Medical importance of biofilms in Candida infections

Many Candida infections involve biofilm formation on implanted devices such as an indwelling catheter, a prosthetic heart valve or a denture. Candida biofilms can be formed in vitro using several model systems. In the simplest of these, organisms are grown on the surfaces of small discs of catheter material or denture acrylic. Biofilms of C. albicans prepared in this way consist of matrix-enclosed microcolonies containing yeasts, hyphae and pseudohyphae, arranged in a bilayer structure. Candida biofilms are resistant to a range of antifungal agents in current clinical use, including amphotericin B and fluconazole. Current research suggests that multiple mechanisms are involved in biofilm drug resistance.     

经皮给药纳米载体及靶向系统治疗类风湿关节炎研究进展

类风湿关节炎(RA)是全世界难治性自身免疫疾病,其治疗药物虽不断发展,但病灶药物浓度达不到有效水平导致药物疗效不理想或存在各种毒副反应,因此,基于新技术、新方法研究开发针对RA的安全、高效新型制剂是必要的.研究表明,纳米技术的运用可提高药物生物利用度,经皮给药可改善口服和注射带来的毒副作用.对近年来基于经皮给药系统治疗...     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Activity of anidulafungin against Candida biofilms

Currently, no standardized method to study the in vitro activity of antifungal agents on biofilms is available, thus, the comparison among different authors is difficult. The studies discussed in this review use the XTT reduction to measure the activity of antifungals on biofilms of 24 h of maturation. To date, biofilm anidulafungin MICs of 47 isolates of Candida spp. (25 Candida albicans, 16 Candida tropicalis, 5 Candida dubliniensis and 1 Candida parapsilosis) have been published. The geometric mean MIC of anidulafungin on biofilms of Candida spp. is of 1.18 microg/ml. Against isolates of species with great capacity of biofilm formation, the geometric mean MIC is 0.325 (C. albicans), 2 (C. parapsilosis) and 0.5 microg/ml (C. dubliniensis). No echinocandin has activity on C. tropicalis biofilms. In addition, anidulafungin can be used for lock therapy of catheters since it is the echinocandin with the least in vitro paradoxical effect.     

In vitro modeling of dental water line contamination and decontamination

The contamination of dental unit water lines (DUWL) is an emerging concern in dentistry. The aim of this study was to use an in vitro DUWL to model microbial contamination and evaluate the decontamination efficacy of tetraacetylethylenediamine (TAED) solutions. A DUWL biofilm model used to simulate clinical conditions was used to generate a range of biofilms in DUWL. Three distinct biofilms were generated: (1) biofilm from water, (2) biofilm from a mix of water + contaminating human commensal bacteria, (3) biofilm from water with contaminating oral bacteria added after biofilm formed. The contaminating oral species used were Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus. Decontamination by simple water flushing or flushing with TAED was evaluated (2, 5 and 10 min intervals). The DUWL tubes were split and samples were plated onto a range of media, incubated and bacteria enumerated. Water flushing did not reduce the number of microorganisms detected. Bacteria were not detected from any of the TAED sampling points for any of the biofilm types tested. Interestingly, if contamination was introduced to new DUWL along with the waterborne species a biofilm was formed containing only the waterborne species. If however, an existing biofilm was present before the introduction of "contaminating" bacteria then these could be detected in the biofilm. This implies that if the DUWL are new or satisfactorily cleaned on a regular basis then the associated cross-contamination aspects are reduced. In conclusion, TAED provides effective control for DUWL biofilms.     

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.     

Local drug and gene delivery through microbubbles and ultrasound

Although gene therapy has great potential as a treatment for diseases, clinical trials are slowed down by the development of a safe and efficient gene delivery system. In this review, we will give an overview of the viral and nonviral vehicles used for drug and gene delivery, and the different nonviral delivery techniques, thereby focusing on delivery through ultrasound contrast agents.The development of ultrasound contrast agents containing encapsulated microbubbles has increased the possibilities not only for diagnostic imaging, but for therapy as well. Microbubbles have been shown to be able to carry drugs and genes, and destruction of the bubbles by ultrasound will result in local release of their contents. Furthermore, ligands can be attached so that they can be targeted to a specific target tissue. The recent advances of microbubbles as vehicles for delivery of drugs and genes will be highlighted.     

Optical and acoustical dynamics of microbubble contrast agents inside neutrophils

Acoustically active microbubbles are used for contrast-enhanced ultrasound assessment of organ perfusion. In regions of inflammation, contrast agents are captured and phagocytosed by activated neutrophils adherent to the venular wall. Using direct optical observation with a high-speed camera and acoustical interrogation of individual bubbles and cells, we assessed the physical and acoustical responses of both phagocytosed and free microbubbles. Optical analysis of bubble radial oscillations during insonation demonstrated that phagocytosed microbubbles experience viscous damping within the cytoplasm and yet remain acoustically active and capable of large volumetric oscillations during an acoustic pulse. Fitting a modified version of the Rayleigh-Plesset equation that describes mechanical properties of thin shells to optical radius-time data of oscillating bubbles provided estimates of the apparent viscosity of the intracellular medium. Phagocytosed microbubbles experienced a viscous damping approximately sevenfold greater than free microbubbles. Acoustical comparison between free and phagocytosed microbubbles indicated that phagocytosed microbubbles produce an echo with a higher mean frequency than free microbubbles in response to a rarefaction-first single-cycle pulse. Moreover, this frequency increase is predicted using the modified Rayleigh-Plesset equation. We conclude that contrast-enhanced ultrasound can detect distinct acoustic signals from microbubbles inside of neutrophils and may provide a unique tool to identify activated neutrophils at sites of inflammation.     

The carbon monoxide releasing molecule CORM-2 attenuates Pseudomonas aeruginosa biofilm formation

Chronic infections resulting from biofilm formation are difficult to eradicate with current antimicrobial agents and consequently new therapies are needed. This work demonstrates that the carbon monoxide-releasing molecule CORM-2, previously shown to kill planktonic bacteria, also attenuates surface-associated growth of the gram-negative pathogen Pseudomonas aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm. CORM-2 treatment has an additive effect when combined with tobramycin, a drug commonly used to treat P. aeruginosa lung infections. CORM-2 inhibited biofilm formation and planktonic growth of the majority of clinical P. aeruginosa isolates tested, for both mucoid and non-mucoid strains. While CORM-2 treatment increased the production of reactive oxygen species by P. aeruginosa biofilms, this increase did not correlate with bacterial death. These data demonstrate that CO-RMs possess potential novel therapeutic properties against a subset of P. aeruginosa biofilm related infections.     

Bacteriophages and Lysins in Biofilm Control

To formulate the optimal strategy of combatting bacterial biofilms, in this review we update current knowledge on the growing problem of biofilm formation and its resistance to antibiotics which has spurred the search for new strategies to deal with this complication. Based on recent findings, the role of bacteriophages in the prevention and elimination of biofilm-related infections has been emphasized. In vitro, ex vivo and in vivo biofilm treatment models with single bacteriophages or phage cocktails have been compared. A combined use of bacteriophages with antibiotics in vitro or in vivo confirms earlier reports of the synergistic effect of these agents in improving biofilm removal. Furthermore, studies on the application of phage-derived lysins in vitro, ex vivo or in vivo against biofilm-related infections are encouraging. The strategy of combined use of phage and antibiotics seems to be different from using lysins and antibiotics. These findings suggest that phages and lysins alone or in combination with antibiotics may be an efficient weapon against biofilm formation in vivo and ex vivo, which could be useful in formulating novel strategies to combat bacterial infections. Those findings proved to be relevant in the prevention and destruction of biofilms occurring during urinary tract infections,orthopedic implant-related infections, periodontal and peri-implant infections. In conclusion, it appears that most efficient strategy of eliminating biofilms involves phages or lysins in combination with antibiotics, but the optimal scheme of their administration requires further studies.     

Sonoporation: Mechanistic insights and ongoing challenges for gene transfer

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.     

Sum of the Parts: Composition and Architecture of the Bacterial Extracellular Matrix

Bacterial biofilms are complex multicellular assemblies that exhibit resistance to antibiotics and contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative data are needed to define the molecular composition of bacterial biofilms. Escherichia coli biofilms are known to contain polysaccharides and functional amyloid fibers termed curli, yet accurate determinations of biofilm composition at the molecular level have been elusive. The ability to define the composition of the extracellular matrix (ECM) is crucial for the elucidation of structure–function relationships that will aid the development of chemical strategies to disrupt biofilms. We have developed an approach that integrates non-perturbative preparation of the ECM with electron microscopy, biochemistry, and solid-state NMR spectroscopy to define the chemical composition of the intact and insoluble ECM of a clinically important pathogenic bacterium—uropathogenic E. coli. Our data permitted a sum-of-all-the-parts analysis. Electron microscopy revealed supramolecular shell-like structures that encapsulated single cells and enmeshed the bacterial community. Biochemical and solid-state NMR measurements of the matrix and constitutive parts established that the matrix is composed of two major components, curli and cellulose, each in a quantifiable amount. This approach to quantifying the matrix composition is widely applicable to other organisms and to examining the influence of biofilm inhibitors. Collectively, our NMR spectra and the electron micrographs of the purified ECM inspire us to consider the biofilm matrix not as an undefined slime, but as an assembly of polymers with a defined composition and architecture.     

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.     

抗菌肽的抗生物膜机理研究进展

生物膜,也称为生物被膜,是指附着于有生命或无生命物体表面被细菌胞外大分子包裹的有组织的细菌群体。与浮游菌相比,生物膜内的细菌对抗生素的耐受性提高了10–1000倍,是造成目前细菌耐药的主要原因之一。作为一种新型抗菌制剂,抗菌肽的使用为生物膜感染的治疗提供了一种新的思路和手段。抗菌肽在抑制生物膜形成、杀灭生物膜内细菌以及消除成熟生物膜的过程中发挥了独特的优势。文中分析了近30年的数据,从细菌生物膜的结构入手,对抗菌肽可能的抗生物膜机理进行了综述,以期为抗菌肽临床治疗生物膜感染提供一定参考。     

Polymer shelled microparticles for a targeted doxorubicin delivery in cancer therapy

Targeting is a main feature supporting any controlled drug delivery modality. Recently we developed poly(vinyl alcohol), PVA, based microbubbles as a potential new ultrasound contrast agent featuring an efficient ultrasound backscattering and a good shelf stability. The chemical versatility of the polymeric surface of this device offers a vast variety of coupling modalities useful for coating and specific targeting. We have designed a conjugation strategy on PVA shelled microbubbles to enable the localization and the drug delivery on tumor cells by modifying the surface of this polymeric ultrasound contrast agent (UCA) with oxidized hyaluronic acid (HAox). After the conversion of the microbubbles into microcapsules, the kinetics of the release of doxorubicin, a well-known antitumor drug, from uncoated and HAox-coated PVA microbubbles and microcapsules was investigated. Cytocompatibility and bioadhesive properties of the HA-modified microparticles were then tested on the HT-29 tumor cell line. Cytotoxicity to HT-29 tumor cells of microcapsules after loading with doxorubicin was studied, evidencing the efficacy of the HAox coating for the delivery of the drug to cells. These features are a prerequisite for a theranostic, that is, diagnostic and therapeutic, use of polymer-based UCAs.     

The evolution of quorum sensing in bacterial biofilms

Bacteria have fascinating and diverse social lives. They display coordinated group behaviors regulated by quorum-sensing systems that detect the density of other bacteria around them. A key example of such group behavior is biofilm formation, in which communities of cells attach to a surface and envelope themselves in secreted polymers. Curiously, after reaching high cell density, some bacterial species activate polymer secretion, whereas others terminate polymer secretion. Here, we investigate this striking variation in the first evolutionary model of quorum sensing in biofilms. We use detailed individual-based simulations to investigate evolutionary competitions between strains that differ in their polymer production and quorum-sensing phenotypes. The benefit of activating polymer secretion at high cell density is relatively straightforward: secretion starts upon biofilm formation, allowing strains to push their lineages into nutrient-rich areas and suffocate neighboring cells. But why use quorum sensing to terminate polymer secretion at high cell density? We find that deactivating polymer production in biofilms can yield an advantage by redirecting resources into growth, but that this advantage occurs only in a limited time window. We predict, therefore, that down-regulation of polymer secretion at high cell density will evolve when it can coincide with dispersal events, but it will be disfavored in long-lived (chronic) biofilms with sustained competition among strains. Our model suggests that the observed variation in quorum-sensing behavior can be linked to the differing requirements of bacteria in chronic versus acute biofilm infections. This is well illustrated by the case of Vibrio cholerae, which competes within biofilms by polymer secretion, terminates polymer secretion at high cell density, and induces an acute disease course that ends with mass dispersal from the host. More generally, this work shows that the balance of competition within and among biofilms can be pivotal in the evolution of quorum sensing.     

Chelated iron sources are inhibitors of Pseudomonas aeruginosa biofilms and distribute efficiently in an in vitro model of drug delivery to the human lung

Aims:  To determine whether chelated sources of ferric iron were efficient inhibitors of biofilm formation in Pseudomonas aeruginosa and might be suitable for drug delivery to the lungs of cystic fibrosis (CF) patients via nebulization.
Methods and Results:  The response of P. aeruginosa biofilms to elevated iron concentrations in the form of eight structurally varied iron chelators in a microtitre plate assay for biofilm production was examined in the lab. Among these iron chelates, picolinic acid and acetohydroxamic acid-chelated iron were able to effectively thwart biofilm production in P. aeruginosa PA14 and in 20 clinical isolates of P. aeruginosa from a local hospital. The chelated iron sources showed excellent distribution in an Anderson cascade impactor model of particle size distribution in the human lung.
Conclusions:  Ferric picolinate and ferric acetohydroxamate are effective anti-biofilm compounds against both lab and clinical strains of P. aeruginosa and are readily nebulized into particles of suitable size for lung delivery.
Significance and Impact of the Study:  The data herein serve both to solidify the growing base of literature correlating high iron levels with biofilm inhibition in P. aeruginosa and to highlight the potential of these chelators as nebulized agents to combat biofilms of P. aeruginosa in CF patients.     

柱[5]芳烃衍生物对细菌抗菌活性及生物被膜抑制的研究进展北大核心

病原体的耐药性很强,其生物被膜(biofilm,BF)的形成是导致耐药性的主要原因之一。生物被膜一旦形成,根除难度很大,会导致患者持久性感染,引发多种慢性疾病,并给全球医疗体系带来沉重负担。柱芳烃(pillararenes)是一类具有独特柱状结构的新型大环化合物,由于其在构建功能化和生物活性材料开发中的潜在应用引起人们广泛的关注。此外,它们在预防和控制抗生素耐药性(antimicrobial resistance,AMR)方面具有广阔的应用前景。本文综述了柱[5]芳烃衍生物对细菌病原菌的抗菌活性,并进一步揭示其在抗菌活性中的抑菌机制,尤其是对生物被膜的抑制作用。在此基础上,探索新的抑菌杀菌策略,用非传统药物以解决抗生素耐药性问题,以期为开发新的抗菌剂防控生物被膜或治疗细菌感染提供理论依据。