A High-Throughput Screen Identifies a New Natural Product with Broad-Spectrum Antibacterial Activity

Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms that grow in the diverse ecosystems of Costa Rica and identified 49 with reproducible antibacterial effects. An extract from an endophytic fungus was further characterized, and this led to the discovery of three novel natural products. One of these, which we named mirandamycin, has broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis. This demonstrates the power of simple high throughput screens for rapid identification of new antibacterial agents from environmental samples

Quantitative analysis of the accumulation, architectural organization, detachment and reseeding of Staphylococcus aureus biofilms under physiological fluid shear conditions

Staphylococcus aureus is an opportunistic gram- positive pathogen responsible for a wide variety of animal and human infections. In humans, it is associated with both superficial and invasive cases of infections, including bacteremia, endocarditis, osteomyelitis, septic arthritis, keratinitis, pneumonia and catheter- related infections. The prevalence of S. aureus as a human pathogen has been attributed to its ability to form specific bonds with a wide variety of extracellular matrix ( ECM) proteins. These binding events contribute significantly to the molecular mechanisms of S. aureus virulence. Additional virulence properties incur from its capacity to colonize surfaces in organized biofilm communities and from the occurrence of secondary metastatic infections caused by bacterial cells detaching from biofilms growing under shear stress. Microbial biofilms have also been associated with the spread of community- acquired bacterial infections and the emergence of resistant bacterial variants. The treatment of biofilm- associated infections costs over $ 1 billion annually in the United States. As a result, the study and characterization of microbial biofilms is rapidly gaining interest in the scientific community. The overall ambition of this project was to investigate the effects of physiologically relevant hydrodynamic forces on the accumulation and proliferation of S. aureus biofilms onto biotic substrates. Additionally, we evaluated the ability of sodium metaperiodate to inhibit the growth of S. aureus biofilms in vitro under both static and dynamic conditions. In the course of these studies, we demonstrated that: 1) hydrodynamic forces and nutrient availability modulate the rate of growth and the internal structure of early S. aureus biofilms grown on biotic surfaces; 2) through the process of erosion, S. aureus biofilms grown under physiologically relevant hydrodynamic conditions release planktonic cells with reduced adhesion avidity to ECM proteins; 3) these eroded planktonic cells demonstrate the potential to initiate secondary biofilm formations; and 4) under hydrodynamic conditions, S. aureus biofilms can withstand antimicrobial challenges that would otherwise be detrimental to sessile cultures grown under static conditions and to individual cells grown in suspension. The current research extended our understanding of the physiological effects of fluid shear forces on the development of S. aureus biofilms. It is essential to establish the principal factors leading to the multilayered accumulation of staphylococcal biofilms in vivo in order to design alternative therapeutic approaches to treating S. aureus infections

Mannitol and the mannitol-specific enzyme IIB subunit activate Vibrio cholerae biofilm formation.

International audienceVibrio cholerae is a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera. V. cholerae use a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport by V. cholerae requires the mannitol-specific EII (EII(Mtl)), which is expressed only in the presence of mannitol. Here we show that mannitol activates V. cholerae biofilm formation and transcription of the vps biofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EII(Mtl) is sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability of V. cholerae to respond to environmental mannitol by forming a biofilm may play an important role in habitat selection

Isolation and identification of three novel natural compounds.

<p>Chromatogram obtained during fractionation of the crude extract CR1223-D showing three peaks corresponding to the three compounds isolated. Compound 1 was further identified as 6-propyl gentisyl alcohol, compound 2 as 5-hydroxy-4-(hydroxymethyl)-2-methyl 2,3-dihydrobenzofuran, and compound 3 as 2-(hydroxymethyl)-3-propyl benzoquinone.</p

Spectrophotometric assay for bacterial sugar fermentation.

<p>Absorbance spectrum of MM^Suc alone (indicator) or incubated with wild-type <i>V. cholerae</i> (WT) or a PTS mutant for 5 hours. The spectra are shown at the left, while the visible color difference is shown in microtiter dish wells at the right. The largest difference in absorbance between MM^Suc incubated with wild-type <i>V. cholerae</i> and that incubated with a PTS mutant is measured at 615 nm (red arrow).</p

Impact of compounds 1, 2, and 3 on <i>V. cholerae</i> sugar fermentation and growth.

<p>The assays were carried out at 30°C in pH-MM^Suc to monitor sugar fermentation by A₆₁₅ (A, B, and C) or in MM^Pyr to monitor bacterial growth by OD₆₁₅ (D, E, and F). Bacteria were exposed to mirandamycin (A and D), compound 2 (B and E) or compound 3 (C and F) at concentrations ranging from 14 to 383 µM. Replicate assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031307#pone.0031307.s002" target="_blank">Figure S2</a>.</p

Cochlin Produced by Follicular Dendritic Cells Promotes Antibacterial Innate Immunity

International audienceCochlin, an extracellular matrix protein, shares homologies with the Factor C, a serine protease found in horseshoe crabs, which is critical for antibacterial responses. Mutations in the COCH gene are responsible for human DFNA9 syndrome, a disorder characterized by neurodegeneration of the inner ear that leads to hearing loss and vestibular impairments. The physiological function of cochlin, however, is unknown. Here, we report that cochlin is specifically expressed by follicular dendritic cells and selectively localized in the fine extracellular network of conduits in the spleen and lymph nodes. During inflammation, cochlin was cleaved by aggrecanases and secreted into blood circulation. In models of lung infection with Pseudomonas aeruginosa and Staphylococcus aureus, Coch(-/-) mice show reduced survival linked to defects in local cytokine production, recruitment of immune effector cells, and bacterial clearance. By producing cochlin, FDCs thus contribute to the innate immune response in defense against bacteria

Minimum inhibitory concentrations (MICs) of mirandamycin and known antibiotics against selected bacterial pathogens.

(*)<p>MIC was determined by Alamar Blue Assay as described in Material and Methods; Mirandamycin (MIR), levofloxacin (LEVO), ampicillin (AMP), imipenem (IMI), bactrim (TMP/SMX), isoniazid (INH), pyrazinamide (PZA), ethambutol: ETH.</p

Representative results for secondary screen.

<p>Bacteria were grown in MM^Pyr, pH-MM^Suc, or pH-MM^Glu. OD₆₁₅ measurements of cultures in MM^Pyr reflect the ability of cells to grow in the presence of extract, while absorbance measurements in pH-MM^Suc and pH-MM^Glu reflect the ability of cells to transport and ferment these sugars in the presence of extract. Data are shown for wild-type <i>V. cholerae</i> and a PTS mutant in the absence of extract (A,B) or for wild-type <i>V. cholere</i> in the presence of extracts that we hypothesize (C) interfere with sugar transport and fermentation, (D) inhibit bacterial growth (bacteriostatic), or (E) kill bacteria (bactericidal).</p