Broad-spectrum Antibiotic Activity Research Articles

Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae

Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis (phz) genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato (Pst) DC3000 and Xanthomonas oryzae pv. oryzae (Xoo) PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-1-carboxylic acid (PCA) of Pst DC3000 accumulated to 13.4 μg L−1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL−1 PCA, while Xoo PXO99A was sensitive to 50 μg mL−1 PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.

Membrane-Targeting DCAP Analogues with Broad-Spectrum Antibiotic Activity against Pathogenic Bacteria.

We performed a structure-activity relationship study of 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), which is an antibacterial agent that disrupts the membrane potential and permeability of bacteria. The stereochemistry of DCAP had no effect on the biological activity of DCAP. The aromaticity and electronegativity of the chlorine-substituted carbazole was required for activity, suggesting that its planar and dipolar characteristics orient DCAP in membranes. Increasing the hydrophobicity of the tail region of DCAP enhanced its antibiotic activity. Two DCAP analogues displayed promising antibacterial activity against the BSL-3 pathogens Bacillus anthracis and Francisella tularensis. Codosing DCAP analogues with ampicillin or kanamycin increased their potency. These studies demonstrate that DCAP and its analogues may be a promising scaffold for developing chemotherapeutic agents that bind to bacterial membranes and kill strains of slow-growing or dormant bacteria that cause persistent infections.

Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP.

BackgroundLipopeptides (LP) are structurally diverse compounds with potent surfactant and broad-spectrum antibiotic activities. In Pseudomonas and other bacterial genera, LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS). To date, relatively little is known about the regulatory genetic network of LP biosynthesis.ResultsThis study provides evidence that the chaperone ClpA, together with the serine protease ClpP, regulates the biosynthesis of the LP massetolide in Pseudomonas fluorescens SS101. Whole-genome transcriptome analyses of clpA and clpP mutants showed their involvement in the transcription of the NRPS genes massABC and the transcriptional regulator massAR. In addition, transcription of genes associated with cell wall and membrane biogenesis, energy production and conversion, amino acid transport and metabolism, and pilus assembly were altered by mutations in clpA and clpP. Proteome analysis allowed the identification of additional cellular changes associated to clpA and clpP mutations. The expression of proteins of the citrate cycle and the heat shock proteins DnaK and DnaJ were particularly affected. Combined with previous findings, these results suggest that the ClpAP complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.ConclusionsCombining transcriptome and proteome analyses provided new insights into the regulation of LP biosynthesis in P. fluorescens and led to the identification of specific missing links in the regulatory pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0367-y) contains supplementary material, which is available to authorized users.

Concise Synthesis of Spergualin-Inspired Molecules With Broad-Spectrum Antibiotic Activity.

There is a growing need to identify new, broad-spectrum antibiotics. The natural product spergualin was previously shown to have promising anti-bacterial activity and a privileged structure, but its challenging synthesis had limited further exploration. For example, syntheses of spergualin and its analogs have been reported in approximately 10 linear steps, with overall yields between 0.3 and 18%. Using the Ugi multi-component reaction, we assembled spergualin-inspired molecules in a single step, dramatically improving the overall yield (20% to 96%). Using this strategy, we generated 43 new analogs and tested them for anti-bacterial activity against two Gram-negative and four Gram-positive strains. We found that the most potent analogue, compound 6, had MIC values between 4 and 32 μg/mL against the six strains, which is a significant improvement on spergualin (MIC ∼ 6.25 to 50 μg/mL). These studies provide a concise route to a broad-spectrum antibiotic with a novel chemical scaffold.

Pseudomonas fluorescens Pf-5 genome-wide mutant screen for resistance to the antimicrobial peptide alfalfa snakin-1.

Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties.

ChemInform Abstract: Dithiolopyrrolones: Biosynthesis, Synthesis, and Activity of a Unique Class of Disulfide‐Containing Antibiotics

AbstractReview: 88 refs.

Clarithromycin dissolution enhancement by preparation of aqueous nanosuspensions using sonoprecipitation technique.

Clarithromycin (CLM) is a member of macrolide family with broad spectrum antibiotic activity. It is practically insoluble in water and its poor solubility is pH dependent. In this study, series of nanosuspensions containing CLM and stabilizer such as HPMC, NaCMC, polysorbate 80, poloxamer 188 and polyvinyl alcohol in various ratios were prepared using sonoprecipitation method. Briefly, CLM was dissolved in acid solution and the pH of solution was raised under sonication and the effects of different stabilizers on particle size of nanoparticles were evaluated. Characterization of nanoparticles in terms of size, polydispersity index, zeta potential, differential scanning calorimetery and dissolution studies was performed. Antimicrobial activity of CLM nanosuspension was compared with coarse powder by using an agar well diffusion method. The results showed that HPMC was more efficient in size reduction of particles and presence of HPMC E5 with ratio of 3:5 to CLM in formulation led to develop the stable nanosuspension with particle size of 340 nm. The obtained nanosuspension successfully showed enhanced dissolution rate and antimicrobial activity.

Dithiolopyrrolones: biosynthesis, synthesis, and activity of a unique class of disulfide-containing antibiotics.

Covering: up to 2014. Dithiolopyrrolone (DTP) group antibiotics were first isolated in the early half of the 20th century, but only recently has research been reawakened by insights gained from the synthesis and biosynthesis of this structurally intriguing class of molecules. DTPs are characterized by an electronically unique bicyclic structure, which contains a compact disulfide bridge between two ene-thiols. Points of diversity within the compound class occur outside of the bicyclic core, at the two amide nitrogens. Such modifications distinguish three of the most well studied members of the class, holomycin, thiolutin, and aureothricin; the DTP core has also more recently been identified in the marine antibiotic thiomarinol, in which it is linked to a marinolic acid moiety, analog of the FDA-approved topical antibiotic Bactroban® (GlaxoSmithKline). Dithiolopyrrolones exhibit relatively broad-spectrum antibiotic activity against many Gram-positive and Gram-negative bacteria, as well as strains of Mycobacterium tuberculosis. Additionally, they have been shown to exhibit potent and selective anti-cancer activity. Despite this promising profile, there is still much unknown about the mechanisms of action for DTPs. Early reports suggested that they inhibit yeast growth at the level of transcription and that this effect is largely responsible for their distinctive microbial static properties; a similar mechanism is supported in bacteria. Elucidation of biosynthetic pathways for holomycin in Streptomyces clavuligerus and Yersinia ruckeri and thiomarinol in Alteromonas rava sp. nov. SANK 73390, have contributed evidence suggesting that multiple mechanisms may be operative in the activity of these compounds. This review will comprehensively cover the history and development of dithiolopyrrolones with particular emphasis on the biosynthesis, synthesis, biological activity and mechanism of action.

Small molecule inhibitors of trans -translation have broad-spectrum antibiotic activity

The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a high-throughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC50 = 0.9 µM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.

Chemical Composition and Antibacterial Activity of Wax from Actiniopteris radiata (Sw.) Link

The wax obtained from whole fern of Actiniopteris radiata (Sw.) Link was analyzed by gas chromatography-mass spectrometry. 37 compounds, out of 38, were identified with the combination of fatty acids, alkanes, triterpenoids and sterols. 9,12,15-octadecatrien-1-ol (18.7 %), Hexa decanoic acid (10.8 %), Hepatacosane (8.3 %), α-amyrin (7.0%), β-sitosterol (6.9 %) and n-Hexadecanoic acid (6.1 %) were the major contents in the wax. Agar well diffusion and micro dilution broth methods were applied to find antibacterial activity against both Gram-positive and Gram-negative bacteria. The wax has shown activity against all selected bacteria, indicating the broad-spectrum antibiotic activity. The maximum activity was observed against S. aureus.

Uncovering the Enzymes that Catalyze the Final Steps in Oxytetracycline Biosynthesis

Tetracyclines are a group of natural products sharing a linearly fused four-ring scaffold, which is essential for their broad-spectrum antibiotic activities. Formation of the key precursor anhydrotetracycline 3 during oxytetracycline 1 biosynthesis has been previously characterized. However, the enzymatic steps that transform 3 into 1, including the additional hydroxylation at C5 and the final C5a-C11a reduction, have remained elusive. Here we report two redox enzymes, OxyS and OxyR, are sufficient to convert 3 to 1. OxyS catalyzes two sequential hydroxylations at C6 and C5 positions of 3 with opposite stereochemistry, while OxyR catalyzes the C5a-C11a reduction using F420 as a cofactor to produce 1. The crystal structure of OxyS was obtained to provide insights into the tandem C6- and C5-hydroxylation steps. The substrate specificities of OxyS and OxyR were shown to influence the relative ratio of 1 and tetracycline 2.

Fatty Acid Conjugation Enhances the Activities of Antimicrobial Peptides

Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

광범위한 항균활성을 보이는 토양 유래 Streptomyces 속 방선균의 분리 및 특성 연구

Three actinobacterial strains exhibiting broad spectrum antibiotic activities were isolated from soil, and characterized. Through the comparative analysis of 16S rRNA genes, the three isolates could be assigned to the genus Streptomyces, as S. tanashiensis, S. nashivillensis, and S. rubiginosohelvolus were found to be the mostly related species, but the strains formed independent phylogenetic lineage. Each strain exhibited different antimicrobial profile against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, Gram-negative bacteria Salmonella typhi, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa, and also fungi Candida tropicalis and Candida krusei. In addition to the antimicrobial profile, the strains also differed in API ZYM test results, which implies that the three strains might produce difference antimicrobial substances.

Beneficial effects of 2,4-diacetylphloroglucinol- producing pseudomonads on the marine alga Saccharina latissima

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 67:239-249 (2012) - DOI: https://doi.org/10.3354/ame01595 Beneficial effects of 2,4-diacetylphloroglucinol- producing pseudomonads on the marine alga Saccharina latissima Kerstin Nagel, Imke Schneemann, Inga Kajahn, Antje Labes, Jutta Wiese, Johannes F. Imhoff* Kieler Wirkstoff-Zentrum (KiWiZ) am Helmholtz-Zentrum für Ozeanforschung (GEOMAR), Am Kiel-Kanal 44, 24106 Kiel, Germany *Email: jimhoff@geomar.de ABSTRACT: Pseudomonas strains were shown to be regularly associated with the brown macroalga Saccharina latissima from the Baltic Sea, studied over several years, and were identified as producers of the antimicrobially active compound 2,4-diacetylphloroglucinol. These findings support the assumption of a stable association between the Pseudomonas spp. strains and S. latissima in the Baltic Sea. The metabolite profile of the Pseudomonas spp. comprised monoacetylphloroglucinol, 2,4-diacetylphloroglucinol, pyoluteorin and several rhizoxins, which exhibited broad-spectrum antibiotic activities against Gram-positive and Gram-negative bacteria as well as against fungi. Because the antibiotic activities included the inhibition of the 2 algal pathogens Pseudoalteromonas elyakovii and Algicola bacteriolytica, we propose a beneficial effect of these marine pseudomonads on their host S. latissima. KEY WORDS: Pseudomonas protegens · Alga–bacteria association · 2,4-diacetylphloroglucinol · Pyoluteorin · Rhizoxin · Brown macroalgae Full text in pdf format PreviousNextCite this article as: Nagel K, Schneemann I, Kajahn I, Labes A, Wiese J, Imhoff JF (2012) Beneficial effects of 2,4-diacetylphloroglucinol- producing pseudomonads on the marine alga Saccharina latissima. Aquat Microb Ecol 67:239-249. https://doi.org/10.3354/ame01595 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 67, No. 3. Online publication date: November 02, 2012 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2012 Inter-Research.

Synthesis and secretory expression of hybrid antimicrobial peptide CecA–mag and its mutants in Pichia pastoris

The hybrid peptide CA(1-7)-M(2-12) gene was designed according to the N-terminal 1-7 amino acid sequence of the antimicrobial peptide cecropin A (CA) and the N-terminal 2-12 amino acid sequence of maganin (M) and synthesized using Pichia pastoris preferred codons. The gene was cloned into pPICZαA and transformed into the P. pastoris recipient bacterium SMD1168, regulated by the alcohol oxidase (AOX). Expression of the cecA-mag hybrid antimicrobial peptide (MW, 1.9 kDa) revealed broad-spectrum antibiotic activity and to the ability to inhibit growth of most G(-) and G(+) bacteria. Three mutants of cecA-mag were designed and synthesized by recombination polymerase chain reaction site-directed mutagenesis to investigate the relationship between the structure and function of this antimicrobial peptide. The inhibition titers of these mutants against Staphylococcus aureus were evaluated using the agar diffusion method. Under the conditions of the same concentration and volume, the bacteriostatic diameters of three cecA-mag mutants were 1.2, 1.2 and 1.5 times, respectively, compared with the diameters of wild-type cecA-mag.

In vitro cytotoxicity of Cu2+, Zn2+, Ag+ and their mixtures on primary human endometrial epithelial cells

BackgroundTo avoid the inherent disadvantages of copper-containing intrauterine device (Cu-IUD) induced by free Cu2+, two other well-performing metal ions, namely, Ag+, with long-effective antimicrobial properties, and Zn2+, as an essential trace element, are being considered for use in the future as multifunctional IUDs. The purpose of this study was to assess the cytotoxicity of these metal ions and their mixtures on primary human endometrial epithelial cells (HEECs) cultured in vitro and to provide several choices of alternative potential materials for creating excellent IUDs in the future. Study DesignWith the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan (MTT-f) production, the cytotoxic effects of single metal ions (Cu2+, Zn2+, Ag+) on HEECs after exposure for 24, 48 or 72 h were investigated, and the synergistic and antagonistic effects of two ions applied simultaneously were also assessed. ResultsThe cytotoxicity of the metal ions on HEECs ranked as follows: Ag+>Cu2+>Zn2+. All combinations of those tested indicated that the Cu2++Zn2+ system exhibited an antagonistic effect absolutely, the Zn2++Ag+ system showed both antagonism and slight synergism, and asynergistic effect was observed in the Cu2++Ag+ system. ConclusionFrom a perspective of favorable biocompatibility, Zn2+ and the Cu2++Zn2+ mixture showed evidence of potential components for use in future IUDs. Although having strong cytotoxicity, Ag+ with its low release rate and broad-spectrum antibiotic activity may also be considered. The study also demonstrated the relative stability of Cu2+ as a classic material of IUD.

Screening and Isolation of Associated Bioactive Microorganisms from Fasciospongia cavernosa from of Visakhapatnam Coast, Bay of Bengal

Nature, especially the marine environment, provides the most effective drugs used in human therapy. Among the metazoans, the marine sponges produce the most potent and highly selective bioactive secondary metabolites. These animals (or their associated symbiotic microorganisms) synthesize secondary metabolites whose activity and selectivity has developed during their long evolutionary history. During the course of exploitation of these resources two marine sponges, Fasciospongia cavernosa doc var.brown (dark brown) Fasciospongia cavernosa doc var.yellow (yellow) collected from the visakhapatnam coast of Bay of Bengal were investigated in order to assess the potential of these microorganisms for the production of antimicrobial compounds. The aqueous and organic extracts of both the sponges showed broad spectrum antibiotic activity. In this study a total of 178 microorganisms were isolated from different parts of two sponges and most of them from middle part of the sponge. The isolates were investigated in order to assess the potential of these microorganisms for the production of antimicrobial compounds. Testing for antimicrobial activities were performed against Gram‐positive (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus) Gram‐negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris), fungi (Candida albicans, Aspergillus niger) and 10 pathogenic organisms. Resulting mean diameter of inhibition zones revealed isolates B4 & B6 were the most potent of all the isolates. The present study has revealed the presence of high numbers of diverse culturable microorganisms associated with the marine sponges from Visakhapatnam Coast of Bay of Bengal as well as their potential to produce bioactive metabolites.

Biophysical Mechanisms of Host Defense Peptide (HDP) Toxicity as Revealed by a Study of Peptoid Mimics of HDPs

We are developing and studying biomimetic N-substituted glycine oligomers (peptoids) with broad-spectrum antibiotic activity against pathogens, including antibiotic-resistant strains of Gram-positive and Gram-negative bacteria. Peptoids are sequence- and length-specific oligomers with high similarity to peptides, which are made on solid phase and resist proteolysis; they are ideal for applications for which stability is desired. We designed peptoid oligomers to mimic features of the natural host defense peptides magainin and LL-37: helical, cationic and amphipathic. A study of over 100 sequence variants allowed us to identify peptoids with potency and broad-spectrum antibacterial activity similar to natural HDPs. The selectivity of antibacterial peptoids and peptides (i.e., their ability to kill bacteria at low-μM concentrations while causing only tolerable damage to host mammalian cells) correlates with molecular hydrophobicity and amphipathicity as well as with self-association in solution. We did biophysical mechanistic assays using peptoids with either high or low antibacterial activity and/or either high or low selectivity, along with selective and non-selective host defense peptides. Comparative vesicle leakage studies, TEM imaging, and soft X-ray tomography of untreated and treated bacteria yield powerful insight into mechanisms of action for both HDPs and their peptoid mimics.

Saccharomyces cerevisiae Genome-Wide Mutant Screen for Sensitivity to 2,4-Diacetylphloroglucinol, an Antibiotic Produced by Pseudomonas fluorescens

2,4-Diacetylphloroglucinol (2,4-DAPG), an antibiotic produced by Pseudomonas fluorescens, has broad-spectrum antibiotic activity, inhibiting organisms ranging from viruses, bacteria, and fungi to higher plants and mammalian cells. The biosynthesis and regulation of 2,4-DAPG in P. fluorescens are well described, but the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a deletion library of Saccharomyces cerevisiae in broth and agar medium supplemented with 2,4-DAPG. We identified 231 mutants that showed increased sensitivity to 2,4-DAPG under both conditions, including 22 multidrug resistance-related mutants. Three major physiological functions correlated with an increase in sensitivity to 2,4-DAPG: membrane function, reactive oxygen regulation, and cell homeostasis. Physiological studies with wild-type yeast validated the results of the mutant screens. The chemical-genetic fitness profile of 2,4-DAPG resembled those of menthol, sodium azide, and hydrogen peroxide determined in previous high-throughput screening studies. Collectively, these findings indicate that 2,4-DAPG acts on multiple basic cellular processes.

Broad-Spectrum Antibiotic Activity of the Arylomycin Natural Products Is Masked by Natural Target Mutations

Novel classes of broad-spectrum antibiotics are needed to treat multidrug-resistant pathogens. The arylomycin class of natural products inhibits a promising antimicrobial target, type I signal peptidase (SPase), but upon initial characterization appeared to lack whole-cell activity against most pathogens. Here, we show that Staphylococcus epidermidis, which is sensitive to the arylomycins, evolves resistance via mutations in SPase and that analogous mutations are responsible for the natural resistance of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We identify diverse bacteria lacking these mutations and demonstrate that most are sensitive to the arylomycins. The results illustrate that the arylomycins have a broad-spectrum of activity and are viable candidates for development into therapeutics. The results also raise the possibility that naturally occurring resistance may have masked other natural product scaffolds that might be developed into therapeutics.