Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

Alkaline protease functionalized hydrothermal synthesis of novel gold nanoparticles (ALPs-AuNPs): A new entry in photocatalytic and biological applications.

Researchers are consistently investigating novel and distinctive methods and materials that are compatible for human life and environmental conditions This study aimed to synthesize gold nanoparticles (ALPs-AuNPs) using for the first time an alkaline protease (ALPs) derived from Phalaris minor seed extract. A series of physicochemical techniques were used to inquire the formation, size, shape and crystalline nature of ALPs-AuNPs. The nanoparticles' ability to degrade methylene blue (MB) through photocatalysis under visible light irradiation was assessed. The findings demonstrated that ALPs-AuNPs exhibited remarkable efficacy by destroying 100 % of MB within a mere 30-minute irradiation period. In addition, the ALPs-AuNPs demonstrated remarkable effectiveness in inhibiting the growth of gram-positive (S. aureus) and gram-negative (E. coli) bacteria. The inhibition zones examined against the two bacterial strains were 23(±0.3) mm and 19(±0.4); 13(±0.3) mm and 11(±0.5) mm under light and dark conditions respectively. The ALPs-AuNPs exhibited significant antioxidant activity by effectively scavenging 88 % of stable and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. As a result, the findings demonstrated that the environmentally friendly ALPs-AuNPs showed a strong potential for MB degradation and bacterial pathogen treatment.

In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

PFKFB3-Meditated Glycolysis via the Reactive Oxygen Species-Hypoxic Inducible Factor 1α Axis Contributes to Inflammation and Proliferation of Staphylococcus aureus in Epithelial Cells.

Mastitis caused by antibiotic-resistant strains of Staphylococcus aureus is a significant concern in the livestock industry due to the economic losses it incurs. Regulating immunometabolism has emerged as a promising approach for preventing bacterial inflammation. To investigate the possibility of alleviating inflammation caused by S aureus infection by regulating host glycolysis, we subjected the murine mammary epithelial cell line (EpH4-Ev) to S aureus challenge. Our study revealed that S aureus can colonize EpH4-Ev cells and promote inflammation through hypoxic inducible factor 1α (HIF1α)-driven glycolysis. Notably, the activation of HIF1α was found to be dependent on the production of reactive oxygen species (ROS). By inhibiting PFKFB3, a key regulator in the host glycolytic pathway, we successfully modulated HIF1α-triggered metabolic reprogramming by reducing ROS production in S aureus-induced mastitis. Our findings suggest that there is a high potential for the development of novel anti-inflammatory therapies that safely inhibit the glycolytic rate-limiting enzyme PFKFB3.

Endemic Mountain Tea (Sideritis dichotoma Huter): Antioxidant and Antibacterial Activities, Mineral Content by ICP-MS and Phytochemical Content by LCHR-MS.

Endemic Sideritis dichotoma Huter, herbal tea, is used for colds, coughs and digestive ailments. Since at least 25 % of the active ingredients of pharmacological drugs are obtained from plants, it is important to investigate many plants, both traditionally used and whose potential hasnt yet been discovered, before they disappear. When the results compared to other Sideritis species, it has better antioxidant activity in DPPH⋅ scavenging activity, Fe3+ and Cu2+ reducing capacity methods and is also rich in minerals, necessary for human body, K, Mg, Ca, P, S. Zone values (10-19,5 mm) obtained on S. aureus, P. aeruginosa, E. coli, S.enteritidis, E. faecalis, P. putida and K. pnemoniae strains are an indication that the plant has antibacterial activity. 27 secondary metabolites were identified by LC-HRMS. Verbascoside, pharmacologically active compound known to have antitumor effect, cytotoxic selectivity and anticancer activity, is the most abundant phenolic in ethanol extract (49297.13 mg/kg).

α-Amylase purified and characterized from fenugreek (Trigonella foenum-graecum) showed substantial anti-biofilm activity against Staphylococcus aureus MTCC740.

Starch hydrolyzing α-amylase from germinated fenugreek (Trigonella foenum-graecum) has been purified 104-fold to apparent electrophoretic homogeneity with a final specific activity of 297.5 units/mg. SDS-PAGE of the final preparation revealed a single protein band of 47.5 kDa, supported by LC/MS analysis and size-exclusion chromatography on the Superdex 200 (ÄKTA-FPLC). α-Amylase exhibited maximum activity at pH 5.5. An activation energy (Ea) of 9.12 kcal/mol was found to exist in the temperature range of 20 to 90 °C. When substrate concentrations were evaluated between 0.5 and 10 mg/mL, the Km and Vmax values for starch were observed to be 1.12 mg/mL and 384.14 µmol/min/mg, respectively. The major substrate starch exhibited high specificity for fenugreek α-amylase. In the presence of EDTA (5 mM), the activity was lost, however, it could be largely reversed with the addition of calcium. Furthermore, an effort was made to assess the ability of fenugreek seed-derived partially purified (DEAE-cellulose enzyme) and purified α-amylase to disperse inside 48 h-old biofilms of Staphylococcus aureus MTCC740. The outcomes clearly demonstrated that the purified and partially purified α-amylase both exhibited strong biofilm dispersion activity.

The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by ß-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the ß-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Deciphering anti-biofilm property of Arthrospira platensis-origin peptides against Staphylococcusaureus.

Arthrospira platensis is a valuable natural health supplement consisting of various types of vitamins, dietary minerals, and antioxidants. Although different studies have been conducted to explore the hidden benefits of this bacterium, its antimicrobial property has been poorly understood. To decipher this important feature, here, we extended our recently introduced optimization algorithm (Trader) for aligning amino acid sequences associated with the antimicrobial peptides (AMPs) of Staphylococcus aureus and A.platensis. As a result, similar amino acid sequences were identified, and several candidate peptides were generated accordingly. The obtained peptides were then filtered based on their potential biochemical and biophysical properties, and their 3D structures were simulated based on homology modeling techniques. Next, to investigate how the generated peptides can interact with S. aureus proteins (i.e., heptameric state of the hly and homodimeric form of the arsB), molecular docking approaches were used. The results indicated that four peptides included better molecular interactions relative to the other generated ones in terms of the number/average length of hydrogen bonds and hydrophobic interactions. Based on the outcomes, it can be concluded that the antimicrobial property of A.platensis might be associated with its capability in disturbing the membrane of pathogens and their functions.

Biological Selenite Reduction, Characterization and Bioactivities of Selenium Nanoparticles Biosynthesised by Pediococcus acidilactici DSM20284.

Selenium (Se) is in great demand as a health supplement due to its superior reactivity and excellent bioavailability, despite selenium nanoparticles (SeNPs) having signs of minor toxicity. At present, the efficiency of preparing SeNPs using lactic acid bacteria is unsatisfactory. Therefore, a probiotic bacterial strain that is highly efficient at converting selenite to elemental selenium is needed. In our work, four selenite-reducing bacteria were isolated from soil samples. Strain LAB-Se2, identified as Pediococcus acidilactici DSM20284, had a reduction rate of up to 98% at ambient temperature. This strain could reduce 100 mg L-1 of selenite to elemental Se within 48 h at pH 4.5-6.0, a temperature of 30-40 °C, and a salinity of 1.0-6.5%. The produced SeNPs were purified, freeze-dried, and subsequently systematically characterised using FTIR, DSL, SEM-EDS, and TEM techniques. SEM-EDS analysis proved the presence of selenium as the foremost constituent of SeNPs. The strain was able to form spherical SeNPs, as determined by TEM. In addition, DLS analysis confirmed that SeNPs were negatively charged (-26.9 mV) with an average particle size of 239.6 nm. FTIR analysis of the SeNPs indicated proteins and polysaccharides as capping agents on the SeNPs. The SeNPs synthesised by P. acidilactici showed remarkable antibacterial activity against E. coli, B. subtilis, S. aureus, and K. pneumoniae with inhibition zones of 17.5 mm, 13.4 mm, 27.9 mm, and 16.2 mm, respectively; they also showed varied MIC values in the range of 15-120 µg mL-1. The DPPH, ABTS, and hydroxyl, and superoxide scavenging activities of the SeNPs were 70.3%, 72.8%, 95.2%, and 85.7%, respectively. The SeNPs synthesised by the probiotic Lactococcus lactis have the potential for safe use in biomedical and nutritional applications.

Copper ions inhibit pentose phosphate pathway function in Staphylococcus aureus.

To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop-). Exposure of the cop- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.

Promising antimicrobial and antibiofilm activities of Orobanche aegyptiaca extract-mediated bimetallic silver-selenium nanoparticles synthesis: Effect of UV-exposure, bacterial membrane leakage reaction mechanism, and kinetic study.

In this research, Orobanche aegyptiaca extract was utilized as an eco-friendly, and cost-effective green route for the construction of bimetallic silver-selenium nanoparticles (Ag-Se NPs). Bimetallic Ag-Se NPs were characterized by XRD, EDX, FTIR, HR-TEM, DLS, SEM/mapping and EDX studies. Antimicrobial, and antibiofilm potentials were tested against some selected pathogenic bacteria and unicellular fungi by ZOI, MIC, effect of UV exposure, and inhibition %. Reaction mechanism was assessed through membrane leakage assay and SEM imaging. HRTEM analysis confirmed the spherical nature and was ranged from 18.1 nm to 72.0 nm, and the avarage particle size is determined to be 30.58 nm. SEM imaging prove that bimetallic Ag-Se NPs presents as a bright particles, and both Ag and Se were distributed equally across O. aegyptiaca extract and Guar gum stabilizers. ZOI results showed that, bimetallic Ag-Se NPs have antimicrobial activity against S. aureus (20.0 nm), E. coli (18.5 nm), P. aeruginosa (12.6 nm), and C. albicans (18.2 nm). In addition, bimetallic Ag-Se NPs were able to inhibit the biofilm formation for S. aureus by 79.48%, for E. coli by 78.79%, for P. aeruginosa by 77.50%, and for C. albicans by 73.73%. Bimetallic Ag-Se NPs are an excellent disinfectant once it had excited by UV light. It was observed that the quantity of cellular protein discharged from S. aureus is directly proportional to the concentration of bimetallic Ag-Se NPs and found to be 244.21 µg/mL after the treatment with 1 mg/mL, which proves the antibacterial characteristics, and explains the creation of holes in the cell membrane of S. aureus producing in the oozing out of the proteins from the S. aureus cytoplasm. Based on the promising properties, they showed superior antimicrobial potential at low concentration (to avoid toxicity) and continued-phase durability, they may use in pharmaceutical and biomedical applications.

In-vitro and In-vivo Identification, Absorbtion and Metabolism Network Analysis of Filifolium sibiricum Flavonoids Dropping Pill by UHPLC-Q-TOF-MS.

BACKGROUND: Filifolium sibiricum flavonoids dropping pill (FSFp), a unique Chinese Filifolii sibirici herba extract preparation, has the potential as an alternative therapy against S. aureus infection (SA) and antiinfection. However, its chemical composition and in vivo metabolism characteristics remain unknown, which limits its clinical application. METHODS: Here, we aimed to understand the in vitro and in vivo material basis of FSFp. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to identify chemicals in FSFp as well as its phase I and phase II reaction metabolites in plasma, urine and feces. RESULTS: A total of 38 chemicals were characterized in FSFp, including 22 flavonoids, 10 organic acids, 3 chromones, 1 aromatic ketone, 1 coumarin, and 1 ligan. After analysis of the drugged bio-samples, a total of 21 compounds were found in urine, and 16 of them were found in feces, but only one was found in plasma. In addition, 56 FSFp-related metabolites were characterized, of which 56 were in urine, 4 in feces, and 8 in plasma. CONCLUSION: This is the first comprehensive research of FSFp on chemical constituents and metabolic profiles. It was expected that this study would offer reliable support for further investigation of FSFp.

A short-chain acyl-CoA synthetase that supports branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids (BCFAs). The branched-chain acyl-CoA precursors, utilized to initiate fatty acid synthesis, are derived from branched-chain ketoacid dehydrogenase (Bkd), a multiprotein complex that converts α-keto acids to their corresponding acyl-CoAs; however, Bkd KO strains still contain BCFAs. Here, we show that commonly used rich medias contain substantial concentrations of short-chain acids, like 2-methylbutyric and isobutyric acids, that are incorporated into membrane BCFAs. Bkd-deficient strains cannot grow in defined medium unless it is supplemented with either 2-methylbutyric or isobutyric acid. We performed a screen of candidate KO strains and identified the methylbutyryl-CoA synthetase (mbcS gene; SAUSA300_2542) as required for the incorporation of 2-methylbutyric and isobutyric acids into phosphatidylglycerol. Our mass tracing experiments show that isobutyric acid is converted to isobutyryl-CoA that flows into the even-chain acyl-acyl carrier protein intermediates in the type II fatty acid biosynthesis elongation cycle. Furthermore, purified MbcS is an ATP-dependent acyl-CoA synthetase that selectively catalyzes the activation of 2-methylbutyrate and isobutyrate. We found that butyrate and isovalerate are poor MbcS substrates and activity was not detected with acetate or short-chain dicarboxylic acids. Thus, MbcS functions to convert extracellular 2-methylbutyric and isobutyric acids to their respective acyl-CoAs that are used by 3-ketoacyl-ACP synthase III (FabH) to initiate BCFA biosynthesis.

Allosteric inhibition of Staphylococcus aureus MenD by 1,4-dihydroxy naphthoic acid: a feedback inhibition mechanism of the menaquinone biosynthesis pathway.

Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus (Sau), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau-MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau-MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau, an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb-MenD and Sau-MenD, feedback inhibition by DHNA is a conserved mechanism in Sau-MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Alkaline "Nanoswords" Coordinate Ferroptosis-like Bacterial Death for Antibiosis and Osseointegration.

Ferroptosis is an iron-dependent cell death and is associated with cancer therapy. Can it play a role in resistance of postoperative infection of implants, especially with an extracellular supplement of Fe ions in a non-cytotoxic dose? To answer this, "nanoswords" of Fe-doped titanite are fabricated on a Ti implant surface to resist bacterial invasion by a synergistic action of ferroptosis-like bacteria killing, proton disturbance, and physical puncture. The related antibiosis mechanism is explored by atomic force microscopy and genome sequencing. The nanoswords induce an increased local pH value, which not only weakens the proton motive force, reducing adenosine triphosphate synthesis of Staphylococcus aureus, but also decreases the membrane modulus, making the nanoswords distort and even puncture a bacterial membrane easily. Simultaneously, more Fe ions are taken by bacteria due to increased bacterial membrane permeability, resulting in ferroptosis-like death of bacteria, and this is demonstrated by intracellular iron enrichment, lipid peroxidation, and glutathione depletion. Interestingly, a microenvironment constructed by these nanoswords improves osteoblast behavior in vitro and bone regeneration in vivo. Overall, the nanoswords can induce ferroptosis-like bacterial death without cytotoxicity and have great promise in applications with clinical implants for outstanding antibiosis and biointegration performance.

Immunomodulation, Fish Health and Resistance to Staphylococcus aureus of Nile Tilapia (Oreochromis niloticus) Fed Diet Supplemented with Zinc Oxide Nanoparticles and Zinc Acetate.

Recently some metal-based nanoparticles have gained serious attention from aquaculture and the fish feed industry as feed supplements. Oral supplementation of zinc oxide nanoparticles (ZnO-NPs) in fish feed, replacing Zn acetate (conventionally used zinc), is suggested as a cost-effective and efficient approach. Our study assessed the response of Nile tilapia, Oreochromis niloticus, fingerlings after its diet supplemented with chemically synthesized ZnO-NPs and zinc acetate under controlled conditions. ZnO-NPs were chemically synthesized and characterized. Tilapia fingerlings with an average body weight of 09.12 ± 1.23 g were randomly distributed into five groups. An 8-week trial was set with control and four experimental groups. Basal diet (D1) was used as control, whereas D2, D3 and D4 comprising 20, 40, and 60 mgkg-1 ZnO-NPs supplementation were experimental diets. Additionally, D5 was composed of a basal diet supplemented with 40 mgkg-1 of conventionally used zinc acetate. Significant improvement (P < 0.05) was found in nanoparticles and Zn acetate supplemented groups as compared to control, while the 40 mgkg-1 Zn-NPs supplemented diet (D3) showed best performance in terms of health parameters, oxidative status and disease resistance. Antioxidant profiling was based on catalase, superoxide dismutase, glutathione's transferase, and malondialdehyde; hematology included Hb, WBCs, RBCs, HCT MCV, MCH and MCHC; immunological parameters comprised IgM, lysozyme activity, phagocytic activity, respiratory burst activity, cholesterol, aspartate aminotransferase, alanine aminotransferase, glucose content, and total serum proteins. We report that the D3 (40 mgkg-1 ZnO-NPs supplementation) significantly (P < 0.05) improved health-related parameters as compared to the other groups. Moreover, D3 also showed significantly decreased mortality percentage when challenged by Staphylococcus aureus, while the Zn acetate supplemented diet group showed better results as compared to control. Overall results suggest the basal diet supplemented with 40 mgkg-1 ZnO-NP for enhanced health parameters, oxidative status, immune response, and disease resistance. Hence, 40mgkg-1 ZnO-NP can be recommended to formulate the practical diet of fish to boost health improvement, immunomodulation, and resistance to bacterial disease.

Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP.

The emergence of methicillin-resistant Staphylococcus aureus isolates highlights the urgent need to develop more antibiotics. ClpP is a highly conserved protease regulated by ATPases in bacteria and in mitochondria. Aberrant activation of  bacterial ClpP is an alternative method of discovering antibiotics, while it remains difficult to develop selective  Staphylococcus aureus ClpP activators that can avoid disturbing Homo sapiens ClpP functions. Here, we use a structure-based design to identify (R)- and (S)-ZG197 as highly selective Staphylococcus aureus ClpP activators. The key structural elements in Homo sapiens ClpP, particularly W146 and its joint action with the C-terminal motif, significantly contribute to the discrimination of the activators. Our selective activators display wide antibiotic properties towards an array of multidrug-resistant staphylococcal strains in vitro, and demonstrate promising antibiotic efficacy in zebrafish and murine skin infection models. Our findings indicate that the species-specific activators of Staphylococcus aureus ClpP are exciting therapeutic agents to treat staphylococcal infections.

Antibacterial Efficacy and Mechanisms of Curcumin-Based Photodynamic Treatment against Staphylococcus aureus and Its Application in Juices.

Antimicrobial Photodynamic Treatment (aPDT) is a non-thermal sterilization technology, which can inactivate common foodborne pathogens. In the present study, photodynamic inactivation on Staphylococcus aureus (S. aureus) with different concentrations of curcumin and light dose was evaluated and the mechanisms were also investigated. The results showed that curcumin-based aPDT could inactivate S. aureus cells by 6.9 log CFU/mL in phosphate buffered saline (PBS). Moreover, the modified Gompertz model presented a good fit at the inactivation data of S. aureus. Photodynamic treatment caused cell membrane damage as revealed by analyzing scanning electron microscopy (SEM) images. Leakage of intracellular constituents further indicated that cell membrane permeability was changed. Flow cytometry with double staining demonstrated that cell membrane integrity and the activity of nonspecific esterase were destroyed. Compared with the control group, intracellular reactive oxygen species (ROS) levels caused by photodynamic treatment significantly increased. Furthermore, curcumin-based aPDT reduced S. aureus by 5 log CFU/mL in juices. The color of the juices was also tested using a Chromatic meter, and it was found that b* values were the most markedly influenced by photodynamic treatment. Overall, curcumin-based aPDT had strong antibacterial activity against S. aureus. This approach has the potential to remove foodborne pathogens from liquid food.

Optimal dose of lactoferrin reduces the resilience of in vitro Staphylococcus aureus colonies.

The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of ß-lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin's method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ ß-lactam antibiotic dose combinations and observed that at low doses (< 0.39 µM), lactoferrin contributes to increased S. aureus growth, but at higher doses (> 6.25 µM), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the ß-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ ß-lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from ß-lactam-induced production of ß-lactamase, which when sufficiently large reduces the population's ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using ß-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of ß-lactams.

Traditional Chinese Medicine Tanreqing Targets Both Cell Division and Virulence in Staphylococcus aureus.

Staphylococcus aureus has been recognized as an important human pathogen and poses a serious health threat worldwide. With the advent of antibiotic resistance, such as the increased number of methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to develop new therapeutical agents. In this study, Chinese traditional medicine Tanreqing (TRQ) has been used as an alternative treating agent against MRSA and we aim to unravel the mode of action of TRQ underlying MRSA inhibition. TRQ treatment affected numerous gene expression as revealed by RNA-seq analysis. Meanwhile, TRQ targeted cell division to inhibit cell growth as shown by illumination microscopy. Besides, we confirmed that TRQ downregulates the expression of virulence factors such as hemolysin and autolysin. Finally, we used a murine model to demonstrate that TRQ efficiently reduces bacterial virulence. Altogether, we have proved TRQ formula to be an effective agent against S. aureus infections.