Diclofenac mitigates virulence of multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that has the ability to cause a wide range of diseases including superficial infection and severe invasive life threatening infections. The pathogenicity of S. aureus is mediated by a group of virulence factors that mediate the colonization and penetration. The antibiotic resistance of S. aureus has evolved due to the abuse of antibiotics rendering the cure of infection very difficult especially with the shortage in new antibiotic production. To combat this shortage, repurposing of FDA-approved drugs against the virulence factors is a new strategy. The analgesic drug Diclofenac was found to have anti-virulence activity against Pseudomonas aeruginosa and Proteus mirabilis. This study aimed to demonstrate the anti-virulence effect of diclofenac against clinical MRSA isolates phenotypically and genotypically using qRT-PCR. In this study, diclofenac showed significant reduction in biofilm formation when compared to controls, the inhibition ranged between 22.67% and 70%. Also, remarkable inhibition of hemolysin activity was found (5.4-66.34%). Additionally, diclofenac has inhibitory activity against the staphyloxanthin production (8-57.2%). The results were confirmed by qRT-PCR that showed significant down-regulation of tested virulence genes. The down-regulation ranged from 43 to 64.05% for SarA, 36.85-64.75% for AgrA, 50-63.2% for hla, 38.55-60.35% for FnbA, 46.75-61.05% for IcaA, 27.55-64% for SigB and 51.05-72.8% for CrtM. In conclusion, diclofenac can be used in combination with antibiotics as anti-virulence agent against MDR-MRSA which will enhance the ability of immune system to eradicate infection.

Callistemon citrinus bioactive metabolites as new inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation.

ETHNOPHARMACOLOGICAL RELEVANCE: The development of new inhibitors of bacterial virulence factors from natural origin has recently received significant attention. Callistemon citrinus Skeels is an important plant of great medicinal value. Its antimicrobial activity is well documented. Although several compounds were isolated from this plant, the actual bioactive compounds responsible for its antimicrobial activity are still unrevealed. AIM OF THE STUDY: To evaluate the effect of C. citrinus crude extract and isolated compounds on methicillin-resistant and sensitive Staphylococcus aureus. MATERIALS AND METHODS: The methylene chloride-methanol extract (MME) of C. citrinus leaves was prepared by Soxhlet apparatus. Biologically guided fractionation of MME was accomplished using several normal and reversed phase silica gel columns. The potency of MME and its isolated compounds against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) was evaluated. In addition, the mechanism of resistance was studied using three virulence factors; antibiofilm activity, inhibition of staphyloxanthin biosynthesis and effect on acid tolerance. Ultrastructural changes in MRSA and MSSA were observed by TEM to understand mode of action of these compounds. RESULTS: Pulverulentone A (C1), 8- desmethyl eucalyptin (C2) and eucalyptin (C3) were isolated from the most bioactive fraction of MME. Confocal scanning laser microscopy images revealed that C. citrinus isolated compounds destroyed the intact architecture of biofilm, thickness and reduced its biomass. Pulverulentone A (C1) showed the most potent anti-biofilm activity up to 71% and 62.3% against MRSA and MSSA, respectively. It also exhibited the highest inhibition of staphyloxanthin biosynthesis of MRSA and MSSA by 55.6% and 54.5%, respectively. The bacterial cell membrane was compromised, losing its integrity and releasing important cellular constituents when exposed to C1-C3 CONCLUSIONS: C. citrinus phenolics and acylphloroglucinols may serve as potential source of plant-based antibacterials and thus could be implicated to control MRSA biofilm formation.

Chitooligosaccharides as Antibacterial, Antibiofilm, Antihemolytic and Anti-Virulence Agent against Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus nosocomial infections with a high mortality rate in human and animals have been reported to associate with bacterial biofilm formation, along with the secretion of numerous virulence factors. Therefore, the inhibition of biofilm formation and attenuation of virulence determinants are considered as a promising solution to combat the spread of S. aureus infections. Modern trends in antibiofilm therapies have opted for the active agents that are biocompatible, biodegradable, non-toxic and cost-effective. Owning the aforementioned properties, chitosan, a natural N-acetylated carbohydrate biopolymer derived from chitin, has been favorably employed. Recently, the chitosan structure has been chemically modified into Chitooligosaccharides (COS) to overcome its limited solubility in water, thus widening chitosan applications in modern antibiofilm research. In the present study, we have investigated the antibacterial, antibiofilm and anti-virulence activities against S. aureus of COS of different molecular weights dissolved in neutral water. METHODS: The study of bactericidal activity was performed using the micro-dilution method while the biofilm inhibition assay was performed using crystal-violet staining method and confirmed by scanning electron microscopic analysis. The inhibition of amyloid protein production was confirmed by Congo Red staining. RESULTS: Results showed that low molecular weight COS exhibited bactericidal activity and reduced the bacterial amylogenesis, hemolytic activity as well as H2O2 resistance properties, while slightly inhibiting biofilm formation. The present study provides a new insight for further applications of the water-soluble COS as a safe and cost-effective drug for the treatment of S. aureus biofilm-associated infections. CONCLUSION: Reducing the molecular weight of chitosan in the form of COS has become an effective strategy to maintain chitosan biological activity while improving its water solubility. The low molecular weight COS investigated in this study have effectively performed antibacterial, antibiofilm and antivirulence properties against S. aureus.

Astaxanthin inhibits homocysteine­induced endothelial cell dysfunction via the regulation of the reactive oxygen species­dependent VEGF­VEGFR2­FAK signaling pathway.

Increased plasma levels of homocysteine (Hcy) can cause severe damage to vascular endothelial cells. Hcy­induced endothelial cell dysfunction contributes to the occurrence and development of human cerebrovascular diseases (CVDs). Our previous studies have revealed that astaxanthin (ATX) exhibits novel cardioprotective activity against Hcy­induced cardiotoxicity in vitro and in vivo. However, the protective effect and mechanism of ATX against Hcy­induced endothelial cell dysfunction requires further investigation. In the present study, treatment of human umbilical vascular endothelial cells (HUVECs) with Hcy inhibited the migration, invasive and tube formation potentials of these cells in a dose­dependent manner. Hcy treatment further induced a time­dependent increase in the production of reactive oxygen species (ROS), and downregulated the expression of vascular endothelial growth factor (VEGF), phosphorylated (p)­Tyr­VEGF receptor 2 (VEGFR2) and p­Tyr397­focal adhesion kinase (FAK). On the contrary, ATX pre­treatment significantly inhibited Hcy­induced cytotoxicity and increased HUVEC migration, invasion and tube formation following Hcy treatment. The mechanism of action may involve the effective inhibition of Hcy­induced ROS generation and the recovery of FAK phosphorylation. Collectively, our findings suggested that ATX could inhibit Hcy­induced endothelial dysfunction by suppressing Hcy­induced activation of the VEGF­VEGFR2­FAK signaling axis, which indicates the novel therapeutic potential of ATX in treating Hcy­mediated CVD.

In vitro and in vivo biofilm inhibitory efficacy of geraniol-cefotaxime combination against Staphylococcus spp.

In humans, the occurrence of bacterial communities in the form of biofilm is considered as a major intrinsic factor accountable for a variety of stubborn infections. Staphylococcus aureus and S. epidermidis have gained considerable attention in clinical settings owing to the formation of intractable and long-lasting biofilms in medical device. The current study has been designed to explain the biofilm inhibitory efficacy of geraniol and cefotaxime combination (GCC) against S. epidermidis and methicillin-resistant S. aureus (MRSA). Biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of GCC against S. epidermidis and MRSA. The minimal biofilm inhibitory concentration of GCC was found to be 100 µg/ml of geraniol and 2 µg/ml of cefotaxime. Further, microscopic analyses ascertained the devastating potential of GCC on the test pathogens' biofilm formation. Besides biofilm inhibition, GCC also suppressed the production of extracellular polymeric substance, slime and staphyloxanthin. More, GCC significantly increased the susceptibility of the test pathogens towards human blood. Further, the results of real time PCR analysis and in vivo assay using Caenorhabditis elegans unveiled the anti-biofilm potentials of GCC. Thus, the present study demonstrates the significant use of polytherapy treatment approaches to overcome the biofilm associated infections of Staphylococcus spp.

Novel Inhibitors of Staphyloxanthin Virulence Factor in Comparison with Linezolid and Vancomycin versus Methicillin-Resistant, Linezolid-Resistant, and Vancomycin-Intermediate Staphylococcus aureus Infections in Vivo.

Our previous work ( Wang et al. J. Med. Chem. 2016 , 59 , 4831 - 4848 ) revealed that effective benzocycloalkane-derived staphyloxanthin inhibitors against methicillin-resistant Staphylococcus aureus (S. aureus) infections were accompanied by poor water solubility and high hERG inhibition and dosages (preadministration). In this study, 92 chroman and coumaran derivatives as novel inhibitors have been addressed for overcoming deficiencies above. Derivatives 69 and 105 displayed excellent pigment inhibitory activities and low hERG inhibition, along with improvement of solubility by salt type selection. The broad and significantly potent antibacterial spectra of 69 and 105 were displayed first with normal administration in the livers and hearts in mice against pigmented S. aureus Newman, Mu50 (vancomycin-intermediate S. aureus), and NRS271 (linezolid-resistant S. aureus), compared with linezolid and vancomycin. In summary, both 69 and 105 have the potential to be developed as good antibacterial candidates targeting virulence factors.

Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence.

The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.

Effect of Illumination Intensity and Inhibition of Carotenoid Biosynthesis on Assembly of Peripheral Light-Gathering Complexes in Purple Sulfur Bacteria C Allochromatium vinosum ATCC 17899.

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain- increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodovibrin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphe- nylamine (DPA) concentrations not exceeding 53 .iM, while at illumination intensity decreased to 500 Ix they could grow at 71 jiM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoine synthetase (resulting in a decrease in the total carotenoid content) and of phyto- ine desturase, which results in formation of neurosporene hydroxy derivatives and ;-carotene. In the cells grown at 500 lx, small amounts of spheroidene and.OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was -15% of the control (without inhibition) for the B800-850 and -20%of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with -carotenoids due to- disintegration of some carotenoid-free complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be Assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.

Anti-infectious agents against MRSA.

Clinically useful antibiotics, ß-lactams and vancomycin, are known to inhibit bacterial cell wall peptidoglycan synthesis. Methicillin-resistant Staphylococcus aureus (MRSA) has a unique cell wall structure consisting of peptidoglycan and wall teichoic acid. In recent years, new anti-infectious agents (spirohexaline, tripropeptin C, DMPI, CDFI, cyslabdan, 1835F03, and BPH-652) targeting MRSA cell wall biosynthesis have been discovered using unique screening methods. These agents were found to inhibit important enzymes involved in cell wall biosynthesis such as undecaprenyl pyrophosphate (UPP) synthase, FemA, flippase, or UPP phosphatase. In this review, the discovery, the mechanism of action, and the future of these anti-infectious agents are described.

Beyond conventional antibiotics for the future treatment of methicillin-resistant Staphylococcus aureus infections: two novel alternatives.

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S. aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S. aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S aureus. These are generally small molecules that inhibit virulence targets in S. aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.

Astaxanthin upregulates heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells.

Astaxanthin (ATX), the most abundant flavonoids in propolis, has been proven to exert neuroprotective property against glutamate-induced neurotoxicity and ischemia-reperfusion-induced apoptosis. Previous study have revealed that ATX can rescue PC12 cells from Aß(25-35)-induced apoptotic death. However, the mechanisms by which ATX mediates its therapeutic effects in vitro are unclear. In the present study, we explored the underlying mechanisms involved in the protective effects of ATX on the Aß(25-35)-induced cytotoxicity in SH-SY5Y cells. Pre-treatment with ATX for 4h significantly reduced the Aß(25-35)-induced viability loss, apoptotic rate and attenuated Aß-mediated ROS production. In addition, ATX inhibited Aß(25-35)-induced lowered membrane potential, decreased Bcl-2/Bax ratio. We also demonstrated that ATX could prevent the activation of p38MAPK kinase pathways induced by Aß. Moreover, we for the first time have revealed the ATX increased antioxidant enzyme heme oxygenase-1 (HO-1) expression in concentration-dependent and time-dependent manners, which were correlated with its protective effect against Aß(25-35)-induced injury. Because the inhibitor of HO-1 activity, ZnPP reversed the protective effect of ATX against Aß(25-35)-induced cell death. We also demonstrated that the specific ERK inhibitor, PD98059, concentration-dependently blocked on ATX-induced HO-1 expression, and meanwhile PD98059 reversed the protective effect of ATX against Aß25-35-induced cell death. Taken together, these findings suggest that astaxanthin can induce HO-1 expression through activation of ERK signal pathways, thereby protecting the SH-SY5Y cells from Aß(25-35)-induced oxidative cell death.

Inhibition of staphyloxanthin virulence factor biosynthesis in Staphylococcus aureus: in vitro, in vivo, and crystallographic results.

The gold color of Staphylococcus aureus is derived from the carotenoid staphyloxanthin, a virulence factor for the organism. Here, we report the synthesis and activity of a broad variety of staphyloxanthin biosynthesis inhibitors that inhibit the first committed step in its biosynthesis, condensation of two farnesyl diphosphate (FPP) molecules to dehydrosqualene, catalyzed by the enzyme dehydrosqualene synthase (CrtM). The most active compounds are phosphonoacetamides that have low nanomolar K(i) values for CrtM inhibition and are active in whole bacterial cells and in mice, where they inhibit S. aureus disease progression. We also report the X-ray crystallographic structure of the most active compound, N-3-(3-phenoxyphenyl)propylphosphonoacetamide (IC(50) = 8 nM, in cells), bound to CrtM. The structure exhibits a complex network of hydrogen bonds between the polar headgroup and the protein, while the 3-phenoxyphenyl side chain is located in a hydrophobic pocket previously reported to bind farnesyl thiodiphosphate (FsPP), as well as biphenyl phosphonosulfonate inhibitors. Given the good enzymatic, whole cell, and in vivo pharmacologic activities, these results should help guide the further development of novel antivirulence factor-based therapies for S. aureus infections.