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    Temporal and stochastic control of Staphylococcus aureus biofilm development. Moormeier Derek E,Bose Jeffrey L,Horswill Alexander R,Bayles Kenneth W mBio Biofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureus biofilms by observing differential expression of the cid and lrg operons during tower formation. In the present study, we examined early biofilm development and identified two new stages (designated "multiplication" and "exodus") that were associated with changes in matrix composition and a distinct reorganization of the cells as the biofilm matured. The initial attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Interestingly, after 6 h of growth, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyperproliferation of the biofilm and disrupting normal tower development. Immediately prior to the exodus event, S. aureus cells carrying a nuc::gfp promoter fusion demonstrated Sae-dependent expression but only in an apparently random subpopulation of cells. In contrast to the existing model for tower development in S. aureus, the results of this study suggest the presence of a Sae-controlled nuclease-mediated exodus of biofilm cells that is required for the development of tower structures. Furthermore, these studies indicate that the differential expression of nuc during biofilm development is subject to stochastic regulatory mechanisms that are independent of the formation of metabolic microniches. Importance: In this study, we provide a novel view of four early stages of biofilm formation by the human pathogen Staphylococcus aureus. We identified an initial nucleoprotein matrix during biofilm development that is DNase I insensitive until a critical point when a nuclease-mediated exodus of the population is induced prior to tower formation. Unlike the previously described dispersal of cells that occurs after tower development, we found that the mechanism controlling this exodus event is dependent on the Sae regulatory system and independent of Agr. In addition, we revealed that the gene encoding the secreted staphylococcal nuclease was expressed in only a subpopulation of cells, consistent with a model in which biofilms exhibit multicellular characteristics, including the presence of specialized cells and a division of labor that imparts functional consequences to the remainder of the population. 10.1128/mBio.01341-14
    Vision for medicine: Staphylococcus aureus biofilm war and unlocking key's for anti-biofilm drug development. Mohammed Yasser Hussein Eissa,Manukumar H M,Rakesh K P,Karthik C S,Mallu P,Qin Hua-Li Microbial pathogenesis The Staphylococcus aureus biofilm-associated burden is challenging to the field of medicine to eradicate or avoid it. Even though a number of S. aureus biofilm mechanisms understood and established the possible ways of biofilm formation but, still need to know more and require a development of new therapeutic strategies. In this viewpoint, we discuss the underlining biofilm mechanism, its existing systems as active therapeutic agents and as vehicles to transport drugs to the site of infection. The step-back in drug development is due to the emergence of antibiotic-resistant S. aureus. The understanding of bacteria/biofilms is an aspect that we likewise summarize for possible drug development for future as medicine against resistant S. aureus was viewed. 10.1016/j.micpath.2018.07.002
    Biofilm Formation of Staphylococcus aureus under Food Heat Processing Conditions: First Report on CML Production within Biofilm. Miao Jian,Lin Shiqi,Soteyome Thanapop,Peters Brian M,Li Yanmei,Chen Huishan,Su Jianyu,Li Lin,Li Bing,Xu Zhenbo,Shirtliff Mark E,Harro Janette M Scientific reports This study aimed to evaluate the Staphylococcus aureus biofilm formation and Nε-carboxymethyl-lysine generation ability under food heat processing conditions including pH (5.0-9.0), temperature (25 °C, 31 °C, 37 °C, 42 °C and 65 °C), NaCl concentration (10%, 15% and 20%, w/v) and glucose concentration (0.5%, 1%, 2%, 3%, 5%, 10%, w/v). S. aureus biofilm genetic character was obtained by PCR detecting atl, ica operon, sasG and agr. Biofilm biomass and metabolic activity were quantified with crystal violet and methyl thiazolyl tetrazolium staining methods. S. aureus biofilm was sensitive to food heat processing conditions with 37 °C, pH 7.0, 2% glucose concentration (w/v) and 10% NaCl concentration (w/v) were favorable conditions. Besides, free and bound Nε-carboxymethyl-lysine level in weak, moderate and strong biofilm were detected by optimized high performance liquid chromatography tandem mass spectrometry. Nε-carboxymethyl-lysine level in S. aureus biofilm possessed a significant gap between strong, moderate and weak biofilm strains. This investigation revealed the biological and chemical hazard of Staphylococcus aureus biofilm to food processing environment. 10.1038/s41598-018-35558-2
    Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm. Xu Yuanxi,Jones John E,Yu Haiqing,Yu Qingsong,Christensen Gordon D,Chen Meng,Sun Hongmin Antimicrobial agents and chemotherapy Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections. 10.1128/AAC.01944-15
    Effects of lipopeptide carboxymethyl chitosan nanoparticles on Staphylococcus aureus biofilm. Jiang X H,Zhou W M,He Y Z,Wang Y,Lv B,Wang X M Journal of biological regulators and homeostatic agents This study aims to evaluate the effect of lipopeptide carboxymethyl chitosan nanoparticles on Staphylococcus aureus biofilm as part of the development of a new anti-biofilm material. The study had three stages. Firstly, we assessed the Staphylococcus aureus capability to form biofilm and enumerated the number of attached bacteria and free bacteria; secondly, we determined the inhibitory effect of different concentrations of Bacillus natto antimicrobial lipopeptid- carboxymethyl chitosan (BNAP-CMCS) nanoparticles added at different times on biofilm formation capability and the numbers of free bacteria and attached bacteria. Lastly, we tested the scavenging effect of BNAP-CMCS nanoparticles on biofilm formation and number of attached bacteria. The results showed that the amount of attached bacteria quickly increased over time and reached the maximum after 24 h of culture. The BNAP-CMCS nanoparticles had the greatest effect on biofilm inhibition at the concentration of 1 MIC, after 8 h of culture, and the effect was dose-dependent. The BNAP-CMCS nanoparticles had decreased also the numbers of free and attached bacteria in a dose-dependent fashion, after 8 hours of culture. The scavenging effect of BNAP-CMCS nanoparticles on free and attached bacteria was maximum at 6 MIC. In conclusion, lipopeptide carboxymethyl chitosan nanoparticles had a good inhibition and scavenging effect on the formation of Staphylococcus aureus biofilm and the growth of surface-attached bacteria.
    Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus. Vinod Kumar Kirubakaran,Lall Chandan,Raj Ratchagadasse Vimal,Vijayachari Paluru Microbiology and immunology It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC-737 showed strong co-aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix-like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin. 10.1111/1348-0421.12679
    Methicillin resistance and the biofilm phenotype in Staphylococcus aureus. McCarthy Hannah,Rudkin Justine K,Black Nikki S,Gallagher Laura,O'Neill Eoghan,O'Gara James P Frontiers in cellular and infection microbiology Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus. 10.3389/fcimb.2015.00001
    Staphylococcus aureus biofilms: recent developments in biofilm dispersal. Lister Jessica L,Horswill Alexander R Frontiers in cellular and infection microbiology Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and represents a significant burden on the healthcare system. S. aureus attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in the persistence of chronic infections. The formation of a biofilm, and encasement of cells in a polymer-based matrix, decreases the susceptibility to antimicrobials and immune defenses, making these infections difficult to eradicate. During infection, dispersal of cells from the biofilm can result in spread to secondary sites and worsening of the infection. In this review, we discuss the current understanding of the pathways behind biofilm dispersal in S. aureus, with a focus on enzymatic and newly described broad-spectrum dispersal mechanisms. Additionally, we explore potential applications of dispersal in the treatment of biofilm-mediated infections. 10.3389/fcimb.2014.00178
    Reserpine attenuates biofilm formation and virulence of Staphylococcus aureus. Parai Debaprasad,Banerjee Malabika,Dey Pia,Mukherjee Samir Kumar Microbial pathogenesis This study investigated the effects of reserpine, the main bioactive compound of Rauwolfia serpentina, on biofilm formation and biofilm-associated virulence factors production in a Gram-positive pathogen, Staphylococcus aureus. Crystal violet assay, MTT assay, Congo red binding, CLSM studies were performed to assess the antibiofilm activity. Molecular docking was performed to explain the possible mode of action, catheter model was used to evaluate its application potential and the combinatorial study was performed in search of an improved therapeutic formulation. Reserpine affected biofilm formation, EPS production, biofilm cell viability and virulence factor production. It could eradicate 72.7% biofilm at ½ × MIC dose and could also stop the metabolic activity of 50.6% bacterial cells in a biofilm. Staphylococcus aureus biofilm- and virulence-regulatory proteins like AgrA, AtlE, Bap, IcaA, SarA and SasG were found to interact with reserpine which might lead to the attenuation of its pathogenicity. Reserpine along with other commercial antibiotics could generate a hightened antibiofilm response, and also eradicated a good percentage of bacterial biofilm from a urinary catheter model. These findings suggested reserpine as a good alternative entity to generate new improved therapeutic formulations. 10.1016/j.micpath.2019.103790
    Alcohol treatment enhances Staphylococcus aureus biofilm development. Redelman Carly V,Maduakolam Chike,Anderson Gregory G FEMS immunology and medical microbiology Staphylococcus aureus forms pathogenic biofilms. Previous studies have indicated that ethanol supplementation during S. aureus biofilm formation results in increased biofilm formation and changes in gene expression. However, the impact of alcohols on preformed S. aureus biofilms has not been studied. In this study, we formed S. aureus biofilms on PVC plastic plates and then treated these preformed biofilms with five different alcohols. We observed that alcohol treatment of preformed S. aureus biofilms led to significant increases in biofilm levels after 24 h of treatment. Many bacteria within these biofilms were found to be alive and metabolically active. Alcohol treatment also resulted in increased transcription of the biofilm-promoting genes icaA and icaD, as well as several antibiotic resistance genes. These results demonstrate that treatment of S. aureus preformed biofilms with alcohols enhances biofilm levels if maintained for extended periods. Thus, alcohols might be of limited usefulness for the eradication of preformed S. aureus biofilms. 10.1111/1574-695X.12005
    Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection. Ibberson Carolyn B,Parlet Corey P,Kwiecinski Jakub,Crosby Heidi A,Meyerholz David K,Horswill Alexander R Infection and immunity Staphylococcus aureus is a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureus biofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureus secretes HysA in order to cleave HA during infection. Through in vitro biofilm studies with HA, the hysA mutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysA expression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysA mutant biofilm infection compared to the S. aureus wild type. Histopathology demonstrated that infection with an hysA mutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureus HysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureus biofilm matrix and HysA is important for dissemination from a biofilm infection. 10.1128/IAI.01418-15
    Staphylococcus aureus biofilm: a complex developmental organism. Moormeier Derek E,Bayles Kenneth W Molecular microbiology Chronic biofilm-associated infections caused by Staphylococcus aureus often lead to significant increases in morbidity and mortality, particularly when associated with indwelling medical devices. This has triggered a great deal of research attempting to understand the molecular mechanisms that control S. aureus biofilm formation and the basis for the recalcitrance of these multicellular structures to antibiotic therapy. The purpose of this review is to summarize our current understanding of S. aureus biofilm development, focusing on the description of a newly-defined, five-stage model of biofilm development and the mechanisms required for each stage. Importantly, this model includes an alternate view of the processes involved in microcolony formation in S. aureus and suggests that these structures originate as a result of stochastically regulated metabolic heterogeneity and proliferation within a maturing biofilm population, rather than a subtractive process involving the release of cell clusters from a thick, unstructured biofilm. Importantly, it is proposed that this new model of biofilm development involves the genetically programmed generation of metabolically distinct subpopulations of cells, resulting in an overall population that is better able to adapt to rapidly changing environmental conditions. 10.1111/mmi.13634
    Staphopains modulate Staphylococcus aureus biofilm integrity. Mootz Joe M,Malone Cheryl L,Shaw Lindsey N,Horswill Alexander R Infection and immunity Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture. 10.1128/IAI.00377-13
    Aspartate inhibits Staphylococcus aureus biofilm formation. Yang Hang,Wang Mengyue,Yu Junping,Wei Hongping FEMS microbiology letters Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. 10.1093/femsle/fnv025
    Propionibacterium acnes biofilm - A sanctuary for Staphylococcus aureus? Tyner Harmony,Patel Robin Anaerobe The purpose of this study was to measure the effect of combined culture of Propionibacterium acnes and Staphylococcus aureus on biofilm formation under different oxygen concentrations. We measured planktonic growth and biofilm formation of P. acnes and S. aureus alone and together under aerobic and anaerobic conditions. Both P. acnes and S. aureus grew under anaerobic conditions. When grown under anaerobic conditions, P. acnes with or without S. aureus formed a denser biomass biofilm than did S. aureus alone. Viable S. aureus was recovered from a16-day old combined P. acnes and S. aureus biofilm, but not a monomicrobial S. aureus biofilm. 10.1016/j.anaerobe.2016.05.014
    Inhibition characteristics of biofilm structure of Staphylococcus aureus. Zhang Liping,Jin Mingxiao,Sun Meng Cellular and molecular biology (Noisy-le-Grand, France) Different extracts have different effects on the biofilm structure of Staphylococcus aureus, and the biofilm structure of Staphylococcus aureus will produce different inhibition reactions. In this study, different experimental reagent extracts were used to analyze the inhibition characteristics of Staphylococcus aureus biofilm structure. The inhibition characteristics of bacterial biofilm structure were obtained by using the same bacteria species and the same experimental environment. The results showed that the chloroform extract had a good inhibitory effect on the biofilm structure, which could effectively inhibit the formation of biofilm; the acetic acid extract had an impact on the formation of biofilm, which was destructive to the biofilm; the petroleum ether extract had no effect on the formation of biofilm, that is, it had no inhibitory effect.
    The ClpCP Complex Modulates Respiratory Metabolism in Staphylococcus aureus and Is Regulated in a SrrAB-Dependent Manner. Mashruwala Ameya A,Eilers Brian J,Fuchs Amanda L,Norambuena Javiera,Earle Carly A,van de Guchte Adriana,Tripet Brian P,Copié Valérie,Boyd Jeffrey M Journal of bacteriology The taphylococcal espiratory egulator (SrrAB) modulates energy metabolism in Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A Δ strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the Δ strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of was decreased in the Δ mutant, and overexpression of suppressed the puromycin sensitivity of the Δ strain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the Δ and Δ mutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis. Oxygen is used as a substrate to derive energy by the bacterial pathogen during infection; however, can also grow fermentatively in the absence of oxygen. To successfully cause infection, must tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism in We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth. 10.1128/JB.00188-19
    Subinhibitory concentrations of tedizolid potently inhibit extracellular toxin production by methicillin-sensitive and methicillin-resistant Staphylococcus aureus. Katahira Eva J,Davidson Stephen M,Stevens Dennis L,Bolz Devin D Journal of medical microbiology PURPOSE:Potent extracellular toxins including alpha-haemolysin, Panton-Valentine leukocidin (PVL) and toxic-shock syndrome toxin 1 (TSST-1) significantly contribute to Staphylococcus aureus pathogenesis, thus, toxin suppression is a primary focus in treatment of staphylococcal disease. S. aureus maintains complex strategies to regulate toxin expression and previous data have demonstrated that subinhibitory concentrations of beta-lactam antibiotics can adversely increase S. aureus exotoxin production. The current study evaluates the effects of subinhibitory concentrations of tedizolid, a second-generation oxazolidinone derivative, on expression of staphylococcal exotoxins in both methicillin-resistant and methicillin-sensitive S. aureus. METHODOLOGY:S. aureus exotoxin expression levels were compared at 12 and 24 h following treatment with tedizolid, linezolid, nafcillin or vehicle control. RESULTS:Our findings show that the level of antibiotic required to alter toxin production was strain-dependent and corresponds with the quantity of toxin produced, but both tedizolid and linezolid could effectively reduce expression of alpha-haemolysin, PVL and TSST-1 toxin at subinhibitory concentrations. In contrast, nafcillin showed less attenuation and, in some S. aureus strains, led to an increase in toxin expression. Tedizolid consistently inhibited toxin production at a lower overall drug concentration than comparator agents. CONCLUSION:Together, our data support that tedizolid has the potential to improve outcomes of infection due to its superior ability to inhibit S. aureus growth and attenuate exotoxin production. 10.1099/jmm.0.000905
    The good side of inflammation: Staphylococcus aureus proteins SpA and Sbi contribute to proper abscess formation and wound healing during skin and soft tissue infections. Gonzalez Cintia D,Ledo Camila,Cela Eliana,Stella Inés,Xu Chunliang,Ojeda Diego S,Frenette Paul S,Gómez Marisa I Biochimica et biophysica acta. Molecular basis of disease Staphylococcus aureus is the most prominent cause of skin and soft tissue infections (SSTI) worldwide. Mortality associated with invasive SSTI is a major threat to public health considering the incidence of antibiotic resistant isolates in particular methicillin resistant S. aureus both in the hospital (HA-MRSA) and in the community (CA-MRSA). To overcome the increasing difficulties in the clinical management of SSTI due to MRSA, new prophylactic and therapeutic approaches are urgently needed and a preventive vaccine would be welcome. The rational design of an anti-S. aureus vaccine requires a deep knowledge of the role that the different bacterial virulence factors play according to the type of infection. In the present study, using a set of isogenic deficient mutants and their complemented strains we determined that the staphylococcal surface proteins SpA and Sbi play an important role in the induction of inflammatory cytokines and chemokines in the skin during SSTI. SpA and Sbi initiate signaling cascades that lead to the early recruitment of neutrophils, modulate their lifespan in the skin milieu and contribute to proper abscess formation and bacterial eradication. Moreover, the expression of SpA and Sbi appear critical for skin repair and wound healing. Thus, these results indicate that SpA and Sbi can promote immune responses in the skin that are beneficial for the host and therefore, should not be neutralized with vaccine formulations designed to prevent SSTI. 10.1016/j.bbadis.2019.07.004
    Timing Is Everything: Impact of Naturally Occurring AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction. Sloan Tim J,Murray Ewan,Yokoyama Maho,Massey Ruth C,Chan Weng C,Bonev Boyan B,Williams Paul Journal of bacteriology Mutations in the polymorphic locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for activation via AIP-dependent autoinduction, thus reducing virulence and maintaining in an -downregulated "colonization" mode. Virulence factor expression in is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins. 10.1128/JB.00409-19
    MgrA Governs Adherence, Host Cell Interaction, and Virulence in a Murine Model of Bacteremia Due to Staphylococcus aureus. Li Liang,Wang Genzhu,Cheung Ambrose,Abdelhady Wessam,Seidl Kati,Xiong Yan Q The Journal of infectious diseases BACKGROUND:MgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. METHODS:In vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model. RESULTS:mgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed. CONCLUSIONS:These results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections. 10.1093/infdis/jiz219
    Finding of Agr Phase Variants in Staphylococcus aureus. Gor Vishal,Takemura Aya J,Nishitani Masami,Higashide Masato,Medrano Romero Veronica,Ohniwa Ryosuke L,Morikawa Kazuya mBio is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated locus with mechanisms resembling phase variation. The revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection. 10.1128/mBio.00796-19
    Disruption of Glycolysis by Nutritional Immunity Activates a Two-Component System That Coordinates a Metabolic and Antihost Response by Staphylococcus aureus. Párraga Solórzano Paola K,Yao Jiangwei,Rock Charles O,Kehl-Fie Thomas E mBio During infection, bacteria use two-component signal transduction systems to sense and adapt to the dynamic host environment. Despite critically contributing to infection, the activating signals of most of these regulators remain unknown. This also applies to the ArlRS two-component system, which contributes to virulence by coordinating the production of toxins, adhesins, and a metabolic response that enables the bacterium to overcome host-imposed manganese starvation. Restricting the availability of essential transition metals, a strategy known as nutritional immunity, constitutes a critical defense against infection. In this work, expression analysis revealed that manganese starvation imposed by the immune effector calprotectin or by the absence of glycolytic substrates activates ArlRS. Manganese starvation imposed by calprotectin also activated the ArlRS system even when glycolytic substrates were present. A combination of metabolomics, mutational analysis, and metabolic feeding experiments revealed that ArlRS is activated by alterations in metabolic flux occurring in the latter half of the glycolytic pathway. Moreover, calprotectin was found to induce expression of staphylococcal leukocidins in an ArlRS-dependent manner. These studies indicated that ArlRS is a metabolic sensor that allows to integrate multiple environmental stresses that alter glycolytic flux to coordinate an antihost response and to adapt to manganese starvation. They also established that the latter half of glycolysis represents a checkpoint to monitor metabolic state in Altogether, these findings contribute to understanding how invading pathogens, such as , adapt to the host during infection and suggest the existence of similar mechanisms in other bacterial species. Two-component regulatory systems enable bacteria to adapt to changes in their environment during infection by altering gene expression and coordinating antihost responses. Despite the critical role of two-component systems in bacterial survival and pathogenesis, the activating signals for most of these regulators remain unidentified. This is exemplified by ArlRS, a global regulator that contributes to virulence and to resisting host-mediated restriction of essential nutrients, such as manganese. In this report, we demonstrate that manganese starvation and the absence of glycolytic substrates activate ArlRS. Further investigations revealed that ArlRS is activated when the latter half of glycolysis is disrupted, suggesting that monitors flux through the second half of this pathway. Host-imposed manganese starvation also induced the expression of pore-forming toxins in an ArlRS-dependent manner. Cumulatively, this work reveals that ArlRS acts as a sensor that links nutritional status, cellular metabolism, and virulence regulation. 10.1128/mBio.01321-19
    Interaction in dual species biofilms between Staphylococcus xylosus and Staphylococcus aureus. Leroy Sabine,Lebert Isabelle,Andant Carine,Talon Régine International journal of food microbiology Staphylococcus xylosus, a coagulase-negative Staphylococcus, is frequently isolated from food products of animal origin and used as a starter culture in these products in which it contributes to their flavour, while Staphylococcus aureus, a coagulase-positive bacterium, causes foodborne intoxication and is implicated in a broad diversity of infections in medical sector, notably in nosocomial infections. S. xylosus and S. aureus are both capable of forming a biofilm and share the same ecological niches, thus we explored their interaction in biofilms with a view to limiting the risks associated with S. aureus. Cell-free supernatants of different strains of S. xylosus were able to inhibit the biofilm formation of S. aureus. The S. xylosus C2a strain released into the supernatant a molecule of molecular weight above 30 kDa that is resistant to proteolytic enzymes and inhibits the formation of S. aureus MW2 biofilm, though the mechanism involved has yet to be elucidated. Furthermore, S. xylosus C2a modified the architecture of S. aureus MW2 in co-culture biofilm. Confocal laser scanning microscopy revealed that S. aureus formed a biofilm with a flat and compact structure while in co-culture with S. xylosus the two species formed large juxtaposed aggregates throughout the period of incubation. This architecture made the S. aureus biofilm more susceptible to detachment. 10.1016/j.ijfoodmicro.2020.108653