contribute to the inability of CP8 vaccines or antibodies to protect against serotype 8 staphylococcal infections.

Structure and Function of Surface Polysaccharides of Staphylococcus aureus.

The major surface polysaccharides of Staphylococcus aureus include the capsular polysaccharide (CP), cell wall teichoic acid (WTA), and polysaccharide intercellular adhesin/poly-ß(1-6)-N-acetylglucosamine (PIA/PNAG). These glycopolymers are important components of the staphylococcal cell envelope, but none of them is essential to S. aureus viability and growth in vitro. The overall biosynthetic pathways of CP, WTA, and PIA/PNAG have been elucidated, and the functions of most of the biosynthetic enzymes have been demonstrated. Because S. aureus CP and WTA (but not PIA/PNAG) utilize a common cell membrane lipid carrier (undecaprenyl-phosphate) that is shared by the peptidoglycan biosynthesis pathway, there is evidence that these processes are highly integrated and temporally regulated. Regulatory elements that control glycopolymer biosynthesis have been described, but the cross talk that orchestrates the biosynthetic pathways of these three polysaccharides remains largely elusive. CP, WTA, and PIA/PNAG each play distinct roles in S. aureus colonization and the pathogenesis of staphylococcal infection. However, they each promote bacterial evasion of the host immune defences, and WTA is being explored as a target for antimicrobial therapeutics. All the three glycopolymers are viable targets for immunotherapy, and each (conjugated to a carrier protein) is under evaluation for inclusion in a multivalent S. aureus vaccine. Future research findings that increase our understanding of these surface polysaccharides, how the bacterial cell regulates their expression, and their biological functions will likely reveal new approaches to controlling this important bacterial pathogen.

Dynamic in vivo mutations within the ica operon during persistence of Staphylococcus aureus in the airways of cystic fibrosis patients.

Cystic fibrosis (CF) is associated with chronic bacterial airway infections leading to lung insufficiency and decreased life expectancy. Staphylococcus aureus is one of the most prevalent pathogens isolated from the airways of CF patients. Mucoid colony morphology has been described for Pseudomonas aeruginosa, the most common pathogen in CF, but not for S. aureus. From the airways of 8 of 313 CF patients (2.5%) mucoid S. aureus isolates (n = 115) were cultured with a mean persistence of 29 months (range 1 month, 126 months). In contrast to non-mucoid S. aureus, mucoid isolates were strong biofilm formers. The upstream region of the ica operon, which encodes the proteins responsible for the synthesis of the polysaccharide intercellular adhesin (PIA), of mucoid isolates was sequenced. Spa-types of mucoid and non-mucoid strains were identical, but differed between patients. Mucoid isolates carried a 5 bp deletion in the intergenic region between icaR and icaA. During long-term persistence, from two patients subsequent non-mucoid isolates (n = 12) with 5 bp deletions were cultured, which did not produce biofilm. Sequencing of the entire ica operon identified compensatory mutations in various ica-genes including icaA (n = 7), icaD (n = 3) and icaC (n = 2). Six sequential isolates of each of these two patients with non-mucoid and mucoid phenotypes were subjected to whole genome sequencing revealing a very close relationship of the individual patient's isolates. Transformation of strains with vectors expressing the respective wild-type genes restored mucoidy. In contrast to the non-mucoid phenotype, mucoid strains were protected against neutrophilic killing and survived better under starvation conditions. In conclusion, the special conditions present in CF airways seem to facilitate ongoing mutations in the ica operon during S. aureus persistence.