Heterogeneous Regulation of StaphylococcusAureus by Different StaphylococcusEpidermidisagr Types in Atopic Dermatitis.

The skin commensal Staphylococcus epidermidis exhibits a protective role in skin inflammation; however, the exact functions of S. epidermidis and their mechanisms in atopic dermatitis (AD) are not fully understood. Here, whole-genome sequencing was conducted on strains of S. epidermidis isolated from pediatric patients with AD and revealed significant strain-level heterogeneity in functional genes. Specific sequence analysis of S. epidermidis identified four types of accessory gene regulator (agr) according to locus variations in the agr operon, which was consistent with the metagenomic data of the contextual microbiota. The number of S. epidermidisagr type I was slightly decreased among AD isolates, whereas agr type IV was hardly detected in AD isolates. Functional experiments showed that strains of S. epidermidisagr types I and IV, but not types II and III, inhibited the expression of S. aureusagr-mediated virulence factors in vitro, suppressed S. aureus epidermal colonization, and attenuated skin inflammation in a mouse model. The delineation of genome signatures of S. epidermidis at the strain level in AD and the quorum-sensing interference between S. epidermidisagr type IV and S. aureus provide a foundation for the modulation of the skin microbiota and the treatment of AD.

The influence of the commensal skin bacterium Staphylococcus epidermidis on the epidermal barrier and inflammation: Implications for atopic dermatitis.

The skin microbiota is a crucial component in maintaining cutaneous barrier function. Staphylococcus epidermidis is considered as a beneficial commensal member of the cutaneous microbiota promoting skin health. However, S. epidermidis is also frequently detectable in the skin of patients with the inflammatory skin disease atopic dermatitis (AD) and some studies reported a significantly higher presence of S. epidermidis in severe AD as compared to mild AD. Therefore, this study aimed to analyse the impact of S. epidermidis on the expression of cutaneous inflammatory mediators and skin barrier molecules. Various S. epidermidis skin-derived isolates activated the proinflammatory transcription factor NF-kappaB and induced expression of AD-associated proinflammatory cytokines in human primary keratinocytes and 3D skin equivalents. Skin barrier molecules such as filaggrin were downregulated by S. epidermidis. In general, AD-derived S. epidermidis strains elicited a higher response than strains derived from the skin of healthy individuals. Taken together, our results provide further evidence that the abundance of S. epidermidis in AD may trigger the inflammatory scenario associated with this disease.

Cec4-Derived Peptide Inhibits Planktonic and Biofilm-Associated Methicillin Resistant Staphylococcus epidermidis.

Staphylococcus epidermidis is part of the normal microbiota that colonizes the skin and mucosal surfaces of human beings. Previous studies suggested that S. epidermidis possessed low virulence, but recent studies confirmed that it can acquire high virulence from Staphylococcus aureus and with the increasing detection of methicillin-resistant S. epidermidis. It has become a major pathogen of graft-associated and hospital-acquired infections. In previous studies, we modified the antimicrobial peptide Cec4 (41 amino acids) and obtained the derived peptide C9 (16 amino acids) showing better antimicrobial activity against S. epidermidis with an MIC value of 8 µg/mL. The peptide has rapid bactericidal activity without detectable high-level resistance, showing certain inhibition and eradication ability on S. epidermidis biofilms. The damage of cell membrane structures by C9 was observed by scanning emission microscopy (SEM) and transmission electron microscopy (TEM). In addition, C9 altered the S. epidermidis cell membrane permeability, depolarization levels, fluidity, and reactive oxygen species (ROS) accumulation and possessed the ability to bind genomic DNA. Analysis of the transcriptional profiles of C9-treated cells revealed changes in genes involved in cell wall and ribosome biosynthesis, membrane protein transport, oxidative stress, and DNA transcription regulation. At the same time, the median lethal dose of C9 in mice was more than 128 mg/kg, and the intraperitoneal administration of 64 mg/kg was less toxic to the liver and kidneys of mice. Furthermore, C9 also showed a certain therapeutic effect on the mouse bacteremia model. In conclusion, C9 may be a candidate drug against S. epidermidis, which has the potential to be further developed as an antibacterial therapeutic agent. IMPORTANCE S. epidermidis is one of the most important pathogens of graft-related infection and hospital-acquired infection. The growing problem of antibiotic resistance, as well as the emergence of bacterial pathogenicity, highlights the need for antimicrobials with new modes of action. Antimicrobial peptides have been extensively studied over the past 30 years as ideal alternatives to antibiotics, and we report here that the derived peptide C9 is characterized by rapid bactericidal and antibiofilm activity, avoiding the development of resistance by acting on multiple nonspecific targets of the cell membrane or cell components. In addition, it has therapeutic potential against S. epidermidis infection in vivo. This study provides a rationale for the further development and application of C9 as an effective candidate antibiotic.

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap.

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation. The B-repeat superdomain of Aap is composed of 5 to 17 Zn2+-binding B-repeats, which undergo rapid, reversible assembly to form dimer and tetramer species. The tetramer can then undergo a conformational change and nucleate highly stable functional amyloid fibrils. In this study, multiple techniques including analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) are used to probe a panel of B-repeat mutant constructs that assemble to distinct oligomeric states to define the structural characteristics of B-repeat dimer and tetramer species. The B-repeat region from Aap forms an extremely elongated conformation that presents several challenges for standard SAXS analyses. Specialized approaches, such as cross-sectional analyses, allowed for in-depth interpretation of data, while explicit-solvent calculations via WAXSiS allowed for accurate evaluation of atomistic models. The resulting models suggest mechanisms by which Aap functional amyloid fibrils form, illuminating an important contributing factor to recurrent staphylococcal infections.

Glucose supply and glycolysis inhibition shape the clinical fate of Staphylococcus epidermidis-infected preterm newborns.

Preterm infants are susceptible to bloodstream infection by coagulase-negative staphylococci (CONS) that can lead to sepsis. Glucose-rich parenteral nutrition is commonly used to support the infants' growth and energy expenditure but may exceed endogenous regulation during infection, causing dysregulated immune response and clinical deterioration. Using a preterm piglet model of neonatal CONS sepsis induced by Staphylococcus epidermidis (S. epidermidis) infection, we demonstrate the delicate interplay between immunity and glucose metabolism to regulate the host infection response. Circulating glucose levels, glycolysis, and inflammatory response to infection are closely connected across the states of tolerance, resistance, and immunoparalysis. Furthermore, high parenteral glucose provision during infection induces hyperglycemia, elevated glycolysis, and inflammation, leading to metabolic acidosis and sepsis, whereas glucose-restricted individuals are clinically unaffected with increased gluconeogenesis to maintain moderate hypoglycemia. Finally, standard glucose supply maintaining normoglycemia or pharmacological glycolysis inhibition enhances bacterial clearance and dampens inflammation but fails to prevent sepsis. Our results uncover how blood glucose and glycolysis control circulating immune responses, in turn determining the clinical fate of preterm infants infected with CONS. Our findings suggest further refinements of the current practice of parenteral glucose supply for preterm infants during infection.

The Ubiquitous Human Skin Commensal Staphylococcus hominis Protects against Opportunistic Pathogens.

Staphylococcus hominis is frequently isolated from human skin, and we hypothesize that it may protect the cutaneous barrier from opportunistic pathogens. We determined that S. hominis makes six unique autoinducing peptide (AIP) signals that inhibit the major virulence factor accessory gene regulator (agr) quorum sensing system of Staphylococcus aureus. We solved and confirmed the structures of three novel AIP signals in conditioned medium by mass spectrometry and then validated synthetic AIP activity against all S. aureus agr classes. Synthetic AIPs also inhibited the conserved agr system in a related species, Staphylococcus epidermidis. We determined the distribution of S. hominis agr types on healthy human skin and found S. hominis agr-I and agr-II were highly represented across subjects. Further, synthetic AIP-II was protective in vivo against S. aureus-associated dermonecrotic or epicutaneous injury. Together, these findings demonstrate that a ubiquitous colonizer of human skin has a fundamentally protective role against opportunistic damage. IMPORTANCE Human skin is home to a variety of commensal bacteria, including many species of coagulase-negative staphylococci (CoNS). While it is well established that the microbiota as a whole maintains skin homeostasis and excludes pathogens (i.e., colonization resistance), relatively little is known about the unique contributions of individual CoNS species to these interactions. Staphylococcus hominis is the second most frequently isolated CoNS from healthy skin, and there is emerging evidence to suggest that it may play an important role in excluding pathogens, including Staphylococcus aureus, from colonizing or infecting the skin. Here, we identified that S. hominis makes 6 unique peptide inhibitors of the S. aureus global virulence factor regulation system (agr). Additionally, we found that one of these peptides can prevent topical or necrotic S. aureus skin injury in a mouse model. Our results demonstrate a specific and broadly protective role for this ubiquitous, yet underappreciated skin commensal.

Glycerol fermentation by skin bacteria generates lactic acid and upregulates the expression levels of genes associated with the skin barrier function.

Commensal bacteria play a major role in multiple skin functions by providing the first layer of defense against pathogens and maintaining the skin barrier. Staphylococcus epidermidis is one of the most common skin commensals. In this study, we showed that S. epidermidis ferments glycerol and uses it as a nutrient, while producing short-chain and organic fatty acids, with the most notable being lactic acid. Lactic acid is an alpha-hydroxy acid that inhibits the growth of pathogenic bacteria, without any negative effect on the commensal bacteria itself. Using in vivo experiments, we validated our in vitro results, showing that the skin microbiome is also capable of doing this. Finally, using 2D and 3D skin culture models, we showed that the fermentation of glycerol, mainly lactic acid, as determined by analytical methods, upregulates the expression levels of several key genes that are associated with the barrier properties of the skin. While the hydration effect of glycerol on the skin is well known, our study shows the overall benefits of glycerol on the skin microbiome, while revealing an alternate mode of action of glycerol for multiple skin benefits.

Submicron topography design for controlling staphylococcal bacterial adhesion and biofilm formation.

Surface topography modification with nano- or micro-textured structures has been an efficient approach to inhibit microbial adhesion and biofilm formation and thereby to prevent biomaterial-associated infection without modification of surface chemistry/bulk properties of materials and without causing antibiotic resistance. This manuscript focuses on submicron-textured patterns with ordered arrays of pillars on polyurethane (PU) biomaterial surfaces in an effort to understand the effects of surface pillar features and surface properties on adhesion and colonization responses of two staphylococcal strains. Five submicron patterns with a variety of pillar dimensions were designed and fabricated on PU film surfaces and bacterial adhesion and biofilm formation of Staphylococcal strains (Staphylococcus epidermidis RP62A and Staphylococcus aureus Newman D2C) were characterized. Results show that all submicron textured surface significantly reduced bacterial adhesion and inhibited biofilm formation, and bacterial adhesion linearly decreased with the reduction in top surface area fraction. Surface wettability did not show a linear correlation with bacterial adhesion, suggesting that surface contact area dominates bacterial adhesion. From this, it appears that the design of textured patterns should minimize surface area fraction to reduce the bacterial interaction with surfaces but in a way that ensures the mechanical strength of pillars in order to avoid collapse. These findings may provide a rationale for design of polymer surfaces for antifouling medical devices.

Involvement of the Iron-Regulated Loci hts and fhuC in Biofilm Formation and Survival of Staphylococcus epidermidis within the Host.

Staphylococcus epidermidis is a major nosocomial pathogen with a remarkable ability to persist on indwelling medical devices through biofilm formation. Nevertheless, it remains intriguing how this process is efficiently achieved under the host's harsh conditions, where the availability of nutrients, such as essential metals, is scarce. Following our previous identification of two iron-regulated loci putatively involved in iron transport, hts and fhuC, we assessed here their individual contribution to both bacterial physiology and interaction with host immune cells. Single deletions of the hts and fhuC loci led to marked changes in the cell iron content, which were partly detrimental for planktonic growth and strongly affected biofilm formation under iron-restricted conditions. Deletion of each of these two loci did not lead to major changes in S. epidermidis survival within human macrophages or in an ex vivo human blood model of bloodstream infection. However, the lack of either hts or fhuC loci significantly impaired bacterial survival in vivo in a murine model of bacteremia. Collectively, this study establishes, for the first time, the pivotal role of the iron-regulated loci hts and fhuC in S. epidermidis biofilm formation and survival within the host, providing relevant information for the development of new targeted therapeutics against this pathogen. IMPORTANCE Staphylococcus epidermidis is one of the most important nosocomial pathogens and a major cause of central line-associated bloodstream infections. Once in the bloodstream, this bacterium must surpass severe iron restriction in order to survive and establish infection. Surprisingly, very little is known about the iron acquisition mechanisms in this species. This study represents the first report on the involvement of the S. epidermidis iron-regulated loci hts and fhuC in biofilm formation under host relevant conditions and, most importantly, in survival within the host. Ultimately, these findings highlight iron acquisition and these loci in particular, as potential targets for future therapeutic strategies against biofilm-associated S. epidermidis infections.

Slippery Liquid-Like Solid Surfaces with Promising Antibiofilm Performance under Both Static and Flow Conditions.

Biofilms are central to some of the most urgent global challenges across diverse fields of application, from medicine to industries to the environment, and exert considerable economic and social impact. A fundamental assumption in anti-biofilms has been that the coating on a substrate surface is solid. The invention of slippery liquid-infused porous surfaces─a continuously wet lubricating coating retained on a solid surface by capillary forces─has led to this being challenged. However, in situations where flow occurs, shear stress may deplete the lubricant and affect the anti-biofilm performance. Here, we report on the use of slippery omniphobic covalently attached liquid (SOCAL) surfaces, which provide a surface coating with short (ca. 4 nm) non-cross-linked polydimethylsiloxane (PDMS) chains retaining liquid-surface properties, as an antibiofilm strategy stable under shear stress from flow. This surface reduced biofilm formation of the key biofilm-forming pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa by three-four orders of magnitude compared to the widely used medical implant material PDMS after 7 days under static and dynamic culture conditions. Throughout the entire dynamic culture period of P. aeruginosa, SOCAL significantly outperformed a typical antibiofilm slippery surface [i.e., swollen PDMS in silicone oil (S-PDMS)]. We have revealed that significant oil loss occurred after 2-7 day flow for S-PDMS, which correlated to increased contact angle hysteresis (CAH), indicating a degradation of the slippery surface properties, and biofilm formation, while SOCAL has stable CAH and sustainable antibiofilm performance after 7 day flow. The significance of this correlation is to provide a useful easy-to-measure physical parameter as an indicator for long-term antibiofilm performance. This biofilm-resistant liquid-like solid surface offers a new antibiofilm strategy for applications in medical devices and other areas where biofilm development is problematic.

Look Who's Talking: Host and Pathogen Drivers of Staphylococcus epidermidis Virulence in Neonatal Sepsis.

Preterm infants are at increased risk for invasive neonatal bacterial infections. S. epidermidis, a ubiquitous skin commensal, is a major cause of late-onset neonatal sepsis, particularly in high-resource settings. The vulnerability of preterm infants to serious bacterial infections is commonly attributed to their distinct and developing immune system. While developmentally immature immune defences play a large role in facilitating bacterial invasion, this fails to explain why only a subset of infants develop infections with low-virulence organisms when exposed to similar risk factors in the neonatal ICU. Experimental research has explored potential virulence mechanisms contributing to the pathogenic shift of commensal S. epidermidis strains. Furthermore, comparative genomics studies have yielded insights into the emergence and spread of nosocomial S. epidermidis strains, and their genetic and functional characteristics implicated in invasive disease in neonates. These studies have highlighted the multifactorial nature of S. epidermidis traits relating to pathogenicity and commensalism. In this review, we discuss the known host and pathogen drivers of S. epidermidis virulence in neonatal sepsis and provide future perspectives to close the gap in our understanding of S. epidermidis as a cause of neonatal morbidity and mortality.

Active Quinolinequinones against Methicillin-Resistant Staphylococcus spp.

Serious bacterial infections could be caused by Gram-positive microorganisms, in particular methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Aiming to address this challenging issue by developing the potent and selective antimicrobial lead structures against methicillin-resistant Staphylococcus spp., herein, we report in vitro evaluation of quinolinequinones (QQ1-QQ10) against the Gram-negative and Gram-positive strains using the broth microdilution technique. The design principle of the quinolinequinones was based on the variation of the structures attached to the 1,4-quinone moiety and substituent(s) within amino phenyl moiety. A series of ten quinolinequinones displayed activity mainly against the Gram-positive strains with a minimal inhibitory concentration (MIC=1.22-1250 mg/L) within the Clinical and Laboratory Standards Institute (CLSI) levels. Interestingly, QQ3, QQ5, and QQ6 displayed equal antibacterial inhibitory activity against S. aureus (MIC=1.22 mg/L), respectively, to the standard positive control Cefuroxime-Na. QQ2, QQ3, and QQ5 had the best inhibitory activity with the MIC value of 1.22 mg/L (4-fold more potent compared reference standard Cefuroxime) against S. epidermidis. On the other hand, QQ3 was the most effective quinolinequinone against fungi, in particular C. albicans. The identified lead quinolinequinones (QQ3 and QQ5) with a comprehensive analysis of structure-activity relationships and further studies showed high activity against methicillin-resistant Staphylococcus spp. It is worth noting that the isopropyl group has importance for excellent bioactivity. Remarkably, the in vitro antibiofilm and bactericidal activities (each of 32 clinically obtained strains of Gram-positive bacteria) of the selected two quinolinequinones (QQ3 and QQ5) have been evaluated for the mode of action in addition to the time-kill curve study.

Cutibacterium acnes and Staphylococcus epidermidis: the unmissable modulators of skin inflammatory response.

Acne is a multifactorial inflammatory dermatose that affects all age categories from teenagers to adults, resulting in important psychological impacts. Multiple hypotheses currently attempt to decrypt the physiopathology of this disease, and four main actors were identified as highly implicated in it: hyperkeratinization of the pilosebaceous follicle, hyperseborrheae, host factors (innate immunity) and skin microbiota. In this letter, we present results illustrating the impact of skin microbiota on inflammatory skin response, and how far the proper balance between each bacterial community, especially C. acnes and S. epidermidis, is crucial to maintain an appropriate inflammatory response on the skin. The data presented in this study demonstrate that within the skin microbiota, an imbalance between Cutibacterium acnes and Staphylococcus epidermidis, is able to induce the activation of inflammation-related markers such as IL-1ra, IL-6, IL-8, G-CSF and the molecules C5/C5a, soluble CD14 MIP-3beta, Serpin E1, VCAM-1 and beta-defensin-2. Moreover, S. epidermidis appears to have a more important role than C. acnes on the induction of inflammation-related markers, particularly on IL-6. This work is the basis of future in vitro studies to further understand acne physiopathology, inspiring the development of future innovative therapies based on skin microbiota modulation.

Heterogeneity of Staphylococcus epidermidis in prosthetic joint infections: time to reevaluate microbiological criteria?

Prosthetic joint infection (PJI) is a feared and challenging to diagnose complication after arthroplasty, with Staphylococcus epidermidis as the major pathogen. One important criteria to define PJI is the detection of phenotypically indistinguishable microorganisms with identical antibiotic susceptibility pattern in at least two different samples. However, owing to phenotypical variation within genetic clones and clonal variation within a phenotype, the criteria may be ambiguous. We investigated the extent of diversity among coagulase-negative staphylococci (CoNS) in PJI and characterised S. epidermidis isolates from PJI samples, specifically multiple S. epidermidis isolates identified in individual PJI patients. We performed a retrospective cohort study on 62 consecutive patients with PJI caused by CoNS from two hospitals in Northern Sweden. In 16/62 (26%) PJIs, multiple S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in samples from 40/62 (65%) of the patients using a combination of pulsed-field gel electrophoresis and multilocus sequence typing. Whole-genome sequencing showed the presence of multiple sequence types (STs) in 7/16 (44%) PJIs where multiple S. epidermidis isolates were available. Within-patient phenotypical variation in the antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%) among isolates with the same ST. The results highlight the ambiguity of S. epidermidis phenotypic characterisation as a diagnostic method in PJI and call for larger systematic studies for determining the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and the therapeutic outcomes in patients.

Viability-Resolved Metagenomics Reveals Antagonistic Colonization Dynamics of Staphylococcus epidermidis Strains on Preterm Infant Skin.

Preterm infants are at increased risk of infections caused by coagulase-negative staphylococci (CoNS) that colonize skin. Technical barriers in sequencing low-microbial-biomass skin swabs from preterm infants hinder attempts to gain a strain-level understanding of CoNS colonization dynamics within their developing skin microbiome. Here, the microbiome of five skin sites and available stool was studied from four preterm infants hospitalized over their first 2 months of life. We used propidium monoazide treatment of samples to enrich for the viable microbiome and metagenomic shotgun sequencing to resolve species and strains. The microbiome of different skin sites overlapped with each other, was dominated by the CoNS species Staphylococcus epidermidis and Staphylococcus capitis, and was distinct from stool. Species diversity on skin increased over time despite antibiotic exposure. Evidence of antagonism between the most common S. epidermidis strains, ST2 and ST59, included negative relationships for species correlation networks and in situ replication rates and that ST2 colonized skin earlier but was often replaced by ST59 over time. Experiments done with reference isolates showed that ST2 produced more biofilm than ST59 on plastic surfaces, which was reduced in mixed culture. We also discovered that a rare S. epidermidis strain, ST5, grew rapidly in stool in association with Stenotrophomonas maltophilia from a suspected episode of infection. Viability treatment of samples and moderate throughput shotgun sequencing provides strain-level information about CoNS colonization dynamics of preterm infant skin that ultimately might be exploited to prevent infections. IMPORTANCE The skin is a habitat for microbes that commonly infect preterm infants, but the use of sequencing for fine-scale study of the microbial communities of skin that develop in these infants has been limited by technical barriers. We treated skin swabs of preterm infants with a photoreactive dye that eliminates DNA from nonviable microbes and then sequenced the remaining DNA. We found that two strains of the most common species, Staphylococcus epidermidis, showed an antagonistic relationship on skin by cooccurring with different species, replicating fastest in different samples, and dominating skin sites at different times. Representatives of these strains also differed in their ability to stick to plastic surfaces-an important pathogenicity trait of this species. Our study shows the feasibility of gaining detailed information about strain colonization dynamics from this difficult-to-sequence body site of preterm infants, which might be used to guide novel approaches to prevent infections.

IL-6 Signaling Protects Zebrafish Larvae during Staphylococcus epidermidis Infection in a Bath Immersion Model.

The host immune responses to Staphylococcus epidermidis, a frequent cause of nosocomial infections, are not well understood. We have established a bath immersion model of this infection in zebrafish (Danio rerio) larvae. Macrophages play a primary role in the host immune response and are involved in clearance of infection in the larvae. S. epidermidis infection results in upregulation of tlr-2 There is marked inflammation characterized by heightened NF-κB signaling and elevation of several proinflammatory cytokines. There is rapid upregulation of il-1b and tnf-a transcripts, whereas an increase in il-6 levels is relatively more delayed. The IL-6 signaling pathway is further amplified by elevation of IL-6 signal transducer (il-6st) levels, which negatively correlates with miRNA dre-miR-142a-5p. Enhanced IL-6 signaling is protective to the host in this model as inhibition of the signaling pathway resulted in increased mortality upon S. epidermidis infection. Our study describes the host immune responses to S. epidermidis infection, establishes the importance of IL-6 signaling, and identifies a potential role of miR-142-5p-il-6st interaction in this infection model.

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.

Effects of Ulva sp. Extracts on the Growth, Biofilm Production, and Virulence of Skin Bacteria Microbiota: Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes Strains.

Ulva sp. is known to be a source of bioactive compounds such as ulvans, but to date, their biological activity on skin commensal and/or opportunistic pathogen bacteria has not been reported. In this study, the effects of poly- and oligosaccharide fractions produced by enzyme-assisted extraction and depolymerization were investigated, for the first time in vitro, on cutaneous bacteria: Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. At 1000 µg/mL, poly- and oligosaccharide fractions did not affect the growth of the bacteria regarding their generation time. Polysaccharide Ulva sp. fractions at 1000 µg/mL did not alter the bacterial biofilm formation, while oligosaccharide fractions modified S. epidermidis and C. acnes biofilm structures. None of the fractions at 1000 µg/mL significantly modified the cytotoxic potential of S. epidermidis and S. aureus towards keratinocytes. However, poly- and oligosaccharide fractions at 1000 µg/mL induced a decrease in the inflammatory potential of both acneic and non-acneic C. acnes strains on keratinocytes of up to 39.8%; the strongest and most significant effect occurred when the bacteria were grown in the presence of polysaccharide fractions. Our research shows that poly- and oligosaccharide Ulva sp. fractions present notable biological activities on cutaneous bacteria, especially towards C. acnes acneic and non-acneic strains, which supports their potential use for dermo-cosmetic applications.

Extracellular DNA released by glycine-auxotrophic Staphylococcus epidermidis small colony variant facilitates catheter-related infections.

Though a definitive link between small colony variants (SCVs) and implant-related staphylococcal infections has been well-established, the specific underlying mechanism remains an ill-explored field. The present study analyzes the role SCVs play in catheter infection by performing genomic and metabolic analyses, as well as analyzing biofilm formation and impacts of glycine on growth and peptidoglycan-linking rate, on a clinically typical Staphylococcus epidermidis case harboring stable SCV, normal counterpart (NC) and nonstable SCV. Our findings reveal that S. epidermidis stable SCV carries mutations involved in various metabolic processes. Metabolome analyses demonstrate that two biosynthetic pathways are apparently disturbed in SCV. One is glycine biosynthesis, which contributes to remarkable glycine shortage, and supplementation of glycine restores growth and peptidoglycan-linking rate of SCV. The other is overflow of pyruvic acid and acetyl-CoA, leading to excessive acetate. SCV demonstrates higher biofilm-forming ability due to rapid autolysis and subsequent eDNA release. Despite a remarkable decline in cell viability, SCV can facilitate in vitro biofilm formation and in vivo survival of NC when co-infected with its normal counterparts. This work illustrates an intriguing strategy utilized by a glycine-auxotrophic clinical S. epidermidis SCV isolate to facilitate biofilm-related infections, and casts a new light on the role of SCV in persistent infections.

Detection and partial characterization of extracellular inducers of persistence in Staphylococcus epidermidis and Staphylococcus aureus.

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci.Hypothesis/Gap Statement. Increases in persisters during mid-log phase growth indicate that quorum-sensing factors might be produced by staphylococci.Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000.Methodology. Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD600s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications.Results. The optimal OD600s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis' PIF activity was decreased by storage at 4 oC but not at 20 oC (16 h), 37 oC (1 h) or 100 oC (15 min). S. aureus' PIF activity was decreased following storage at 4 oC (2 weeks) and after boiling at 100 oC for 5 min but not after incubation at 37 oC (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis. PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells.Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600 -based PIF assay.