Identification of Micrococcin P2-Derivatives as Antibiotic Candidates against Two Gram-Positive Pathogens.

Thiopeptides exhibit potent antimicrobial activity against Gram-positive pathogens by inhibiting bacterial protein synthesis. Micrococcins are among the structurally simpler thiopeptides, but they have not been exploited in detail. This research involved a computational simulation of micrococcin P2 (MP2) docking in parallel with the structure-activity relationship (SAR) studied. The incorporation of particular nitrogen heterocycles in the side chain of MP2 enhances the antimicrobial activity. Micrococcin analogues 6c and 6d thus proved to be more effective against impetigo and C. difficile infection (CDI), respectively, as compared to current first-line treatments. Compound 6c also showed a shorter treatment period than that of a first-line treatment for impetigo. This may be attributed to its ability to downregulate pro-inflammatory cytokines. Compound 6d had no observed recurrence for C. difficile and exerted a minimal impact on the beneficial gut microbiome. Their pharmacokinetic properties and low toxicity profile make these compounds ideal candidates for the treatment of impetigo and CDI and validate their involvement in preclinical development.

Reversible Mg-Metal Batteries Enabled by a Ga-Rich Protective Layer through One-Step Interface Engineering.

Practical applications of Mg-metal batteries (MMBs) have been plagued by a critical bottleneck─the formation of a native oxide layer on the Mg-metal interface─which inevitably limits the use of conventional nontoxic electrolytes. The major aim of this work was to propose a simple and effective way to reversibly operate MMBs in combination with Mg(TFSI)2-diglyme electrolyte by forming a Ga-rich protective layer on the Mg metal (GPL@Mg). Mg metal was carefully reacted with a GaCl3 solution to trigger a galvanic replacement reaction between Ga3+ and Mg, resulting in the layering of a stable and ion-conducting Ga-rich protective film while preventing the formation of a native insulating layer. Various characterization tools were applied to analyze GPL@Mg, and it was demonstrated to contain inorganic-rich compounds (MgCO3, Mg(OH)2, MgCl2, Ga2O3, GaCl3, and MgO) roughly in a double-layered structure. The artificial GPL on Mg was effective in greatly reducing the high polarization for Mg plating and stripping in diglyme-based electrolyte, and the stable cycling was maintained for over 200 h. The one-step process suggested in this work offers insights into exploring a cost-effective approach to cover the Mg-metal surface with an ion-conducting artificial layer, which will help to practically advance MMBs.

Combination of Lactobacillus plantarum HAC03 and Garcinia cambogia Has a Significant Anti-Obesity Effect in Diet-Induced Obesity Mice.

Obesity is a major global health problem which is associated with various diseases and psychological conditions. Increasing understanding of the relationship between obesity and gut microbiota has led to a worldwide effort to use microbiota as a treatment for obesity. However, several clinical trials have shown that obesity treatment with single strains of probiotics did not achieve as significant results as in animal studies. To overcome this limitation, we attempted to find a new combination that goes beyond the effects of probiotics alone by combining probiotics and a natural substance that has a stronger anti-obesity effect. In this study, we used a diet-induced obesity mouse (DIO) model to investigate the effects of combining Lactobacillus plantarum HAC03 with Garcinia cambogia extract, as compared to the effects of each substance alone. Combining L. plantarum HAC03 and G. cambogia, treatment showed a more than two-fold reduction in weight gain compared to each substance administered alone. Even though the total amount administered was kept the same as for other single experiments, the combination treatment significantly reduced biochemical markers of obesity and adipocyte size, in comparison to the treatment with either substance alone. The treatment with a combination of two substances also significantly decreased the gene expression of fatty acid synthesis (FAS, ACC, PPARγ and SREBP1c) in mesenteric adipose tissue (MAT). Furthermore, 16S rRNA gene sequencing of the fecal microbiota suggested that the combination of L. plantarum HAC03 and G. cambogia extract treatment changed the diversity of gut microbiota and altered specific bacterial taxa at the genus level (the Eubacterium coprostanoligenes group and Lachnospiraceae UCG group) and specific functions (NAD salvage pathway I and starch degradation V). Our results support that the idea that the combination of L. plantarum HAC03 and G. cambogia extract has a synergistic anti-obesity effect by restoring the composition of the gut microbiota. This combination also increases the abundance of bacteria responsible for energy metabolism, as well as the production of SCFAs and BCAAs. Furthermore, no significant adverse effects were observed during the experiment.

Revisiting Lithium- and Sodium-Ion Storage in Hard Carbon Anodes.

The galvanostatic lithiation/sodiation voltage profiles of hard carbon anodes are simple, with a sloping drop followed by a plateau. However, a precise understanding of the corresponding redox sites and storage mechanisms is still elusive, which hinders further development in commercial applications. Here, a comprehensive comparison of the lithium- and sodium-ion storage behaviors of hard carbon is conducted, yielding the following key findings: 1) the sloping voltage section is presented by the lithium-ion intercalation in the graphitic lattices of hard carbons, whereas it mainly arises from the chemisorption of sodium ions on their inner surfaces constituting closed pores, even if the graphitic lattices are unoccupied; 2) the redox sites for the plateau capacities are the same as those for the closed pores regardless of the alkali ions; 3) the sodiation plateau capacities are mostly determined by the volume of the available closed pore, whereas the lithiation plateau capacities are primarily affected by the intercalation propensity; and 4) the intercalation preference and the plateau capacity have an inverse correlation. These findings from extensive characterizations and theoretical investigations provide a relatively clear elucidation of the electrochemical footprint of hard carbon anodes in relation to the redox mechanisms and storage sites for lithium and sodium ions, thereby providing a more rational design strategy for constructing better hard carbon anodes.

Antibacterial Activity against Clinical Isolates and In Vivo Efficacy of Coralmycins.

Coralmycins, such as coralmycin A and DH-coralmycin A, have novel molecular skeletons and have been reported to exhibit potent antibacterial activity against standard Gram-positive bacterial strains. Here, the in vitro antibacterial activity against an extensive clinical isolate collection, time-kill kinetics, pharmacokinetics (PK), and in vivo efficacy of coralmycins were studied. Coralmycin A showed potent antibacterial activity with an MIC90 of 1 mg/L against 73 clinical methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci isolates, which was 2-8 times higher than the corresponding activities of DH-coralmycin A, vancomycin, daptomycin, and linezolid, and against 73 vancomycin-resistant Enterococcus and Streptococcus pneumoniae isolates, which was 4-16 times higher than the corresponding activities of DH-coralmycin A, daptomycin, and linezolid. Pharmacokinetic analysis after i.v. injection showed that coralmycins have a moderate volume of distribution and moderate-to-high clearance in mice. The coralmycin A and DH-coralmycin A bioavailability values were 61.3% and 11.7%, respectively, after s.c. administration. In a mouse respiratory tract infection model, coralmycin A showed bacteriostatic and bactericidal in vivo efficacies at an s.c. administration of 4 and 100 mg/kg bid, respectively; these efficacies were similar to those of vancomycin at 4 and 20 mg/kg bid, respectively. The present findings indicate that coralmycin A has great potential as a new class of antibiotic for treating infections caused by multidrug-resistant Gram-positive bacteria.

Micrococcin P2 Targets Clostridioides difficile.

Clostridioides difficile infection is a global public health threat. Extensive in vitro assays using clinical isolates have identified micrococcin P2 (MP2, 1) as a particularly effective anti-C. difficile agent. MP2 possesses a mode of action that differs from other antibiotics and pharmacokinetic properties that render it especially promising. Its time-kill studies have been investigated using hypervirulent C. difficile ribotype 027. DSS (dextran sulfate sodium)-induced in vivo mouse studies with that strain indicate that 1 is better than vancomycin and fidaxomicin. Thus, micrococcin P2 is a valuable platform to be exploited for the development of new anti-C. difficile antibiotics.

Nitro-Group-Containing Thiopeptide Derivatives as Promising Agents to Target Clostridioides difficile.

The US Centers for Disease Control and Prevention (CDC) lists Clostridioides difficile as an urgent bacterial threat. Yet, only two drugs, vancomycin and fidaxomicin, are approved by the FDA for the treatment of C. difficile infections as of this writing, while the global pipeline of new drugs is sparse at best. Thus, there is a clear and urgent need for new antibiotics against that organism. Herein, we disclose that AJ-024, a nitroimidazole derivative of a 26-membered thiopeptide, is a promising anti-C. difficile lead compound. Despite their unique mode of action, thiopeptides remain largely unexploited as anti-infective agents. AJ-024 combines potent in vitro activity against various strains of C. difficile with a noteworthy safety profile and desirable pharmacokinetic properties. Its time-kill kinetics against a hypervirulent C. difficile ribotype 027 and in vivo (mouse) efficacy compare favorably to vancomycin, and they define AJ-024 as a valuable platform for the development of new anti-C. difficile antibiotics.

Antibacterial Activity of LCB10-0200 against Klebsiella pneumoniae.

Klebsiella pneumoniae is one of the important clinical organisms that causes various infectious diseases, including urinary tract infections, necrotizing pneumonia, and surgical wound infections. The increase in the incidence of multidrug-resistance K. pneumoniae is a major problem in public healthcare. Therefore, a novel antibacterial agent is needed to treat this pathogen. Here, we studied the in vitro and in vivo activities of a novel antibiotic LCB10-0200, a siderophore-conjugated cephalosporin, against clinical isolates of K. pneumoniae. In vitro susceptibility study found that LCB10-0200 showed potent antibacterial activity against K. pneumoniae, including the beta-lactamase producing strains. The in vivo efficacy of LCB10-0200 was examined in three different mouse infection models, including systemic, thigh, and urinary tract infections. LCB10-0200 showed more potent in vivo activity than ceftazidime in the three in vivo models against the drug-susceptible and drug-resistant K. pneumoniae strains. Taken together, these results show that LCB10-0200 is a potential antibacterial agent to treat infection caused by K. pneumoniae.

Selenium Nanoparticles as Candidates for Antibacterial Substitutes and Supplements against Multidrug-Resistant Bacteria.

In recent years, multidrug-resistant (MDR) bacteria have increased rapidly, representing a major threat to human health. This problem has created an urgent need to identify alternatives for the treatment of MDR bacteria. The aim of this study was to identify the antibacterial activity of selenium nanoparticles (SeNPs) and selenium nanowires (SeNWs) against MDR bacteria and assess the potential synergistic effects when combined with a conventional antibiotic (linezolid). SeNPs and SeNWs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), zeta potential, and UV-visible analysis. The antibacterial effects of SeNPs and SeNWs were confirmed by the macro-dilution minimum inhibitory concentration (MIC) test. SeNPs showed MIC values against methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant enterococci (VRE) at concentrations of 20, 80, 320, and >320 µg/mL, respectively. On the other hand, SeNWs showed a MIC value of >320 µg/mL against all tested bacteria. Therefore, MSSA, MRSA, and VRSA were selected for the bacteria to be tested, and SeNPs were selected as the antimicrobial agent for the following experiments. In the time-kill assay, SeNPs at a concentration of 4X MIC (80 and 320 µg/mL) showed bactericidal effects against MSSA and MRSA, respectively. At a concentration of 2X MIC (40 and 160 µg/mL), SeNPs showed bacteriostatic effects against MSSA and bactericidal effects against MRSA, respectively. In the synergy test, SeNPs showed a synergistic effect with linezolid (LZD) through protein degradation against MSSA and MRSA. In conclusion, these results suggest that SeNPs can be candidates for antibacterial substitutes and supplements against MDR bacteria for topical use, such as dressings. However, for use in clinical situations, additional experiments such as toxicity and synergistic mechanism tests of SeNPs are needed.

Grapefruit Seed Extract as a Natural Derived Antibacterial Substance against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria are increasing due to the abuse and misuse of antibiotics, and nosocomial infections by MDR bacteria are also increasing. The aim of this study was to identify new substances that can target MDR bacteria among 12 plant extracts that are known to have antibacterial effects. The experiments were performed by the disk diffusion test and microdilution minimum inhibitory concentration (MIC) test, as described by the Clinical and Laboratory Standards Institute (CLSI). By screening against methicillin-sensitive Staphylococcus aureus (MSSA), grapefruit seed extract (GSE) was selected from 12 plant extracts for subsequent experiments. GSE showed antibacterial effects against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) in the disk diffusion test. Even at the lowest concentration, GSE showed antibacterial activity in the microdilution MIC test. As a result, we can conclude that GSE is a naturally derived antibacterial substance that exhibits a favorable antibacterial effect even at a very low concentration, so it is a good candidate for a natural substance that can be used to prevent or reduce nosocomial infections as coating for materials used in medical contexts or by mixing a small amount with other materials.

Hierarchically Nanoporous 3D Assembly Composed of Functionalized Onion-Like Graphitic Carbon Nanospheres for Anode-Minimized Li Metal Batteries.

Despite the recent attention for Li metal anode (LMA) with high theoretical specific capacity of ≈3860 mA h g-1 , it suffers from not enough practical energy densities and safety concerns originating from the excessive metal load, which is essential to compensate for the loss of Li sources resulting from their poor coulombic efficiencies (CEs). Therefore, the development of high-performance LMA is needed to realize anode-minimized Li metal batteries (LMBs). In this study, high-performance LMAs are produced by introducing a hierarchically nanoporous assembly (HNA) composed of functionalized onion-like graphitic carbon building blocks, several nanometers in diameter, as a catalytic scaffold for Li-metal storage. The HNA-based electrodes lead to a high Li ion concentration in the nanoporous structure, showing a high CE of ≈99.1%, high rate capability of 12 mA cm-2 , and a stable cycling behavior of more than 750 cycles. In addition, anode-minimized LMBs are achieved using a HNA that has limited Li content (≈0.13 mg cm-2 ), corresponding to 6.5% of the cathode material (commercial NCM622 (≈2 mg cm-2 )). The LMBs demonstrate a feasible electrochemical performance with high energy and power densities of ≈510 Wh kgelectrode -1 and ≈2760 W kgelectrode -1 , respectively, for more than 100 cycles.

Hierarchically Nanoporous Pyropolymers Derived from Waste Pinecone as a Pseudocapacitive Electrode for Lithium Ion Hybrid Capacitors.

The non-aqueous asymmetric lithium ion hybrid capacitor (LIHC) is a tactical energy storage device composed of a faradic and non-faradic electrode pair, which aims to achieve both high energy and great power densities. On the other hand, the different types of electrode combinations cause severe imbalances in energy and power capabilities, leading to poor electrochemical performance. Herein, waste pinecone-derived hierarchically porous pyropolymers (WP-HPPs) were fabricated as a surface-driven pseudocapacitive electrode, which has the advantages of both faradic and non-faradic electrodes. The unique materials properties of WP-HPPs possessing high effective surface areas and hierarchically open nanopores led to high specific capacities of ~412 mA h g-1 and considerable rate/cycling performance as a cathode for LIHCs. In particular, nanometer-scale pores, approximately 3 nm in size, plays a key role in the pseudocapacitive charge storage behaviors because open nanopores can transport solvated Li-ions easily into the inside of complex carbon structures and a large specific surface area can be provided by the effective active surface for charge storage. In addition, WP-HPP-based asymmetric LIHCs assembled with a pseudocapacitive counterpart demonstrated feasible electrochemical performance, such as maximum specific energy and specific power of ~340 Wh kg-1 and ~11,000 W kg-1, respectively, with significant cycling stability.

Pbp1-Interacting Protein Mkt1 Regulates Virulence and Sexual Reproduction in Cryptococcus neoformans.

The Mkt1-Pbp1 complex promotes mating-type switching by regulating the translation of HO mRNA in Saccharomyces cerevisiae. Here, we performed in vivo immunoprecipitation assays and mass spectrometry analyses in the human fungal pathogen Cryptococcus neoformans to show that Pbp1, a poly(A)-binding protein-binding protein, interacts with Mkt1 containing a PIN like-domain. Association of Pbp1 with Mkt1 was confirmed by co-immunoprecipitation assays. Results of spot dilution growth assays showed that unlike pbp1 deletion mutant strains, mkt1 deletion mutant strains were not resistant to heat stress compared with wild-type. However, similar to the pbp1 deletion mutant strains, the mkt1 deletion mutants exhibited both, defective dikaryotic hyphal production and reduced pheromone gene (MFα1) expression during mating. In addition, deletion of mkt1 caused attenuated virulence in a murine intranasal inhalation model. Taken together, our findings reveal that Mkt1 plays a crucial role in sexual reproduction and virulence in C. neoformans.

Intensification of Pseudocapacitance by Nanopore Engineering on Waste-Bamboo-Derived Carbon as a Positive Electrode for Lithium-Ion Batteries.

Nanoporous carbon, including redox-active functional groups, can be a promising active electrode material (AEM) as a positive electrode for lithium-ion batteries owing to its high electrochemical performance originating from the host-free surface-driven charge storage process. This study examined the effects of the nanopore size on the pseudocapacitance of the nanoporous carbon materials using nanopore-engineered carbon-based AEMs (NE-C-AEMs). The pseudocapacitance of NE-C-AEMs was intensified, when the pore diameter was ≥2 nm in a voltage range of 1.0~4.8 V vs Li+/Li under the conventional carbonate-based electrolyte system, showing a high specific capacity of ~485 mA·h·g-1. In addition, the NE-C-AEMs exhibited high rate capabilities at current ranges from 0.2 to 4.0 A·g-1 as well as stable cycling behavior for more than 300 cycles. The high electrochemical performance of NE-C-AEMs was demonstrated by full-cell tests with a graphite nanosheet anode, where a high specific energy and power of ~345 Wh·kg-1 and ~6100 W·Kg-1, respectively, were achieved.

Differential effects of alkyl gallates on quorum sensing in Pseudomonas aeruginosa.

Virulence factors and biofilms constitute attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Among alkyl gallates, propyl gallate (PG) and octyl gallate (OG) are used as food preservatives. Here we found that alkyl gallates differentially affect virulence, biofilm formation, and quorum sensing (QS) in Pseudomonas aeruginosa. Ethyl gallate (EG), PG, and butyl gallate (BG) inhibited biofilm formation and virulence factors including elastase, pyocyanin, and rhamnolipid, in P. aeruginosa without affecting cell viability by antagonizing the QS receptors LasR and RhlR. PG exhibited the most potent activity. Interestingly, hexyl gallate (HG) inhibited the production of rhamnolipid and pyocyanin but did not affect elastase production or biofilm formation. Notably, OG inhibited the production of rhamnolipid and pyocyanin but stimulated elastase production and biofilm formation. Analysis of QS signaling molecule production and QS gene expression suggested that HG inhibited RhlR, while OG activated LasR but inhibited PqsR. This mechanism was confirmed using QS mutants. Additionally, PG prevented the virulence of P. aeruginosa in Caenorhabditis elegans and a mouse model. This is the first report of the differential effects of alkyl gallates on QS systems and PG has great potential as an inhibitor of the virulence and biofilm formation of P. aeruginosa.

Catalytic Pyroprotein Seed Layers for Sodium Metal Anodes.

We report a pyroprotein seed layer (PSL, ∼100 nm in thickness)-coated Cu foil electrode (PSL-Cu) demonstrating highly reversible Na metal storage behavior with a mean Coulombic efficiency (CE) of ∼99.96% over 300 cycles in a glyme-based electrolyte. Via a synergistic effect with the electrolyte, the carbonaceous thin film containing numerous nucleophilic active sites guides the homogeneous Na metal deposition/stripping process with the formation of numerous catalytic seeds, resulting in remarkably stable cycling and a low Na metal nucleation overpotential of ∼10 mV. In addition, the CE deviation values of the PSL-Cu electrode were ∼0.43% in several cell tests, demonstrating its reliable cycling behavior with low cell-to-cell variation. The practicality of PSL-Cu was further demonstrated via full-cell experiments with a polyanion cathode, in which it achieved a high specific power density and energy density of 3,800 W kg-1 and ∼402 W h kg-1, respectively. This work provides a simple process for the fabrication of a Na metal anode.

Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic.

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans.

Mon1 is a guanine nucleotide exchange factor subunit that activates the Ypt7 Rab GTPase and is essential for vacuole trafficking and autophagy in eukaryotic organisms. Here, we identified and characterized the function of Mon1, an ortholog of Saccharomyces cerevisiae Mon1, in a human fungal pathogen, Cryptococcus neoformans. Mutation in mon1 resulted in hypersensitivity to thermal stress. The mon1 deletion mutant exhibited increased sensitivity to cell wall and endoplasmic reticulum stress. However, the mon1 deletion mutant showed more resistance to the antifungal agent fluconazole. In vivo studies demonstrated that compared to the wild-type strain, the mon1 deletion mutant attenuated virulence in the Galleria mellonella insect model. Moreover, the mon1 deletion mutant was avirulent in the murine inhalation model. These results demonstrate that Mon1 plays a crucial role in stress survival and pathogenicity in C. neoformans.

Had1 Is Required for Cell Wall Integrity and Fungal Virulence in Cryptococcus neoformans.

Calcineurin modulates environmental stress survival and virulence of the human fungal pathogen Cryptococcus neoformans Previously, we identified 44 putative calcineurin substrates, and proposed that the calcineurin pathway is branched to regulate targets including Crz1, Pbp1, and Puf4 in C. neoformans In this study, we characterized Had1, which is one of the putative calcineurin substrates belonging to the ubiquitously conserved haloacid dehalogenase ß-phosphoglucomutase protein superfamily. Growth of the had1∆ mutant was found to be compromised at 38° or higher. In addition, the had1∆ mutant exhibited increased sensitivity to cell wall perturbing agents, including Congo Red and Calcofluor White, and to an endoplasmic reticulum stress inducer dithiothreitol. Virulence studies revealed that the had1 mutation results in attenuated virulence compared to the wild-type strain in a murine inhalation infection model. Genetic epistasis analysis revealed that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways that orchestrate stress survival and fungal virulence. Overall, our results demonstrate that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C. neoformans.

In Vivo Activity of LCB 01-0699, a Prodrug of LCB 01-0648, against Staphylococcus aureus.

LCB01-0648 is a novel oxazolidinone compound that shows potent antibacterial activities against most Gram-positive cocci, including the multi-drug resistant Staphylococcusaureus. In this study, in vivo activity of LCB01-0699, a LCB01-0648 prodrug, against S.aureus was evaluated in comparison with that of Linezolid. The results of the systemic infection study demonstrated that LCB01-0699 was more potent than Linezolid against methicillin-susceptible and -resistant S. aureus strains. The in vivo efficacy of LCB01-0699 against methicillin-susceptible and -resistant S. aureus strains in a skin infection model showed more potent activity than Linezolid. LCB01-0699 shows potent in vivo activity against methicillin-susceptible and -resistant S. aureus strains, suggesting that LCB01-0699 would be a novel candidate for the treatment of these infectious diseases caused by S. aureus.