Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

Staphylococcus epidermidis bacteriocin A37 kills natural competitors with a unique mechanism of action.

Many bacteria produce antimicrobial compounds such as lantibiotics to gain advantage in the competitive natural environments of microbiomes. Epilancins constitute an until now underexplored family of lantibiotics with an unknown ecological role and unresolved mode of action. We discovered production of an epilancin in the nasal isolate Staphylococcus epidermidis A37. Using bioinformatic tools, we found that epilancins are frequently encoded within staphylococcal genomes, highlighting their ecological relevance. We demonstrate that production of epilancin A37 contributes to Staphylococcus epidermidis competition specifically against natural corynebacterial competitors. Combining microbiological approaches with quantitative in vivo and in vitro fluorescence microscopy and cryo-electron tomography, we show that A37 enters the corynebacterial cytoplasm through a partially transmembrane-potential-driven uptake without impairing the cell membrane function. Upon intracellular aggregation, A37 induces the formation of intracellular membrane vesicles, which are heavily loaded with the compound and are essential for the antibacterial activity of the epilancin. Our work sheds light on the ecological role of epilancins for staphylococci mediated by a mode of action previously unknown for lantibiotics.

Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus.

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.

Electrogenic Staphylococcus epidermidis colonizes nasal cavities and alleviates IL-6 progression induced by the SARS2-CoV nucleocapsid protein.

AIM: Certain probiotic bacteria have been shown to possess an immunomodulatory effect and a protective effect on influenza infections. Using the Staphylococcus epidermidis K1 colonized mice model, we assessed the effect of nasal administration of glycerol or flavin mononucleotide (FMN) on the production of interleukin (IL)-6 mediated by the severe acute respiratory syndrome coronavirus 2 (SARS2-CoV) nucleocapsid protein (NPP). METHODS AND RESULTS: FMN, one of the key electron donors for the generation of electricity facilitated by S. epidermidis ATCC 12228, was detected in the glycerol fermentation medium. Compared to the S. epidermidis ATCC 12228, the S. epidermidis K1 isolate showed significant expression of the electron transfer genes, including pyruvate dehydrogenase (pdh), riboflavin kinase (rk), 1,4-dihydroxy-2-naphthoate octaprenyltransferase (menA), and type II NADH quinone oxidoreductase (ndh2). Institute of cancer research (ICR) mice were intranasally administered with S. epidermidis K1 with or without pretreatment with riboflavin kinase inhibitors, then nasally treated with glycerol or FMN before inoculating the NPP. Furthermore, J774A.1 macrophages were exposed to NPP serum and then treated with NPP of SARS2-CoV. The IL-6 levels in the bronchoalveolar lavage fluid (BALF) of mice and macrophages were quantified using a mouse IL-6 enzyme-linked immunosorbent assay kit. CONCLUSIONS: Here, we report that nasal administration of NPP strongly elevates IL-6 levels in both BALF and J774A.1 macrophages. It is worth noting that NPP-neutralizing antibodies can decrease IL-6 levels in macrophages. The nasal administration of glycerol or FMN to S. epidermidis K1-colonized mice results in a reduction of NPP-induced IL-6 production.

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

Antibacterial activity of a complex bacteriocin secreted by Staphylococcus epidermidis against Porphyromonas gingivalis.

OBJECTIVE: To characterize the inhibitory activity of a novel bacteriocin produced by Staphylococcus epidermidis against this periodontal pathogen. DESIGN: The bacteriocin activity was evaluated by the agar diffusion method over a lawn of P. gingivalis ATCC 33277. The bacteriocin was purified by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) and Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry (MALDI-TOF-MS). In addition, the bacteriocin host specificity, production on different media cultures and susceptibility to enzymes, pH, and heat treatment were determined. RESULTS: The bacteriocin BAC 14990 was selective to P. gingivalis, suggesting a narrow activity range. The production during the growth curve indicated that S. epidermidis had a continued production of this antimicrobial, showing the highest concentration in the stationary phase. The purification of BAC 14990 showed that bacteriocin had a molecular mass of 5795 Da. BAC 14990 was partially resistant to the treatment with proteinase K and papain, however, was fully susceptible to amylase treatment indicating the presence of sugar residues in the protein, suggesting a conjugated type of bacteriocin. Also, this diffusible inhibitory substance was heat and pH treatment resistant. CONCLUSIONS: The results indicate the isolation of a new staphylococcal complex bacteriocin that is able to eliminate a Gram-negative bacterium. These results could contribute to the development of treatments directed against pathogens in mixed communities, as is the case with oral diseases.

1H, 15N, and 13C chemical shift backbone resonance NMR assignment of the accumulation-associated protein (Aap) lectin domain from Staphylococcus epidermidis.

Staphylococcus epidermidis is the leading causative agent for hospital-acquired infections, especially device-related infections, due to its ability to form biofilms. The accumulation-associated protein (Aap) of S. epidermidis is primarily responsible for biofilm formation and consists of two domains, A and B. It was found that the A domain is responsible for the attachment to the abiotic/biotic surface, whereas the B domain is responsible for the accumulation of bacteria during biofilm formation. One of the parts of the A domain is the Aap lectin, which is a carbohydrate-binding domain having 222 amino acids in its structure. Here we report the near complete backbone chemical shift assignments for the lectin domain, as well as its predicted secondary structure. This data will provide a platform for future NMR studies to explore the role of lectin in biofilm formation.

Stability and Resistance to Proteolysis of Enterotoxins SEC and SEL Produced by Staphylococcus epidermidis and Staphylococcus aureus.

The only staphylococcal enterotoxins produced by Staphylococcus epidermidis include SECepi and SELepi, whereas Staphylococcus aureus produces orthologous SECs and SEL having different sequences. We compared S. epidermidis and S. aureus SECs and SELs in terms of resistance to proteolysis and both, thermal and chemical stability. We show that SECepi and SELepi produced by S. epidermidis have similar resistance to proteolysis if compared with their respective orthologues produced by S. aureus. Studied S. epidermidis and S. aureus SEC variants incubated with pepsin at pH 2.0 were found to be more resistant to proteolysis than SELs. SELs turned out to be more resistant than SECs to proteolysis with trypsin at pH 8.0. SECepi was found to be more resistant to thermal denaturation if compared with its S. aureus orthologues. The S. epidermidis and S. aureus SEC variants were found to have higher thermal stability than SELs. Our data indicate that, due to their high stability, the enterotoxins SECepi and SELepi produced in food by S. epidermidis may pose a food safety risk comparable with that posed by S. aureus enterotoxins.

Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

The endoglycosidase activity of Dispersin B is mediated through electrostatic interactions with cationic poly-ß-(1→6)-N-acetylglucosamine.

Bacterial biofilms consist of bacterial cells embedded within a self-produced extracellular polymeric substance (EPS) composed of exopolysaccharides, extra cellular DNA, proteins and lipids. The enzyme Dispersin B (DspB) is a CAZy type 20 ß-hexosaminidase enzyme that catalyses the hydrolysis of poly-N-acetylglucosamine (PNAG), a major biofilm polysaccharide produced by a wide variety of biofilm-forming bacteria. Native PNAG is partially de-N-acetylated, and the degree of deacetylation varies between species and dependent on the environment. We have previously shown that DspB is able to perform both endo- and exo-glycosidic bond cleavage of PNAG depending on the de-N-acetylation patterns present in the PNAG substrate. Here, we used a combination of synthetic PNAG substrate analogues, site-directed mutagenesis and in vitro biofilm dispersal assay to investigate the molecular basis for the endo-glycosidic cleavage activity of DspB and the importance of this activity for dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. We found that D242 contributes to the endoglycosidase activity of DspB through electrostatic interactions with cationic substrates in the -2 binding site. A DspBD242N mutant was highly deficient in endoglycosidase activity while maintaining exoglycosidase activity. When used to disperse S. epidermidis biofilms, this DspBD242N mutant resulted in an increase in residual biofilm biomass after treatment when compared to wild-type DspB. These results suggest that the de-N-acetylation of PNAG in S. epidermidis biofilms is not uniformly distributed and that the endoglycosidase activity of DspB is required for efficient biofilm dispersal.

Structures of an active type III-A CRISPR effector complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.

Evaluation of the Effects of Ag, Cu, ZnO and TiO2 Nanoparticles on the Expression Level of Oxidative Stress-Related Genes and the Activity of Antioxidant Enzymes in Escherichia coli, Bacillus cereus and Staphylococcus epidermidis.

Although the molecular response of bacteria exposed to metal nanoparticles (NPs) is intensively studied, many phenomena related to their survival, metal uptake, gene expression and protein production are not fully understood. Therefore, this work aimed to study Ag-NPs, Cu-NPs, ZnO-NPs and TiO2-NPs-induced alterations in the expression level of selected oxidative stress-related genes in connection with the activity of antioxidant enzymes: catalase (CAT), peroxidase (PER) and superoxide dismutase (SOD) in Escherichia coli, Bacillus cereus and Staphylococcus epidermidis. The methodology used included: the extraction of total RNA and cDNA synthesis, the preparation of primers for selected housekeeping and oxidative stress genes, RT-qPCR reaction and the measurements of CAT, PER and SOD activities. It was established that the treatment of E. coli and S. epidermidis with NPs resulted mainly in the down-regulation of targeted genes, whilst the up-regulation of genes was confirmed in B. cereus. The greatest differences in the relative expression levels of tested genes occurred in B. cereus and S. epidermidis treated with TiO2-NPs, while in E. coli, they were observed under ZnO-NPs exposure. The changes found were mostly related to the expression of genes encoding proteins with PER and CAT-like activity. Among NPs, ZnO-NPs and Cu-NPs increased the activity of antioxidants in E. coli and B. cereus. In turn, TiO2-NPs had a major effect on enzymes activity in S. epidermidis. Considering all of the collected results for tested bacteria, it can be emphasised that the impact of NPs on the antioxidant system functioning was dependent on their type and concentration.

The expression of glycosyltransferases sdgA and sdgB in Staphylococcus epidermidis depends on the conditions of biofilm formation.

The Staphylococcus aureus SdrG protein is glycosylated by SdgA and SdgB for protection against its degradation by the neutrophil cathepsin G. So far, there is no information about the role of Staphylococcus epidermidis SdgA or SdgB in biofilm-forming; therefore, the focus of this work was to determine the distribution and expression of the sdrG, sdgA and sdgB genes in S. epidermidis under in vitro and in vivo biofilm conditions. The frequencies of the sdrG, sdgA and sdgB genes were evaluated by PCR in a collection of 75 isolates. Isolates were grown in dynamic (non-biofilm-forming) or static (biofilm-forming) conditions. The expression of sdrG, sdgA and sdgB was determined by RT-qPCR in cells grown under dynamic conditions (CGDC), as well as in planktonic and sessile cells from a biofilm and cells adhered to a catheter implanted in Balb/c mice. The sdrG and sdgB genes were detected in 100% of isolates, while the sdgA gene was detected in 71% of the sample (p < 0.001). CGDC did not express sdrG, sdgA and sdgB mRNAs. Planktonic and sessile cells expressed sdrG and sdgB, and the same was observed in cells adhered to the catheter. In particular, one isolate, capable of inducing a biofilm under treatment with cathepsin G, expressed sdrG and sdgB in planktonic and sessile cells and cells adhering to the catheter. This suggests that bacteria require biofilm conditions as an important factor for the transcription of the sdgA, sdgB and sdrG genes.

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation.

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 ΔspoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 ΔspoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 ΔspoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions.

Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system.

CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called 'spacers', into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.

The Extracellular Vesicles from the Commensal Staphylococcus Epidermidis ATCC12228 Strain Regulate Skin Inflammation in the Imiquimod-Induced Psoriasis Murine Model.

Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis' EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis' EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis' EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble ß1/ß2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.

Insight into the Antibacterial Activity of Selected Metal Nanoparticles and Alterations within the Antioxidant Defence System in Escherichia coli, Bacillus cereus and Staphylococcus epidermidis.

The antimicrobial activity of nanoparticles (NPs) is a desirable feature of various products but can become problematic when NPs are released into different ecosystems, potentially endangering living microorganisms. Although there is an abundance of advanced studies on the toxicity and biological activity of NPs on microorganisms, the information regarding their detailed interactions with microbial cells and the induction of oxidative stress remains incomplete. Therefore, this work aimed to develop accurate oxidation stress profiles of Escherichia coli, Bacillus cereus and Staphylococcus epidermidis strains treated with commercial Ag-NPs, Cu-NPs, ZnO-NPs and TiO2-NPs. The methodology used included the following determinations: toxicological parameters, reactive oxygen species (ROS), antioxidant enzymes and dehydrogenases, reduced glutathione, oxidatively modified proteins and lipid peroxidation. The toxicological studies revealed that E. coli was most sensitive to NPs than B. cereus and S. epidermidis. Moreover, NPs induced the generation of specific ROS in bacterial cells, causing an increase in their concentration, which further resulted in alterations in the activity of the antioxidant defence system and protein oxidation. Significant changes in dehydrogenases activity and elevated lipid peroxidation indicated a negative effect of NPs on bacterial outer layers and respiratory activity. In general, NPs were characterised by very specific nano-bio effects, depending on their physicochemical properties and the species of microorganism.

Biofilm formation of Staphylococcus epidermidis imaged using atmospheric scanning electron microscopy.

Staphylococcus epidermidis are gram-positive bacteria that form a biofilm around implanted devices and develop an infection into a chronic state. Recently, it has been revealed that microvesicles have important roles in biofilm formation and intercellular communication among bacteria. However, biofilm formation of Staphylococcus epidermidis, and its relation to microvesicle secretion, is poorly understood because of the difficulty required to preserve the delicate water-rich morphology of biofilm for high-resolution observations. Here, we successfully imaged the microvesicles secreted from Staphylococcus epidermidis and the subsequent process of their integration into biofilm using liquid-phase imaging using atmospheric scanning electron microscopy (ASEM). In the biofilm, cells were connected by nanotube-like structures attached by microvesicles, and surrounded by extracellular polymeric substances. Cells cultured in the ASEM specimen holder were aldehyde-fixed and stained using positively charged nanogold labelling and/or using National Center for Microscopy and Imaging Research method. The samples immersed in aqueous radical scavenger glucose buffer were imaged by the inverted SEM of ASEM. Information regarding the morphologies of microvesicles, nanotube-like fibrils, and biofilm formed by Staphylococcus epidermidis is expected to be useful to elucidate the biological mechanism of biofilm formation and to develop a medicine against biofilms and their associated infections.

The Multitasking Surface Protein of Staphylococcus epidermidis: Accumulation-Associated Protein (Aap).

The stratum corneum is the outermost layer of the epidermis and is thus directly exposed to the environment. It consists mainly of corneocytes, which are keratinocytes in the last stage of differentiation, having neither nuclei nor organelles. However, they retain keratin filaments embedded in filaggrin matrix and possess a lipid envelope which protects the body from desiccation. Despite the desiccated, nutrient-poor, and acidic nature of the skin making it a hostile environment for most microorganisms, this organ is colonized by commensal microbes. Among the classic skin commensals are Propionibacterium acnes and coagulase-negative staphylococci (CoNS) with Staphylococcus epidermidis as a leading species. An as-yet-unanswered question is what enables S. epidermis to colonize skin so successfully. In their recent article, P. D. Fey and his colleagues (P. Roy, A. R. Horswill, and P. D. Fey, mBio 12:e02908-20, 2021, https://doi.org/10.1128/mBio.02908-20) have brought us one step closer to answering this question.

Patient-specific effects of soluble factors from Staphylococcus aureus and Staphylococcus epidermidis biofilms on osteogenic differentiation of primary human osteoblasts.

Due to the frequency of biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis in orthopedics, it is crucial to understand the interaction between the soluble factors produced by prokaryotes and their effects on eukaryotes. Our knowledge concerning the effect of soluble biofilm factors (SBF) and their virulence potential on osteogenic differentiation is limited to few studies, particularly when there is no direct contact between prokaryotic and eukaryotic cells. SBF were produced by incubating biofilm from S. aureus and S. epidermidis in osteogenic media. Osteoblasts of seven donors were included in this study. Our results demonstrate that the detrimental effects of these pathogens do not require direct contact between prokaryotic and eukaryotic cells. SBF produced by S. aureus and S. epidermidis affect the metabolic activity of osteoblasts. However, the effect of SBF derived from S. aureus seems to be more pronounced compared to that of S. epidermidis. The influence of SBF of S. aureus and S. epidermidis on gene expression of COL1A1, ALPL, BGLAP, SPP1, RUNX2 is bacteria-, patient-, concentration-, and incubation time dependent. Mineralization was monitored by staining the calcium and phosphate deposition and revealed that the SBF of S. epidermidis markedly inhibits calcium deposition; however, S. aureus shows a less inhibitory effect. Therefore, these new findings support the hypotheses that soluble biofilm factors affect the osteogenic processes substantially, particularly when there is no direct interaction between bacteria and osteoblast.