Structure‐Based Optimization of the Terminal Tripeptide in Glycopeptide Dendrimer Inhibitors of Pseudomonas aeruginosa Biofilms Targeting LecA

The galactopeptide dendrimer GalAG2 ((β‐Gal‐OC₆H₄CO‐Lys‐Pro‐Leu)₄(Lys‐Phe‐Lys‐Ile)₂Lys‐His‐Ile‐NH₂) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA‐KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide–lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X‐ray crystallography. Two of these tripeptides, GalA‐KPY (dissociation constant (K_D)=2.7 μM ) and GalA‐KRL (K_D=2.7 μM ), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2 , particularly G2KPY ((β‐Gal‐OC₆H₄CO‐Lys‐Pro‐Tyr)₄(Lys‐Phe‐Lys‐Ile)₂Lys‐His‐Ile‐NH₂). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine‐tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions.     

Highly Active Bidirectional Electron Transfer by a Self‐Assembled Electroactive Reduced‐Graphene‐Oxide‐Hybridized Biofilm

Low extracellular electron transfer performance is often a bottleneck in developing high‐performance bioelectrochemical systems. Herein, we show that the self‐assembly of graphene oxide and Shewanella oneidensis MR‐1 formed an electroactive, reduced‐graphene‐oxide‐hybridized, three‐dimensional macroporous biofilm, which enabled highly efficient bidirectional electron transfers between Shewanella and electrodes owing to high biomass incorporation and enhanced direct contact‐based extracellular electron transfer. This 3D electroactive biofilm delivered a 25‐fold increase in the outward current (oxidation current, electron flux from bacteria to electrodes) and 74‐fold increase in the inward current (reduction current, electron flux from electrodes to bacteria) over that of the naturally occurring biofilms.     

Spatially Resolved Confocal Resonant Raman Microscopic Analysis of Anode‐Grown Geobacter sulfurreducens Biofilms

When grown on the surface of an anode electrode, Geobacter sulfurreducens forms a multi‐cell thick biofilm in which all cells appear to couple the oxidation of acetate with electron transport to the anode, which serves as the terminal metabolic electron acceptor. Just how electrons are transported through such a biofilm from cells to the underlying anode surface over distances that can exceed 20 microns remains unresolved. Current evidence suggests it may occur by electron hopping through a proposed network of redox cofactors composed of immobile outer membrane and/or extracellular multi‐heme c‐type cytochromes. In the present work, we perform a spatially resolved confocal resonant Raman (CRR) microscopic analysis to investigate anode‐grown Geobacter biofilms. The results confirm the presence of an intra‐biofilm redox gradient whereby the probability that a heme is in the reduced state increases with increasing distance from the anode surface. Such a gradient is required to drive electron transport toward the anode surface by electron hopping via cytochromes. The results also indicate that at open circuit, when electrons are expected to accumulate in redox cofactors involved in electron transport due to the inability of the anode to accept electrons, nearly all c‐type cytochrome hemes detected in the biofilm are oxidized. The same outcome occurs when a comparable potential to that measured at open circuit (?0.30 V vs. SHE) is applied to the anode, whereas nearly all hemes are reduced when an exceedingly negative potential (?0.50 V vs. SHE) is applied to the anode. These results suggest that nearly all c‐type cytochrome hemes detected in the biofilm can be electrochemically accessed by the electrode, but most have oxidation potentials too negative to transport electrons originating from acetate metabolism. The results also reveal a lateral heterogeneity (x–y dimensions) in the type of c‐type cytochromes within the biofilm that may affect electron transport to the electrode.     

Searching Hit Potential Antimicrobials in Natural Compounds Space against Biofilm Formation

Biofilms are communities of microorganisms that can colonize biotic and abiotic surfaces and thus play a significant role in the persistence of bacterial infection and resistance to antimicrobial. About 65% and 80% of microbial and chronic infections are associated with biofilm formation, respectively. The increase in infections by multi-resistant bacteria instigates the need for the discovery of novel natural-based drugs that act as inhibitory molecules. The inhibition of diguanylate cyclases (DGCs), the enzyme implicated in the synthesis of the second messenger, cyclic diguanylate (c-di-GMP), involved in the biofilm formation, represents a potential approach for preventing the biofilm development. It has been extensively studied using PleD protein as a model of DGC for in silico studies as virtual screening and as a model for in vitro studies in biofilms formation. This study aimed to search for natural products capable of inhibiting the Caulobacter crescentus enzyme PleD. For this purpose, 224,205 molecules from the natural products ZINC15 database, have been evaluated through molecular docking and molecular dynamic simulation. Our results suggest trans-Aconitic acid (TAA) as a possible starting point for hit-to-lead methodologies to obtain new inhibitors of the PleD protein and hence blocking the biofilm formation.     

Antibacterial Activity of Pharmaceutical-Grade Rose Bengal: An Application of a Synthetic Dye in Antibacterial Therapies

Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has been studied for the treatment of solid tumor cancers. To date, the antibacterial activity of rose bengal has been sporadically reported; however, these data have been generated with a commercial grade of rose bengal, which contains major uncontrolled impurities generated by the manufacturing process (80–95% dye content). A high-purity form of rose bengal formulation (HP-RBf, >99.5% dye content) kills a battery of Gram-positive bacteria, including drug-resistant strains at low concentrations (0.01–3.13 μg/mL) under fluorescent, LED, and natural light in a few minutes. Significantly, HP-RBf effectively eradicates Gram-positive bacterial biofilms. The frequency that Gram-positive bacteria spontaneously developed resistance to HP-RB is extremely low (less than 1 × 10⁻¹³). Toxicity data obtained through our research programs indicate that HP-RB is feasible as an anti-infective drug for the treatment of skin and soft tissue infections (SSTIs) involving multidrug-resistant (MDR) microbial invasion of the skin, and for eradicating biofilms. This article summarizes the antibacterial activity of pharmaceutical-grade rose bengal, HP-RB, against Gram-positive bacteria, its cytotoxicity against skin cells under illumination conditions, and mechanistic insights into rose bengal’s bactericidal activity under dark conditions.     

XPS analysis of bio‐organic systems

A survey of the literature is made for the XPS analysis of food products (mainly spray‐dried powders, which reveal a considerable surface enrichment in lipids) and of microorganisms and related systems (extracellular polymer substances and biofilms). This survey is used as a background for discussions and recommendations regarding methodology. Sample preparation methods reviewed are freeze drying, analysis of frozen hydrated specimens and adsorption of surface‐active biocompounds on model substrates. Peak decomposition is a way to increase the wealth of information provided by the XPS spectra. It should be performed after a check that sample charge stabilization is satisfactory. Moreover, ensuring the precision needed to make comparisons within sets of samples may involve a trade‐off between imposing constraints and generating information. The examination of correlations between spectral data in the light of chemical guidelines is useful to validate or improve peak decomposition and component assignment, and may also upgrade the chemical information regarding speciation. Further upgrading may be achieved by expressing marker XPS data in terms of concentrations of compounds of interest. Different methods of computation are discussed, providing a composition in terms of ingredients, classes of biochemical compounds, or various organic and inorganic compounds. As an alternative or complement to this deterministic approach, multivariate analysis of suitable spectral windows provides spectral components, which may be used for comparing samples, and which may have a direct chemical relevance or be used to identify features of interest. Copyright © 2011 John Wiley & Sons, Ltd.     

3‐D spatio–temporal structures of biofilms in a water channel

We develop a numerical predictive tool for multiphase fluid mixtures consisting of biofilms grown in a viscous fluid matrix by implementing a second‐order finite difference discretization of the multiphase biofilm model developed recently on a general purpose graphic processing unit. With this numerical tool, we study a 3‐D biomass–flow interaction resulting in biomass growth, structure formation, deformation, and detachment phenomena in biofilms grown in a water channel in quiescent state and subject to a shear flow condition, respectively. The numerical investigation is limited in the viscous regime of the biofilm–solvent mixture. In quiescent flows, the model predicts growth patterns consistent with experimental findings for single or multiple adjacent biofilm colonies, the so‐called mushroom shape growth pattern. The simulated biomass growth both in density and thickness matches very well with the experimentally grown biofilm in a water channel. When shear is imposed at a boundary, our numerical studies reproduce wavy patterns, pinching, and streaming phenomena observed in biofilms grown in a water channel. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of Pathogenic Bacterial and Yeast Biofilms Using the Combination of Synchrotron ATR-FTIR Microspectroscopy and Chemometric Approaches

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms’ heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200–950 cm⁻¹) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements.     

复合垂直流构建湿地中生物膜的空间分布、特性和降解性能

对复合垂直流构建湿地基质不同层次进行采样，测定了生物膜的空间分布、脱氢酶活性、呼吸强度、间隙水总有机碳、发育时间及五氯酚的去除率等指标．结果表明，基质生物膜发育良好，可挥发性固体均值达到0．0218．从下行池整体看，生物膜生物量、呼吸强度、脱氢酶活性及有机污染物去除能力都较上行池高，下行池为污水中有机物的主要降解空间．不同层次生物膜的空间分布存在着差别，生物膜主要积累在0～10cm表层，其脱氢酶活性、呼吸强度高于其他各层次，各指标在变化趋势上存在明显的一致性．生物膜的发育时间直接影响着其对有机碳的降解能力，从10天到70天，降解率提高了5倍．其生物膜基质对五氯酚的去除率是无生物膜基质的1．5倍．