Immunotherapeutic approaches against Staphylococcus aureus

Staphylococcus aureus is a major cause of life-threatening infections such as bacteremia and endocarditis. Unfortunately, many strains of this bacterial species have become resistant to certain antibiotics, including methicillin and amoxicillin. These strains are known as methicillin-resistant S. aureus (MRSA). Therefore, the prophylactic and therapeutic potential of antistaphylococcal vaccines is currently being explored with priority. In animal models, (passive) immunization with (antibodies directed against) certain S. aureus surface components, staphylococcal toxins and capsular polysaccharides protects against S. aureus colonization or infection. However, immunization studies performed in humans show less promising results. So far, not a single antistaphylococcal vaccine successfully passed clinical trials. This article focuses on the results that were obtained with immunotherapeutic approaches directed against S. aureus in animal and human studies. In addition, it is discussed whether effective immunization approaches against S. aureus are feasible in humans.     

Dengue virus infections and anti-dengue virus activities of Andrographis paniculata

Dengue is a debilitating disease that poses a perpetual threat to human health and increases the global economic burden every year. Despite advances in medical sciences, dengue virus(DENV) infects approximately 200 million people every year. To date, no effective antiviral is valiable to treat DENV in individuals despite great efforts in accomplishing these goals. Numerous approaches have been used in the search for dengue antiviral like screening of combinatorial compounds against DENV enzymes and structurebased computational discovery. In recent years, investigators have turned their focus into medicinal plants, trying to identify compounds that can be used as dengue antiviral. Nature represents a great reservoir of potential substances that can be explored with the aim of discovering new drugs that can be either used directly as pharmaceuticals or can provide drug leads, which can be scrutinized further for the development of new anti-dengue natural product. Many previous investigations have dealt with numerous plant extracts or bioactive principles for their antiviral property as they normally considered being safer when compared to synthetic drugs. Andrographis paniculata belongs to family Acanthaceae and is generally known as ‘king of bitters'. Diverse bioactive compounds from this plant such as diterpenes, flavonoids, xanthones, noriridoides and other miscellaneous compounds have exhibited their potential as therapeutics for various chronic as well as infectious diseases. This review is based on literature review on scientific journals, books and electronic sources, which highlights the pathogenesis of DENV and describe an assortment of bioactive principles that have been possessing antiviral potential, which include dengue and discuss the therapeutic efficacy and mechanism of action of Andrographis paniculata. However, a detailed and more comprehensive clinical trial on mammalian tissues and organs is needed in future studies.     

Comparison of Pouzolzia indica methanolic extract and Virkon against cysts of Acanthamoeba spp

The present study was conducted to investigate the morphological and structural changes of Acanthamoeba cysts after being treated with various concentrations of Pouzolzia indica methanolic extract fraction 3 (methanol eluted) and Virkon solution. Changes in the Acanthamoeba cysts were detected by light microscopy, scanning electron microscopy and transmission electron microscopy. The results show Acanthamoeba cysts were killed by Pouzolzia indica methanolic extract fraction 3 at a concentration of 1:8 and by Virkon solution at a concentration of 0.25%, with a minimal cysticidal concentration (MCC) by 24 hours. Both agents caused similar structural damage to Acanthamoeba cysts in the same sequence. Step by step structural alterations occurred within the cyst. First, the cyst shrank, collapsed and had clumping of cytoplasmic stuctures inside the cyst walls. Second, the cysts began to bulge, swell, have a decrease in wrinkles in the cyst walls and spill the cytoplasmic contents into the environment. Finally, the cyst walls broke into small pieces. This study may be beneficial to compare with future studies of pharmaceutical agents against Acanthamoeba keratitis.     

Endotoxin-induced suppression of pulmonary antibacterial defenses against Staphylococcus aureus

In order to evaluate the effect of endotoxin on lung host defenses, Sprague-Dawley rats were intravenously injected with either placebo or 5 mg/kg of Escherichia coli lipopolysaccharide B. Two hours after treatment, animals were challenged with Staphylococcus aureus by either low dose aerosol inhalation or high dose intratracheal instillation of the bacteria into the lungs. Quantitative lung bacteriologic examination and bronchoalveolar lavage (BAL) for total and differential cell counts were performed immediately (zero hour) and at 4 h after bacterial challenge. Lung phagocytic defenses against aerosolized S. aureus challenges are provided solely by the alveolar macrophage (AM) in the absence of inflammation. In aerosol-challenged control rats, 20.6 +/- 2.0% of the initial deposited bacterial challenge remained viable in the lung at 4 h. Animals pretreated with endotoxin, however, showed a significant decrease in pulmonary bactericidal activity (31.3 +/- 3.4% bacteria remaining at 4 h), indicating a defect in alveolar macrophage (AM) function. Further assessment of the bactericidal oxidative metabolism of endotoxin-treated AM by luminol-enhanced chemiluminescence indicated an increased production of free radical oxygen species when compared with control nontreated cells in both the unstimulated (66 +/- 4 versus 38 +/- 7 x 10(3) cpm in control) and stimulated (250.5 +/- 17.1 versus 147.1 +/- 6.2 x 10(3) cpm in control) states. Total and differential cell counts in both control and endotoxin-treated aerosol-challenged rats were similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of bactericidal activity of five antibiotics against Staphylococcus aureus

Ten volunteers were given each of five antibiotics, sequentially, until steady state was reached. Peak and trough sera were then drawn, and bactericidal titers were determined to two different isolates of Staphylococcus aureus, both sensitive in vitro to all antibiotics tested. The antibiotics were cephalexin, trimethoprim/sulfamethoxazole (TMP/SMZ), clindamycin, dicloxacillin, and ciprofloxacin. Mean peak serum bactericidal titers (SBT) were significantly higher for cephalexin than for dicloxacillin, ciprofloxacin, and TMP/SMZ (P less than .05). The difference between cephalexin and clindamycin did not achieve statistical significance. Dicloxacillin, clindamycin, and ciprofloxacin were not statistically different from each other. Mean SBT for TMP/SMZ was less than 1:2, significantly less than that achieved by the other antibiotics. Only clindamycin achieved a trough SBT greater than 1:2. This was statistically significant compared with each of the other antibiotics.     

Therapies against virulence products of Staphylococcus aureus and Pseudomonas aeruginosa

Methicillin-resistant STAPHYLOCOCCUS AUREUS (MRSA) and PSEUDOMONAS AERUGINOSA are key pathogens in hospitals (particularly intensive care units), in long-term care facilities, and in outpatients with specific comorbidities and risk factors. Both MRSA and P. AERUGINOSA display resistance to a wide array of antibiotics. Further, both bacteria contain a variety of virulence products or systems that make it difficult to treat associated infections. Within the past several years, community-acquired MRSA containing virulence factors [particularly the Panton-Valentine leukocidin (PVL) gene] has emerged globally. Given the limited number of novel antibiotics to treat antibiotic-resistant organisms, there is growing interest in treating bacterial infections by targeting specific virulence products or systems. This article reviews potential therapeutic targets in the virulence systems of these two bacteria that are responsible for a large number of serious infections in critically ill patients.     

Protective role of standardized Feronia limonia stem bark methanolic extract against carbon tetrachloride induced hepatotoxicity

Objective. This study evaluates hepatoprotective potential of Feronia limonia stem bark (FSB) extracts and fractions using experimental models.Materials and methods. Activity levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and cell viability were evaluated in HepG2 cells treated with carbon tetrachloride (CCl4) in presence or absence of FL extracts or fractions. Also, plasma markers of hepatic damage, hepatic antioxidants, lipid peroxidation and histopathological alterations were assessed in rats treated with CCl4 alone or in combination with 200 or 400 mg/kg bodyweight (BW) of FSB-7 or 25 mg/kg BW of silymarin.Results. In vitro co-supplementation of FSB extracts or fractions recorded varying degree of hepatoprotective potentials. Also, pre-supplementation of FSB methanolic extract (FSB-7) followed by CCl4 treatment significantly prevented hepatic damage and depletion of cellular antioxidants. Also, CCl4+ FSB-7 group showed minimal distortion in the histoarchitecture of liver and results were comparable to that of CCl4+ silymarin treated rats.Conclusion. This inventory is the first scientific report on hepatoprotective potential of FSB methanolic extract.     

Mechanism of Staphylococcus aureus peptidoglycan O-acetyltransferase A as an O-acyltransferase

The O-acetylation of exopolysaccharides, including the essential bacterial cell wall polymer peptidoglycan, confers resistance to their lysis by exogenous hydrolases. Like the enzymes catalyzing the O-acetylation of exopolysaccharides in the Golgi of animals and fungi, peptidoglycan O-acetyltransferase A (OatA) is predicted to be an integral membrane protein comprised of a membrane-spanning acyltransferase-3 (AT-3) domain and an extracytoplasmic domain; for OatA, these domains are located in the N- and C-terminal regions of the enzyme, respectively. The recombinant C-terminal domain (OatA_C) has been characterized as an SGNH acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatA_N) or its homologs associated with other acyltransferases. We report herein the experimental determination of the topology of Staphylococcus aureus OatA_N, which differs markedly from that predicted in silico. We present the biochemical characterization of OatA_N as part of recombinant OatA and demonstrate that acetyl-CoA serves as the substrate for OatA_N. Using in situ and in vitro assays, we characterized 35 engineered OatA variants which identified a catalytic triad of Tyr-His-Glu residues. We trapped an acetyl group from acetyl-CoA on the catalytic Tyr residue that is located on an extracytoplasmic loop of OatA_N. Further enzymatic characterization revealed that O-acetyl-Tyr represents the substrate for OatA_C. We propose a model for OatA action involving the translocation of acetyl groups from acetyl-CoA across the cytoplasmic membrane by OatA_N and their subsequent intramolecular transfer to OatA_C for the O-acetylation of peptidoglycan via the concerted action of catalytic Tyr and Ser residues.

The O-acetylation of cell wall and extracellular polysaccharides is a common theme in nature for their protection from lysis and degradation. Examples include the sialic acid residues of glycoproteins that comprise the glycocalyx of eukaryotic cells (1), xylan of the secondary cell walls of plants (2), and the peptidoglycan (PG), secondary cell wall polysaccharides and biofilm components (e.g., cellulose, alginate) of bacterial cell walls or extracellular matrices (3, 4). PG is an essential component of bacterial cell walls, contributing to both cell shape and protection. Although over 50 chemotypes of PG have been identified (5), the glycan structure is conserved among bacteria. It is composed of repeating units of β-1,4–linked N-acetylglucosaminyl (GlcNAc) and N-acetylmuramoyl residues (MurNAc), and adjacent glycan strands are cross-linked together through peptides that stem from MurNAc residues.PG O-acetylation occurs at the C-6 hydroxyl of MurNAc residues in both gram-positive and gram-negative bacteria (reviewed in ref. 6). Though rare, it can also occur at GlcNAc residues, such as in some lactobacilli (7). Initially described in 1958 (8), this modification is now known to occur in a large number of bacteria, particularly pathogens. For example, in a comprehensive study of the staphylococci, only pathogenic species (including Staphylococcus aureus) were found to produce O-acetylated PG (9). O-acetylation is a nonstoichiometric modification, and its extent can range from 20 to 70% depending on the organism, environmental conditions, and age of the culture (10–12). With Enterococcus faecalis, for example, PG O-acetylation levels increase by 10 to 40% as cultures enter the stationary phase and a further 10 to 16% as cells enter the viable but nonculturable state (13). It is not yet known with any bacterium if the O-acetyl moieties are dispersed throughout the PG sacculus or localized to particular regions (6).The main pathobiological role of PG O-acetylation is the inhibition of lysozyme, an enzyme of the innate immunity system that hydrolyzes the glycosidic linkage between MurNAc and GlcNAc residues causing bacterial cells to lyse. The C-6 hydroxyl of MurNAc is important for the coordination of PG into the substrate-binding cleft of lysozyme (14) and so its O-acetylation sterically hinders this interaction (15, 16). With Listeria monocytogenes, in addition to providing resistance to lysozyme, PG O-acetylation is important for its growth in macrophages, conferring resistance to bacteriocins and β-lactam antibiotics and limiting the innate immune response (17). The ability of PG O-acetylation to modulate the immune system is of particular interest, as during S. aureus infection, the O-acetylation of PG limits T helper cell priming required to develop an effective protective response to systemic infection (18). Hence, PG O-acetylation is considered important for virulence in this (18–20) and numerous other pathogens (21), including L. monocytogenes (17), Streptococcus pneumoniae (22), E. faecalis (23), Neisseria meningitidis (24), Neisseria gonorrhoeae (25, 26), and Helicobacter pylori (27).As with other cell wall and many glycocalyx components, the O-acetylation of PG is a postbiosynthetic modification. It occurs after the incorporation of GlcNAc-MurNAc-pentapeptide (Lipid II) precursors into the existing sacculus (28–31). Accordingly, acetyl groups must therefore be translocated across the cytoplasmic membrane from a cytoplasmic donor to the cell wall. Two distinct enzymatic systems have been identified in bacteria for this purpose, both of which are now thought to be the ancestors for homologous systems that evolved in eukaryotes (2). The system operating in gram-positive bacteria, which was first discovered in S. aureus (20), is analogous to those involved with the O-acetylation of exopolysaccharides in the Golgi of animals and fungi (2). In each of these cells, a single bimodal protein (named O-acetyltransferase A [OatA] in gram-positive bacteria) is postulated to catalyze both the translocation and transfer reactions. With OatA, its N-terminal domain (OatA_N) is predicted to be a member of Acyl_transf_3 (AT-3) Acyltransferases (pfam PF01757; InterPro IPR002656) family of proteins and also as an acyltransferase_3/putative acetyl-CoA transporter (Transporter Classification Database number TC 9.B.97). As such, OatA_N is thought to span the cytoplasmic membrane and function as an O-acyltransferase to shuttle acetyl groups from an unknown source for presentation to the C-terminal extracellular domain (OatA_C), which subsequently transfers them onto MurNAc residues (4).Our earlier structural and biochemical studies of OatA_C from both S. aureus (32) and S. pneumoniae (33, 34) demonstrated that this domain functions like an SGNH hydrolase in which the C-6 hydroxyl group of muramoyl residues serves as the acceptor for PG O-acetylation instead of water as for a hydrolase. Nothing, however, is known mechanistically nor structurally about the cognate OatA_N or other members of AT-3, and little insight has been gained from studies on any other system translocating acetyl groups across a membrane for their transfer to a glycan. The concept of the two-step coordinated reaction for the overall O-acetylation of extracytoplasmic glycans was first proposed by Higa et al. (35) in their study of a sialyl O-acetyltransferase embedded in the Golgi membrane of the rat liver. These early investigators used a chemical modification approach to identify the participation of an essential histidyl and lysyl residues for the first step translocation reaction. More recently, a site-directed mutagenesis of the gene encoding Salmonella Typhimurium O-antigen O-acetyltransferase A (OafA) confirmed the importance of an Arg-X-X-Arg motif (36) identified previously in this enzyme (37) and a Shigella flexneri homolog, Oac (38). The Arg and His residues in an Arg/Lys-X₁₀-His motif were also found to be important for function in OafA, and they are proposed to interact with acetyl-CoA, assuming that this metabolite serves as the acetyl donor for the enzyme (36). Pearson et al. (36) also observed that, while this latter motif is present in all AT-3 homologs, the Arg-X-X-Arg motif together with a Gly-Gly-Phe-(Val/Ile/Leu)-Gly-Val-Asp-(Ile/Val/Leu) motif are found uniquely in AT-3 domains fused to a cognate SGNH domain. In addition to OatA and the O-antigen and O-acetyltransferases noted above, members of this subfamily of enzymes are present in other important bacterial pathogens. Examples include OatB and Lot3 that O-acetylate GlcNAc in Lactobacillus PG and N. meningitidis lipooligosaccharides, respectively. In the intervening years between these studies, little other knowledge has been gained.Herein, we present a biochemical characterization of the AT-3 domain of OatA. We experimentally determined the topology of S. aureus OatA_N (SaOatA_N), which we find to differ significantly from that predicted in silico. Using two novel assays (one each in situ and in vitro) coupled with site-specific replacements of consensus amino acids, we identified catalytically essential Arg, His, and Tyr residues. Based on these data, combined with our identification of two acetyl intermediates, we propose a mechanism for the translocation of acetyl groups from cytoplasmic acetyl-CoA to an external loop of SaOatA_N that serves as the acetyl donor for the O-acetylation of PG by OatA_C.     

Studies on effects of Andrographis paniculata (Burm.f.) and Andrographis lineata nees (Family: Acanthaceae) extracts against two mosquitoes Culex quinquefasciatus (Say.) and Aedes aegypti (Linn.)

ObjectiveTo investigate the studies on effects of Andrographis paniculata (A. paniculata) (Burm.f.) and Andrographis lineata (A. lineata) nees (Family: Acanthaceae) extracts against two mosquitoes Culex quinquefasciatus (Cx. quinquefasciatus) (Say.) and Aedes aegypti (Ae. aegypti) (Linn.).MethodsThe aqueous and petroleum ether extracts of two plant species, A. paniculata and A. lineate were examined against the larvae of A. aegypti (L.) and Cx. quinquefasciatus with gradually increasing concentration ie. from 50 to 200 ppm of solvent extracts and to test their activity in combination with each other.ResultsIn a 24 h bioassay experiment with plant extracts, highest mortalities were recorded at 200 ppm of concentrations for leaves of A. lineta and A. paniculata individually. For combination effect, only 150 ppm of the mixture of solvent extracts of petroleum ether: aqueous (1:1) extracts showed 100% mortality after 24 h of exposure.ConclusionsThe results show that, insecticides of plant combination is ecofriend and has better larvicidal activity compared to individual extracts.     

Effect of gentiana violet against methicillin-resistant Staphylococcus aureus (MRSA)]

The bactericidal effect of gentiana violet against MRSA isolated from clinical specimens was studied both in vitro and in vivo. The results obtained are as follows: 1) Minimum bactericidal concentration (MBC) of gentiana violet to MRSA was between 0.0025% and 0.08% and the MBC was not influenced even if 25% human whole serum exists in the medium. 2) The number of cells were 2.1 x 10(7) CFU/ml in medium which decreased to 5.4 x 10(4) CFU/ml within 5 min by existing 0.1% gentiana violet in the medium as the final concentration, and also decreased under 10(3) CFU/ml 15 min later. 3) Minimum inhibitory concentration (MIC) of gentiana violet to MRSA was between 0.00015% and 0.00063%, and the inhibitory activity was not influenced even if gentiana violet was incubated with the bacteria in the medium for 72 hr at 37 degrees C. 4) By using an ointment containing 0.1% gentiana violet to 12 cases of patients with the MRSA infected skin lesions, MRSA was eliminated completely from the infected areas of the skin within 4 weeks. 5) The side effects of gentiana violet were not observed in all cases during the use of the ointment containing gentiana violet. It is suggested that gentiana violet may be one of the useful drugs for the treatment of the skin lesions infected with MRSA.     

Bactericidal kinetics of 3 lactoferricins against Staphylococcus aureus and Escherichia coli

Bovine lactoferricin is an antimicrobial, cationic peptide generated upon gastric pepsin cleavage of bovine lactoferrin. We investigated the bactericidal effects of native lactoferricin [Lfcin B(17-41)], a shortened derivative [Lfcin B(17-31)] and the all-D-amino acid counterpart of Lfcin B(17-31) against Escherichia coli and Staphylococcus aureus. The results revealed different activities for the peptides against Gram-positive and -negative bacteria. D-Lfcin B(17-31) was the most efficient peptide against E. coli. The same peptide showed improved activity against S. aureus, D-Lfcin B(17-31) showed a significant better efficacy when compared to the L-form, but not when compared to Lfcin B(17-41). There was no correlation between the bactericidal concentrations and the time needed to achieve maximum effect. This indicates the importance of structural differences between the peptides and/or bacteria and implies that the simple thesis of I antibacterial target is not valid for lactoferricin.     

Host defenses against Staphylococcus aureus infection require recognition of bacterial lipoproteins

Toll-like receptors and other immune-signaling pathways play important roles as sensors of bacterial pattern molecules, such as peptidoglycan, lipoprotein, or teichoic acid, triggering innate host immune responses that prevent infection. Immune recognition of multiple bacterial products has been viewed as a safeguard against stealth infections; however, this hypothesis has never been tested for Staphylococcus aureus, a frequent human pathogen. By generating mutations that block the diacylglycerol modification of lipoprotein precursors, we show here that S. aureus variants lacking lipoproteins escape immune recognition and cause lethal infections with disseminated abscess formation, failing to elicit an adequate host response. Thus, lipoproteins appear to play distinct, nonredundant roles in pathogen recognition and host innate defense mechanisms against S. aureus infections.     

Traditional medicines and their in-vitro proof against Staphylococcus aureus in Pakistan

Objective:To gather the fragmented literature on ethnobotany,phytochemistry and in-vitro activities of medicinal plants of Pakistan being used against common infections caused by Staphylococcus aureus(S.aureus).Methods: A large number of published and unpublished research studies related to the ethnomedicinal,phytochemical and anti-S.aureus activity of medicinal flora of Pakistan published from 1990-2018 were reviewed using online bibliographic databases such as PubMed,Web of Science,Science Direct,ResearchGate and libraries.Results: S.aureus can cause many human ailments including endocarditis,staphylococcal scalded skin syndrome,septic arthritis,respiratory problems with an estimated infection rate of 25%-35% across the globe.This review comprised of 86 medicinal plants.Data showed that people mostly used leaves(50%) for the preparation of traditional medicines.Correlation analysis on the reviewed data revealed that methanolic extract concentrations of medicinal plants was highly significantly positive correlated(r=0.8;P0.01) with the S.aureus zone of inhibitions.S.aureus reportedly showed complete resistant to the commonly used antibiotic erythromycin.Isolated compounds like altheahexacosanyl lactone,cinnamaldehyde,niloticane,gobicusin A,asparacosin A,muzanzagenin,isoagatharesinol,friedelin,inophynone and eugenol were active against S.aureus.This study provided in-vitro proof for the flora of Pakistan used against different infections caused by S.aureus.Conclusions: Antibacterial agents from natural sources could be more effective against bacterial pathogens and will be helpful in minimizing the adverse effects of synthetic drugs,and hence provides a base for the pharmaceutical industries.     

Antimicrobial activity and synergism of Sami-Hyanglyun-Hwan with ciprofloxacin against methicillin-resistant Staphylococcus aureus

Objective: To investigate the antibacterial activity of SHHextracted with either water or ethanol against methicillin-resistant Staphylococcus aureus(MRSA) and combinatory antimicrobial effect with ciprofloxacin(CIP) by time kill assay and checkerboard dilution test. Methods: The antibacterial activity determined by broth dilution method indicated that the antibacterial activity of Sami-Hyanglyun-Hwan(SHH) water extract(SHHW) and SHH ethanol extract(SHHE) ranged from 250 to 2000 μg/m L and 125 to 1000 μg/m L against MRSA, respectively. Results: In the checkerboard method, the combinations of SHHE with CIP had a partial synergistic or synergistic effect against MRSA. The time-kill curves showed that a combined SHHE and CIP treatment reduced the bacterial counts dramatically after 24 h. Conclusions: The present study demonstrates the therapeutic ability of SHHE against MRSA infections.