Synthesis of novel ciprofloxacin-avibactam conjugates for the development of second-generation non-ß-lactam-ß-lactamase inhibitors.

To overcome the antibiotic resistance challenge, we synthesized a novel class of conjugates based on ciprofloxacin and avibactam, covalently linked by diverse amino acids. In vitro studies of these conjugates have shown improved antibacterial efficacy of avibactam when used alone against some ESKAPE pathogens, i.e., S. aureus, E. coli, and A. baumannii. Further, ceftazidime was screened in combination with all conjugates and found to be less synergistically effective than avibactam-ceftazidime co-dosing against K. pneumoniae and E. coli bacterial strains. Subsequently, the top-ranked active conjugates were investigated against the commercially available ß-lactamase-II (Penicillinase from Bacillus cereus) through in vitro studies. These studies elucidated two conjugates i.e, 9 (IC50 = 1.69±0.35 nM) and 24b (IC50 = 57.37±5.39 nM), which have higher inhibition profile than avibactam (IC50 = 141.08±12.20 nM). These outcomes allude to avibactam integration with ciprofloxacin is a novel and fruitful approach to discovering clinically valuable next-generation non-ß-lactam-ß-lactamase inhibitors.

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.

Chalcone-Derived Lactones: Synthesis, Whole-Cell Biotransformation, and Evaluation of Their Antibacterial and Antifungal Activity.

Four compounds with lactone moiety were synthesized from chalcone 1 in three- or four-step synthesis. γ-Bromo-δ-lactone 5 was the only product of bromolactonization of acid 4 whereas bromolactonization of ester 3, apart from lactone 5 also afforded its isomer 6 and two diastereoisomeric δ-hydroxy-γ-lactones 7 and 8. Lactone 8 was also obtained in 88% yield as a product of simultaneous dehalogenation and translactonization of γ-bromo-δ-lactone 5 by Penicillum frequentans AM 359. Chalcone-derived lactones 5-8 were subjected to the tests on antimicrobial activity and the results compared with activity of starting chalcone 1. Obtained lactones 5-8 in most cases limited the growth of tested bacterial and fungal strains. The highest activity was found for δ-hydroxy-γ-lactone 8 which completely inhibited the growth of Staphylococcus aureus, Fusarium graminearum, Aspergillus niger, and Alternaria sp. The introduction of lactone moiety into chalcone scaffold significantly improved antimicrobial activity of the compound: γ-bromo-δ-lactone 6 and δ-hydroxy-γ-lactone 8 were significantly stronger growth inhibitors of S. aureus and F. graminearum. In the case of the latter, a clear positive effect of the lactone function on the antifungal activity was also observed for γ-bromo-δ-lactone 5.

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.

Staphylococcus aureus persisters are associated with reduced clearance in a catheter-associated biofilm infection.

Background: Staphylococcus aureus causes a wide variety of infections, many of which are chronic or relapsing in nature. Antibiotic therapy is often ineffective against S. aureus biofilm-mediated infections. Biofilms are difficult to treat partly due to their tolerance to antibiotics, however the underlying mechanism responsible for this remains unknown. One possible explanation is the presence of persister cells-dormant-like cells that exhibit tolerance to antibiotics. Recent studies have shown a connection between a fumC (fumarase C, a gene in the tricarboxylic acid cycle) knockout strain and increased survival to antibiotics, antimicrobial peptides, and in a Drosophila melanogaster model. Objective: It remained unclear whether a S. aureus high persister strain would have a survival advantage in the presence of innate and adaptive immunity. To further investigate this, a fumC knockout and wild type strains were examined in a murine catheter-associated biofilm model. Results: Interestingly, mice struggled to clear both S. aureus wild type and the fumC knockout strains. We reasoned both biofilm-mediated infections predominantly consisted of persister cells. To determine the persister cell population within biofilms, expression of a persister cell marker (Pcap5A::dsRED) in a biofilm was examined. Cell sorting of biofilms challenged with antibiotics revealed cells with intermediate and high expression of cap5A had 5.9-and 4.5-fold higher percent survival compared to cells with low cap5A expression. Based on previous findings that persisters are associated with reduced membrane potential, flow cytometry analysis was used to examine the metabolic state of cells within a biofilm. We confirmed cells within biofilms had reduced membrane potential compared to both stationary phase cultures (2.5-fold) and exponential phase cultures (22.4-fold). Supporting these findings, cells within a biofilm still exhibited tolerance to antibiotic challenge following dispersal of the matrix through proteinase K. Conclusion: Collectively, these data show that biofilms are largely comprised of persister cells, and this may explain why biofilm infections are often chronic and/or relapsing in clinical settings.

Harzianic Acid Activity against Staphylococcus aureus and Its Role in Calcium Regulation.

Staphylococcus aureus is a Gram-positive bacterium, which can be found, as a commensal microorganism, on the skin surface or in the nasal mucosa of the human population. However, S. aureus may become pathogenic and cause severe infections, especially in hospitalized patients. As an opportunistic pathogen, in fact, S. aureus interferes with the host Ca2+ signaling, favoring the spread of the infection and tissue destruction. The identification of novel strategies to restore calcium homeostasis and prevent the associated clinical outcomes is an emerging challenge. Here, we investigate whether harzianic acid, a bioactive metabolite derived from fungi of the genus Trichoderma, could control S. aureus-induced Ca2+ movements. First, we show the capability of harzianic acid to complex calcium divalent cations, using mass spectrometric, potentiometric, spectrophotometric, and nuclear magnetic resonance techniques. Then, we demonstrate that harzianic acid significantly modulates Ca2+ increase in HaCaT (human keratinocytes) cells incubated with S. aureus. In conclusion, this study suggests harzianic acid as a promising therapeutical alternative against diseases associated with Ca2+ homeostasis alteration.

Functional membrane microdomains and the hydroxamate siderophore transporter ATPase FhuC govern Isd-dependent heme acquisition in Staphylococcus aureus.

Sufficient access to transition metals such as iron is essential for bacterial proliferation and their active limitation within host tissues effectively restricts infection. To overcome iron limitation, the invasive pathogen Staphylococcus aureus uses the iron-regulated surface determinant (Isd) system to acquire hemoglobin-derived heme. While heme transport over the cell wall is well understood, its transport over the membrane is hardly investigated. In this study, we show the heme-specific permease IsdF to be energized by the general ATPase FhuC. Additionally, we show that IsdF needs appropriate location within the membrane for functionality. The membrane of S. aureus possesses special compartments (functional membrane microdomains [FMMs]) to organize membrane complexes. We show IsdF to be associated with FMMs, to directly interact with the FMM scaffolding protein flotillin A (FloA) and to co-localize with the latter on intact bacterial cells. Additionally, Isd-dependent bacterial growth required FMMs and FloA. Our study shows that Isd-dependent heme acquisition requires a highly structured cell envelope to allow coordinated transport over the cell wall and membrane and it gives the first example of a bacterial nutrient acquisition system that depends on FMMs.

Interactions between metabolism and growth can determine the co-existence of Staphylococcus aureus and Pseudomonas aeruginosa.

Most bacteria exist and interact within polymicrobial communities. These interactions produce unique compounds, increase virulence and augment antibiotic resistance. One community associated with negative healthcare outcomes consists of Pseudomonas aeruginosa and Staphylococcus aureus. When co-cultured, virulence factors secreted by P. aeruginosa reduce metabolism and growth in S. aureus. When grown in vitro, this allows P. aeruginosa to drive S. aureus toward extinction. However, when found in vivo, both species can co-exist. Previous work has noted that this may be due to altered gene expression or mutations. However, little is known about how the growth environment could influence the co-existence of both species. Using a combination of mathematical modeling and experimentation, we show that changes to bacterial growth and metabolism caused by differences in the growth environment can determine the final population composition. We found that changing the carbon source in growth media affects the ratio of ATP to growth rate for both species, a metric we call absolute growth. We found that as a growth environment increases the absolute growth for one species, that species will increasingly dominate the co-culture. This is due to interactions between growth, metabolism, and metabolism-altering virulence factors produced by P. aeruginosa. Finally, we show that the relationship between absolute growth and the final population composition can be perturbed by altering the spatial structure in the community. Our results demonstrate that differences in growth environment can account for conflicting observations regarding the co-existence of these bacterial species in the literature, provides support for the intermediate disturbance hypothesis, and may offer a novel mechanism to manipulate polymicrobial populations.

Infections caused by multiple types of bacteria are tough to treat. For example, co-infections with Staphylococcus aureus and Pseudomonas aeruginosa are so difficult to cure they may persist for years in humans and cause serious illness. But when these two types of bacteria are grown together in the laboratory, P. aeruginosa kills off all the S. aureus. Learning why these two types of bacteria can coexist in people but not in the laboratory may lead to new treatments to clear infections. It may also help scientists grow beneficial bacteria mixes that break down pollution or produce biofuels. Pajon and Fortoul et al. show that interactions between bacterial metabolism and growth rate determine whether S. aureus and P. aeruginosa coexist. In the experiments, they grew both types of bacteria in different environments with different food sources. They measured their growth and metabolism and how many bacteria of each species survived over time. Then, they used their data to develop a mathematical model and tested its predictions in the laboratory again. The type of bacteria that had more energy also grew faster and outcompeted the other species. Measuring the growth rate of the two species allowed the scientists to predict which one would win out and what the tipping point would be. Physically disrupting the mix of bacteria disrupted this relationship. These results may help explain what allows these bacteria to coexist in some settings but not others. It may enable scientists to develop new ways to treat infections with P. aeruginosa and S. aureus that work by manipulating growth in the two species. Bacterial growth and metabolism are known to drive antibacterial resistance. Studies in mice using drugs or other therapies to manipulate growth and metabolism may help scientists thwart these resistance mechanisms. The results may also help scientists design and grow beneficial multispecies bacteria communities.

Effects of 5α-dihydrotestosterone on the modulation of monocyte/macrophage response to Staphylococcus aureus: an in vitro study.

BACKGROUND: Staphylococcus aureus (S. aureus) is a pathogen responsible for a wide range of clinical manifestations and potentially fatal conditions. There is a paucity of information on the influence of androgens in the immune response to S. aureus infection. In this study, we evaluated the influence of the hormone 5α-dihydrotestosterone (DHT) on mouse peritoneal macrophages (MPMs) and human peripheral blood monocytes (HPBMs) induced by S. aureus. METHODS: An in vitro model of MPMs from BALB/c sham males, orchiectomised (OQX) males, and females was used. Cells were inoculated with 10 µL of S. aureus, phage-type 80 or sterile saline (control) for 6 h. The MPMs of OQX males and females were pre-treated with 100 µL of 10-2 M DHT for 24 h before inoculation with S. aureus. The concentration of the cytokines TNF-α, IL-1α, IL-6, IL-8, and IL-10; total nitrites (NO-2); and hydrogen peroxide (H2O2) were measured in the supernatant of MPM cultures. In addition, the toll-like receptor 2 (TLR2) and nuclear factor kappa B (NF-kB) genes that are involved in immune responses were analysed. For the in vitro model of HPBMs, nine men and nine women of childbearing age were selected and HPBMs were isolated from samples of the volunteers' peripheral blood. In women, blood was collected during the periovulatory period. The HPBMs were inoculated with S. aureus for 6 h and the supernatant was collected for the analysis of cytokines TNF-α, IL-6, IL-12; and GM-CSF, NO-2, and H2O2. The HPBMs were then removed for the analysis of 84 genes involved in the host's response to bacterial infections by RT-PCR array. GraphPad was used for statistical analysis with a p value < 0.05. RESULTS: Our data demonstrated that MPMs from sham males inoculated with S. aureus displayed higher concentrations of inflammatory cytokines and lower concentrations of IL-10, NO-2, and H2O2 when compared with MPMs from OQX males and females. A similar result was observed in the HPBMs of men when compared with those of women. Previous treatment with DHT in women HPBMs increased the production of pro-inflammatory cytokines and decreased the levels of IL-10, NO-2, and H2O2. The analysis of gene expression showed that DHT increased the activity of the TLR2 and NF-kB pathways in both MPMs and HPBMs. CONCLUSIONS: We found that DHT acts as an inflammatory modulator in the monocyte/macrophage response induced by S. aureus and females exhibit a better immune defence response against this pathogen.

A unique C-type lectin, Ladderlectin, from large yellow croaker (Larimichthys crocea) is involved in bacterial cell membrane damage.

Ladderlectin is unique C-type lectin because it has been so far found only in teleost fish. In this study, large yellow croaker (Larimichthys crocea) Ladderlecin (LcLL) sequence was identified and characterized. LcLL encodes a polypeptide of 186 amino acids that includes a signal peptide and a C-type lectin-like domains (CTLD) with two sugar-binding motifs of WSD and EPN. Tissues distribution analysis revealed that LcLL is a ubiquitous gene, with the highest expression in head kidney and gill. Subcellular localization showed that LcLL was in cytoplasm and nucleus of HEK 293T cells. Transcripts of LcLL were significantly up regulated after immune challenge with P. plecoglossicida. In contrast to this, a sharp down-regulation occurred after Scuticociliatida infection. Moreover, recombinant LcLL (rLcLL) was prepared and exhibited hemagglutination on L. crocea and N. albiflora erythrocytes in a Ca2+-dependent manner, which can be only inhibited by LPS. rLcLL showed a strong ability of binding to Gram + bacteria (M. lysodeikticus, S. aureus, B. subtilis) and Gram-bacteria (P. plecoglossicida, E. coli, V. Vulnificus, V. harveyi, V. alginolyticus, V. parahaemolyticus. A. hydrophila, and E. tarda), and could agglutinate all tested bacteria except for P. plecoglossicida. Further study showed that rLcLL promoted the gathered bacteria death through damaging cell membrane based on PI staining and SEM observation. However, rLcLL does neither kill bacteria directly nor have complement-activating activities. Altogether, these results demonstrated that LcLL played a vital role in L. crocea innate immune towards bacterial and parasitic challenge.

A Pilot Study to Evaluate Genipin in Staphylococcus aureus and Pseudomonas aeruginosa Keratitis Models: Modulation of Pro-Inflammatory Cytokines and Matrix Metalloproteinases.

Infectious keratitis is a vision-threatening microbial infection. The increasing antimicrobial resistance and the fact that severe cases often evolve into corneal perforation necessitate the development of alternative therapeutics for effective medical management. Genipin, a natural crosslinker, was recently shown to exert antimicrobial effects in an ex vivo model of microbial keratitis, highlighting its potential to serve as a novel treatment for infectious keratitis. This study aimed to evaluate the antimicrobial and anti-inflammatory effects of genipin in an in vivo model of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) keratitis. Clinical scores, confocal microscopy, plate count, and histology were carried out to evaluate the severity of keratitis. To assess the effect of genipin on inflammation, the gene expression of pro- and anti-inflammatory factors, including matrix metalloproteinases (MMPs), were evaluated. Genipin treatment alleviated the severity of bacterial keratitis by reducing bacterial load and repressing neutrophil infiltration. The expression of interleukin 1B (IL1B), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 15 (IL15), tumor necrosis factor-α (TNF-α), and interferon γ (IFNγ), as well as MMP2 and MMP9, were significantly reduced in genipin-treated corneas. Genipin promoted corneal proteolysis and host resistance to S. aureus and P. aeruginosa infection by suppressing inflammatory cell infiltration, regulating inflammatory mediators, and downregulating the expression of MMP2 and MMP9.

Staphylococcus aureus induces tolerance in human monocytes accompanied with expression changes of cell surface markers.

Exposure of human monocytes to lipopolysaccharide (LPS) or other pathogen-associated molecular pattern (PAMPs) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance (ET), associated with the pathogenesis of sepsis. In this study, we aimed to characterize the cellular state of human monocytes from healthy donors stimulated with Staphylococcus aureus in comparison to TLR2-specific ligands. We analyzed S. aureus induced gene expression changes after 2 and 24 hours by amplicon sequencing (RNA-AmpliSeq) and compared the pro-inflammatory response after 2 hours with the response in re-stimulation experiments. In parallel, glycoprotein expression changes in human monocytes after 24 hours of S. aureus stimulation were analyzed by proteomics and compared to stimulation experiments with TLR2 ligands Malp-2 and Pam3Cys and TLR4 ligand LPS. Finally, we analyzed peripheral blood monocytes of patients with S. aureus bloodstream infection for their ex vivo inflammatory responses towards S. aureus stimulation and their glycoprotein expression profiles. Our results demonstrate that monocytes from healthy donors stimulated with S. aureus and TLR ligands of Gram-positive bacteria entered the tolerant cell state after activation similar to LPS treatment. In particular reduced gene expression of pro-inflammatory cytokines (TNF, IL1ß) and chemokines (CCL20, CCL3, CCL4, CXCL2, CXCL3 and CXCL8) could be demonstrated. Glycoprotein expression changes in monocytes tolerized by the different TLR agonists were highly similar while S. aureus-stimulated monocytes shared some of the PAMP-induced changes but also exhibited a distinct expression profile. 11 glycoproteins (CD44, CD274, DSC2, ICAM1, LAMP3, LILRB1, PTGS2, SLC1A3, CR1, FGL2, and HP) were similarly up- or downregulated in all four comparisons in the tolerant cell state. Monocytes from patients with S. aureus bacteremia revealed preserved pro-inflammatory responsiveness to S. aureus stimulation ex vivo, expressed increased CD44 mRNA but no other glycoprotein of the tolerance signature was differentially expressed.

Divalent metal ion binding to Staphylococcus aureus FeoB transporter regions.

Transition metal ions such as iron, copper, zinc, manganese or, nickel are essential in many biological processes. Bacteria have developed a number of mechanisms for their acquisition and transport, in which numerous of proteins and smaller molecules are involved. One of the representatives of these proteins is FeoB, which belongs to the Feo (ferrous ion transporter) family. Although ferrous iron transport system is widespread among microorganisms, it is still poorly described in Gram-positive pathogens, such as Staphylococcus aureus. In this work, combined potentiometric and spectroscopic studies (UV-Vis, CD and EPR) were carried out to determine Cu(II), Fe(II) and Zn(II) binding modes to FeoB fragments (Ac-IDYHKLMK-NH2, Ac-ETSHDKY-NH2, and Ac-SFLHMVGS-NH2). For the first time iron(II) complexes with peptides were characterized by potentiometry. All studied ligands are able to form a variety of thermodynamically stable complexes with transition metal ions. It was concluded that among the studied systems, the most effective metal ion binding is observed for the Ac-ETSHDKY-NH2 peptide. Moreover, comparing preferences of all ligands towards different metal ions, copper(II) complexes are the most stable ones at physiological pH.

Proteomic landscape of extracellular vesicles in human retinal cells infected with Staphylococcus aureus and Pseudomonas aeruginosa: Role in endophthalmitis.

Extracellular Vesicles (EVs) have evolved as a promising entity for developing diagnostic and therapeutic biomarkers. We profiled global EV proteome of EVs from Human retinal cells (ARPE-19) infected with S. aureus and P. aeruginosa. EVs were isolated by ultracentrifugation and subjected to LC-MS/MS for proteome analysis. In S. aureus infection, sequest identified 864 proteins, of which 81 were differentially expressed in comparison to control. Similarly, in P. aeruginosa infection, of 516 proteins identified, 86 were differentially expressed. Additionally, 38 proteins were exclusive to infected sets. KEGG and Gene Ontology revealed crucial dysregulated pathways involving proteins such as complement cascades, annexins and calpain-2, all playing major role in the pathogenesis of the disease. This study provides insight into the global EV proteome of S. aureus and P. aeruginosa endophthalmitis with their functional correlation and distinctive pattern of expression. Calpain-2 and C8a are attractive biomarkers for bacterial endophthalmitis.

The Two-Track Investigation of Fibronectin Binding Protein A of Staphylococcus aureus from Bovine Mastitis as a Potential Candidate for Immunodiagnosis: A Pilot Study.

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Transcription tuned by S-nitrosylation underlies a mechanism for Staphylococcus aureus to circumvent vancomycin killing.

Treatment of Staphylococcus aureus infections is a constant challenge due to emerging resistance to vancomycin, a last-resort drug. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to a cysteine thiol, mediates redox-based signaling for eukaryotic cellular functions. However, its role in bacteria is largely unknown. Here, proteomic analysis revealed that S-nitrosylation is a prominent growth feature of vancomycin-intermediate S. aureus. Deletion of NO synthase (NOS) or removal of S-nitrosylation from the redox-sensitive regulator MgrA or WalR resulted in thinner cell walls and increased vancomycin susceptibility, which was due to attenuated promoter binding and released repression of genes involved in cell wall metabolism. These genes failed to respond to H2O2-induced oxidation, suggesting distinct transcriptional responses to alternative modifications of the cysteine residue. Furthermore, treatment with a NOS inhibitor significantly decreased vancomycin resistance in S. aureus. This study reveals that transcriptional regulation via S-nitrosylation underlies a mechanism for NO-mediated bacterial antibiotic resistance.

Yttrium Oxide Nanoparticle-Loaded, Self-Assembled Peptide Gel with Antibacterial, Anti-Inflammatory, and Proangiogenic Properties for Wound Healing.

Chronic wounds are a major healthcare challenge owing to their complex healing mechanism and number of impediments to the healing process, like infections, unregulated inflammation, impaired cellular functions, poor angiogenesis, and enhanced protease activity. Current topical care strategies, such as surgical debridement, absorption of exudates, drug-loaded hydrogels for infection and inflammation management, and exogenous supply of growth factors for angiogenesis and cell proliferation, slow the progression of wounds and reduce patient suffering but suffer from low overall cure rates. Therefore, we have developed a proteolytically stable, multifunctional nanoparticle loaded-peptide gel with inherent anti-inflammatory, antibacterial, and pro-angiogenic properties to provide a favorable wound healing milieu by restoring impaired cellular functions. We have fabricated a self-assembled, lauric acid-peptide conjugate gel, LA-LLys-DPhe-LLys-NH2, loaded with yttrium oxide (Y2O3) nanoparticles (NLG). Gel formed a nanofibrous structure, and nanoparticles were passively entrapped within the network. The surface morphology, stability, viscoelastic, and self-healing characteristics of gels were characterized. It showed a high stability against degradation by proteolytic enzymes and highly potent antibacterial activities against E. coli and S. aureus due to the presence of positively charged side chains of lysine in the peptide chain. It also exhibited an excellent antioxidant activity as well as ability to stimulate cell proliferation in murine fibroblast (L929) cells and human umbilical vein endothelial cells (HUVECs). The incorporation of nanoparticles promoted angiogenesis by upregulating pro-angiogenic genes, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF2), and epidermal growth factor (EGFR), and the gel caused complete wound closure in cells. In summary, the Y2O3 nanoparticle-loaded lauric acid-peptide conjugate gel is able to elicit the desired tissue regeneration responses and, therefore, has a strong potential as a matrix for the treatment of chronic wounds.

Complementary spectroscopy studies and potential activities of levan-type fructan produced by Bacillus paralicheniformis ND2.

This study aimed at the production of marine bacterial exopolysaccharides (EPS) as biodegradable and nontoxic biopolymers, competing the synthetic derivatives, with detailed structural and conformational analyses using spectroscopy techniques. Twelve marine bacterial bacilli were isolated from the seawater of Mediterranean Sea, Egypt, then screened for EPS production. The most potent isolate was identified genetically as Bacillus paralicheniformis ND2 by16S rRNA gene sequence of ~99 % similarity. Plackett-Burman (PB) design identified the optimization conditions of EPS production, which yielded the maximum EPS (14.57 g L-1) with 1.26-fold increase when compared to the basal conditions. Two purified EPSs namely NRF1 and NRF2 with average molecular weights (Mw¯) of 15.98 and 9.70 kDa, respectively, were obtained and subjected for subsequent analyses. FTIR and UV-Vis reflected their purity and high carbohydrate contents while EDX emphasized their neutral type. NMR identified the EPSs as levan-type fructan composed of ß-(2-6)-glycosidic linkage as a main backbone, and HPLC explained that the EPSs composed of fructose. Circular dichroism (CD) suggested that NRF1 and NRF2 had identical structuration with a little variation from the EPS-NR. The EPS-NR showed antibacterial activity with the maximum inhibition against S. aureus ATCC 25923. Furthermore, all the EPSs revealed a proinflammatory action through dose-dependent increment of expression of proinflammatory cytokine mRNAs, IL-6, IL-1ß and TNFα.

Identification of a Small Molecule Compound Active against Antibiotic-Tolerant Staphylococcus aureus by Boosting ATP Synthesis.

Antibiotic tolerance poses a threat to current antimicrobial armamentarium. Bacteria at a tolerant state survive in the presence of antibiotic treatment and account for persistence, relapse and recalcitrance of infections. Antibiotic treatment failure may occur due to antibiotic tolerance. Persistent infections are difficult to treat and are often associated with poor prognosis, imposing an enormous burden on the healthcare system. Effective strategies targeting antibiotic-tolerant bacteria are therefore highly warranted. In this study, small molecule compound SA-558 was identified to be effective against Staphylococcus aureus that are tolerant to being killed by conventional antibiotics. SA-558 mediated electroneutral transport across the membrane and led to increased ATP and ROS generation, resulting in a reduction of the population of antibiotic-tolerant bacteria. In a murine chronic infection model, of which vancomycin treatment failed, we demonstrated that SA-558 alone and in combination with vancomycin caused significant reduction of MRSA abundance. Our results indicate that SA-558 monotherapy or combinatorial therapy with vancomycin is an option for managing persistent S. aureus bacteremia infection and corroborate that bacterial metabolism is an important target for counteracting antibiotic tolerance.

Peptidomic analysis of the host-defense peptides in skin secretions of the Amazon River frog Lithobates palmipes (Ranidae).

Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.