Generation Of Antimicrobials Research Articles

Disruption of N-acyl-homoserine lactone-specific signalling and virulence in clinical pathogens by marine sponge bacteria.

SummaryIn recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche‐specific substrates, in wide‐ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell–cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth‐limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture‐based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and Paracoccus JM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.

Na+-NQR (Na+-translocating NADH:ubiquinone oxidoreductase) as a novel target for antibiotics.

The recent breakthrough in structural studies on Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae creates a perspective for the systematic design of inhibitors for this unique enzyme, which is the major Na+ pump in aerobic pathogens. Widespread distribution of Na+-NQR among pathogenic species, its key role in energy metabolism, its relation to virulence in different species as well as its absence in eukaryotic cells makes this enzyme especially attractive as a target for prospective antibiotics. In this review, the major biochemical, physiological and, especially, the pharmacological aspects of Na+-NQR are discussed to assess its 'target potential' for drug development. A comparison to other primary bacterial Na+ pumps supports the contention that NQR is a first rate prospective target for a new generation of antimicrobials. A new, narrowly targeted furanone inhibitor of NQR designed in our group is presented as a molecular platform for the development of anti-NQR remedies.

Bactericidal potential of silver nanoparticles synthesized using cell-free extract of Comamonas acidovorans: in vitro and in silico approaches

The need to overcome human threats from pathogenic microbes, development of nanomaterials have been provoked for a new generation of antimicrobials. In the present study, biosynthesis of silver nanoparticles (AgNPs) was acquired using Comamonas acidovorans extract within 72 h under static condition. Electron microscopy studies revealed that the size of AgNPs was ranging from 6–53 nm and had spherical, oval and irregular shapes with smooth surfaces. Prepared AgNPs interacted with proteins, carbohydrates and other aromatic molecules. Biosynthesized AgNPs were bactericidal, which significantly inhibited pathogenic microbes, i.e., Streptococcus pyogenes, Staphylococcus aureus and Escherichia coli. Higher concentrations of AgNPs (20 μg ml−1) inhibited 92–98% growth of all tested bacteria within 24 h. AgNPs–protein network studies carried out to recognize the protein interactions with AgNPs and to understand probable bactericidal mechanisms. AgNPs may penetrate into cell through membrane proteins and damage them by modifying amino acids. Due to AgNPs–protein interactions, dysfunctions in enzymes obstruct certain metabolic processes, which cause the bacteria to die eventually. In certain pathogenic microbes, cue and cus systems detoxify Ag+ ions, transport through transporter proteins and expel them to the extracellular space, which are mainly responsible for Ag resistance.

Green synthesis and characterization of silver nanoparticles using Alcea rosea flower extract as a new generation of antimicrobials

Green synthesis of silver nanoparticles (AgNPs) was developed by treating Ag+ with Alcea rosea flower extract. AgNO3 concentration, flower extract quantity, and reaction temperature were found to be significant factors in the bioreduction reaction. Synthesized AgNPs were almost spherical in shape with an average diameter of 7.2 nm. Fourier transform infrared spectroscopy (FTIR) analysis revealed that oxygen-bearing functional groups in the A. rosea flower extract are responsible for reduction of Ag+. The minimum inhibitory concentration (MIC) of AgNPs against a Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was determined to be 37.5 ?g/ml.

Computational resources and tools for antimicrobial peptides.

Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immune system, protect against pathogens including bacteria, fungi, viruses, and parasites. In general, AMPs are relatively small peptides (<10 kDa) with cationic nature and amphipathic structure and have modes of action different from traditional antibiotics. Up to now, there are more than 19 000 AMPs that have been reported, including those isolated from nature sources or by synthesis. They have been considered to be promising substitutes of conventional antibiotics in the quest to address the increasing occurrence of antibiotic resistance. However, most AMPs have modest direct antimicrobial activity, and their mechanisms of action, as well as their structure-activity relationships, are still poorly understood. Computational strategies are invaluable assets to provide insight into the activity of AMPs and thus exploit their potential as a new generation of antimicrobials. This article reviews the advances of AMP databases and computational tools for the prediction and design of new active AMPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Three Novel Lantibiotics, Ticins A1, A3, and A4, Have Extremely Stable Properties and Are Promising Food Biopreservatives.

Lantibiotics are antimicrobial peptides with potential applications as the next generation of antimicrobials in the food industry and/or the pharmaceutical industry. Nisin has successfully been used as a food preservative for over 40 years, but its major drawback is its limited stability under neutral and alkaline pH conditions. To identify alternatives with better biochemical properties, we screened more than 100 strains of the Bacillus cereus group. Three novel lantibiotics, ticins A1 (4,062.98 Da), A3 (4,048.96 Da), and A4 (4,063.02 Da), which were highly thermostable (121°C for 30 min) and extremely pH tolerant (pH 2.0 to 9.0), were identified in Bacillus thuringiensis BMB3201. They all showed potent antimicrobial activities against all tested Gram-positive bacteria and greater activities than those of nisin A against Bacillus cereus and Listeria monocytogenes, two important foodborne pathogens. These three novel lantibiotics, with their extremely stable properties and potent antimicrobial activities, have the potential for use as biopreservatives.

Unconventional screening approaches for antibiotic discovery.

The dramatic rise in microbial drug resistance in recent years has led to ongoing searches for novel drugs to add to the armory against infectious disease. Nevertheless, a paucity of new antibacterial drugs in discovery and development pipelines using traditional approaches has prompted a variety of unconventional and disruptive strategies for antibacterial drug discovery. Herein, we review recent nontraditional approaches that have been piloted for early drug discovery efforts. These unique methodologies open new avenues for finding the next generation of antimicrobials.

English

Nanotechnology is rapidly growing with nanoparticles produced and utilized in a wide range of pharmaceutical and commercial products throughout the world. In this study, fig (Ficus carica) leaf extracts were used for ecofriendly extracellular synthesis of stable silver nanoparticles (AgNPs) by treating an aqueous silver nitrate (1 mM) solution and using the plant F. carica leaf extracts as reducing agents. The bioreduced silver nanoparticles were characterized by ultra violet visible (UV-Vis) spectrophotometer, Fourier transform infra-red (FTIR) spectroscopy and transmission electron microscopy (TEM). The average particle size ranged from 5 to 40 nm. The particle size could be controlled by changing the reaction temperature, leaf broth concentration and AgNO3 concentration. Further, these biologically synthesized nanoparticles concentration of 50 &micro;l were found to be highly effective and exhibited maximum microbial activity with mean zone of inhibition 20.33&plusmn;1.00 mm and 18.00&plusmn;1.00 against pseudomonas aeruginosa and Aspergillus fumigatus isolated from human corneal ulcer patients. This environmentally friendly green synthesis is an eco-friendly approach to conventional chemical synthesis and can potentially be used in various areas such as food, cosmetics, and medical applications and hope the recent technology can provide next generation of anti-microbials. &nbsp; Key words: Ficus carica, silver nanoparticles, characterization, antimicrobial activity.

Reversion of antibiotic resistance by inhibiting mecA in clinical methicillin-resistant Staphylococci by antisense phosphorothioate oligonucleotide.

Methicillin-resistant Staphylococci (MRS), methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) have become a challenging problem in nosocomial infections and are connected with high morbidity and mortality rates. This is due to the increasing incidence of resistance to virtually all β-lactams and a wide variety of antimicrobials. The spread of MRS severely limits therapeutic options and generates the need for novel antibiotics that are able to combat MRS infections. One method of inhibiting bacterial growth is by blocking the expression of conserved bacterial genes and provides potential new avenues for generating a new generation of antimicrobials. The mecA gene is highly conserved among Staphylococcal species, and this makes it an ideal target for antisense inhibition. We had identified a target sequence (854-871 nt) within the mecA mRNA coding region that is particularly sensitive to antisense inhibition. The anti-mecA PS-ODN04 oligonucleotide was encapsulated into an anionic liposome. MRSA01 and MRSE01 clinical strains treated with this antisense sequence became susceptible to existing β-lactam antibiotics, and their growth was inhibited by oxacillin in vitro and in vivo. PS-ODN04 reduced the bacterial titers in the blood of mice infected with MRSA01 and MRSE01 and significantly improved their survival rate. Our data offer a possible new strategy for treating MRS infections.

Aedesin: structure and antimicrobial activity against multidrug resistant bacterial strains.

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.

Evaluation of plant-mediated Silver nanoparticles synthesis and its application in postharvest Physiology of cut Flowers

Today the use of silver nanoparticles is becoming increasingly widespread due to their wide applications as antimicrobial agent. Green synthesis of silver nanoparticles (SNPs) using the petal extract of saffron (Crocus sativus) as a reducing agent from 5mM AgNO3 has been investigated in this work. Diverse petal extracts quantities and reaction times were used for the synthesis of SNPs. The resulting SNPs were characterized by means of UV-Vis, XRD and FTIR techniques. SNPs were synthesized rapidly within 30min of incubation period and synthesized SNPs showed an absorption peak at 380-400nm in the UV-Vis spectrum. XRD spectrum confirmed the formation of metallic silver, too. Green synthesized SNPs were used as antimicrobial agent against three bacterial genera of Bacillus, Pseudomonas and Acinetobacter which contaminate preservative solution of cut-flowers, too. According to the results biosynthesized SNPs using saffron petals successfully controlled these bacteria and have made them promising candidates as new generation of antimicrobials. This route is rapid, simple without any hazardous chemicals and economical to synthesized SNPs.

Glycopeptide antibiotic biosynthesis

Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

Green Synthesis of Silver Nanoparticles with High Fungicidal Activity from Olive Seed Extract

Silver nanoparticles in the form of silver based chemicals trace back their origin to time immemorial since the dilute forms of silver nitrate were used in place of antibiotics before they dominated the field of medicine. But, it has now become necessary to explore the anti-microbial properties of silver based chemicals again due to the microbes gaining resistance against the wide range of present day antibiotics. The advancements in the field of medicine and technology started to coalesce to combat the adaptability of microbes as they successfully become tolerant to antibiotics and it manifested in the form a current technology, Nanomedicine. Nanomedicine deals about the medicines at a nano scale to rarefy the intensity of medicine to unaffected tissues and reduce the volume of medicine used. In the present context, our attempt is to develop potential anti-microbial particles in the form of silver nanoparticles by using the biological phenomena which we call Green synthesis an eco-friendly approach to conventional chemical synthesis. The enzymatic machinery of the olive seeds has been exploited to produce silver nanoparticles and test their efficacy as antifungal agents before we characterized their physical properties using UV-Vis, TEM, and FTIR analysis. The efficacy of these particles as antagonists on fungal pathogen Aspergillus niger a causative agent of Aspergillosis in human beings and is promising and they have a lot of scope for the purpose and hope the technology leads the next generation of anti-microbials.

Ab Initio Design of Potent Anti-MRSA Peptides Based on Database Filtering Technology

To meet the challenge of antibiotic resistance worldwide, a new generation of antimicrobials must be developed. This communication demonstrates ab initio design of potent peptides against methicillin-resistant Staphylococcus aureus (MRSA). Our idea is that the peptide is very likely to be active when the most probable parameters are utilized in each step of the design. We derived the most probable parameters (e.g., amino acid composition, peptide hydrophobic content, and net charge) from the antimicrobial peptide database by developing a database filtering technology (DFT). Different from classic cationic antimicrobial peptides usually with high cationicity, DFTamP1, the first anti-MRSA peptide designed using this technology, is a short peptide with high hydrophobicity but low cationicity. Such a molecular design made the peptide highly potent. Indeed, the peptide caused bacterial surface damage and killed community-associated MRSA USA300 in 60 min. Structural determination of DFTamP1 by NMR spectroscopy revealed a broad hydrophobic surface, providing a basis for its potency against MRSA known to deploy positively charged moieties on the surface as a mechanism for resistance. Our ab initio design combined with database screening led to yet another peptide with enhanced potency. Because of the simple composition, short length, stability to proteases, and membrane targeting, the designed peptides are attractive leads for developing novel anti-MRSA therapeutics. Our database-derived design concept can be applied to the design of peptide mimicries to combat MRSA as well.

Targeting protein-protein interactions using methods of cheminformatics

We have recently mapped the protein interaction networks of methicillin-resistant Staphylococcus aureus that revealed its scale-free organization with characteristic presence of highly-connected hub proteins that are critical for bacterial survival [1]. Here we report the discovery of highly selective nanomolar inhibitors for one such hub target - staphylococcal pyruvate kinase. The lead compound has been identified through synergetic combination of methods of high-throughput screening and cheminformatics; its further synthetic modifications resulted in much improved antimicrobial properties. Further lead optimization yielded drug candidates with picomolar activity against methicillin-resistant Staphylococcus aureus. Considering a notable lack of recent reports on novel antibacterial targets and cognate antibacterial compounds, this study provides a valuable perspective on the development of a new generation of antimicrobials. Equally noteworthy, the results of the current work highlight the importance of cheminformatics-driven exploration of chemical space around initial high throughput screening hits.

Cheminformatics-Driven Discovery of Selective, Nanomolar Inhibitors for Staphylococcal Pyruvate Kinase

We have recently mapped the protein interaction network of methicillin-resistant Staphylococcus aureus (MRSA), which revealed its scale-free organization with characteristic presence of highly connected hub proteins that are critical for bacterial survival. Here we report the discovery of inhibitors that are highly potent against one such hub target, staphylococcal pyruvate kinase (PK). Importantly, the developed compounds demonstrate complete selectivity for the bacterial enzyme compared to all human orthologues. The lead 91nM inhibitor IS-130 has been identified through ligand-based cheminformatic exploration of a chemical space around micromolar hits initially generated by experimental screening. The following crystallographic study resulted in identification of a tetrameric MRSA PK structure where IS-130 is bound to the interface between the protein's subunits. This newly described binding pocket is not present in otherwise highly similar human orthologues and can be effectively utilized for selective inhibition of bacterial PK. The following synthetic modifications of IS-130, guided by structure-based molecular modeling, resulted in the development of MRSA PK inhibitors with much improved antimicrobial properties. Considering a notable lack of recent reports on novel antibacterial targets and cognate antibacterial compounds, this study provides a valuable perspective on the development of a new generation of antimicrobials. Equally noteworthy, the results of the current work highlight the importance of rigorous cheminformatics-based exploration of the results of high-throughput experiments.

Biological nanofactories facilitate spatially selective capture and manipulation of quorum sensing bacteria in a bioMEMS device

The emergence of bacteria that evade antibiotics has accelerated research on alternative approaches that do not target cell viability. One such approach targets cell-cell communication networks mediated by small molecule signaling. In this report, we assemble biological nanofactories within a bioMEMS device to capture and manipulate the behavior of quorum sensing (QS) bacteria as a step toward modifying small molecule signaling. Biological nanofactories are bio-inspired nanoscale constructs which can include modules with different functionalities, such as cell targeting, molecular sensing, product synthesis, and ultimately self-destruction. The biological nanofactories reported here consist of targeting, sensing, synthesis and, importantly, assembly modules. A bacteria-specific antibody constitutes the targeting module while a genetically engineered fusion protein contains the sensing, synthesis and assembly modules. The nanofactories are assembled on chitosan electrodeposited within a microchannel of the bioMEMS device; they capture QS bacteria in a spatially selective manner and locally synthesize and deliver the "universal" small signaling molecule autoinducer-2 (AI-2) at the captured cell surface. The nanofactory based AI-2 delivery is demonstrated to alter the progression of the native AI-2 based QS response of the captured bacteria. Prospects are envisioned for utilizing our technique as a test-bed for understanding the AI-2 based QS response of bacteria as a means for developing the next generation of antimicrobials.

High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Bacterial type IV secretion systems in human disease

Type IV secretion (T4S) systems are versatile machines involved in many processes relevant to bacterial virulence, such as horizontal DNA transfer and effector translocation into human cells. A recent workshop organized by the International University of Andalousia in Baeza, Spain, covered most aspects of bacterial T4S relevant to human disease, ranging from the structural and mechanistic analysis of the T4S systems to the physiological roles of the translocated effector proteins in subverting cellular functions in infected humans. This review reports the highlights from this workshop, which include the first visualization of a T4S system core complex spanning both membranes of Gram-negative bacteria, the identification of the first host receptors for T4S systems, the identification and characterization of novel T4S effector proteins, the analysis of the molecular function of effector proteins in subverting human cellular functions and an analysis of the role of T4S systems in the evolution of pathogenic bacteria. Our increasing knowledge of the biology of T4S systems improves our ability to exploit them as biotechnological tools or to use them as novel targets for a new generation of antimicrobials.

Current and novel antibiotics against resistant Gram-positive bacteria

The challenge posed by resistance among Gram-positive bacteria, epitomized by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and vancomycin-intermediate and -resistant S. aureus (VISA and VRSA) is being met by a new generation of antimicrobials. This review focuses on the new β-lactams with activity against MRSA (ceftobiprole and ceftaroline) and on the new glycopeptides (oritavancin, dalbavancin, and telavancin). It will also consider the role of vancomycin in an era of existing alternatives such as linezolid, daptomycin and tigecycline. Finally, compounds in early development are described, such as iclaprim, friulimicin, and retapamulin, among others.