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Front Matter |
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Abstract
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Methods for Dynamic Investigations of Surface-Attached In Vitro Bacterial and Fungal Biofilms |
Claus Sternberg,Thomas Bjarnsholt,Mark Shirtliff |
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Abstract
Three dynamic models for the investigation of in vitro biofilm formation are described in this chapter. In the 6-well plate assay presented here, the placing of the plate on a rotating platform provides shear, thereby making the system dynamic with respect to the static microtiter assay..The second reported model, especially suitable for harvesting high amounts of cells for transcriptomic or proteomic investigations, is based on numerous glass beads placed in a flask incubated with shaking on a rotating platform, thus increasing the surface area for biofilm formation. Finally, the flow-cell system, that is the driving model for elucidating the biofilm-forming process in vitro as well as the biofilm tolerance towards antibiotics and host defense components, is illustrated here.
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Aqueous Two-Phase System Technology for Patterning Bacterial Communities and Biofilms |
Mohammed Dwidar,Shuichi Takayama,Robert J. Mitchell |
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Abstract
We describe a novel method which makes use of polymer-based aqueous two-phase systems to pattern bacterial communities inside Petri dishes. This method allows us to culture submillimeter-sized bacterial communities in spatially distinct spots while maintaining a degree of chemical connectedness to each other through the aqueous phase. Given sufficient time, these bacterial cultures develop biofilms, each corresponding to the footprint of the droplet spot. This method can be used to study the interactions between bacterial communities and biofilms spotted adjacent to each other. Furthermore, it can be extended to study the interactions between different bacterial communities and an underlying epithelial cell layer.
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Quorum Sensing in Gram-Positive Bacteria: Assay Protocols for Staphylococcal , and Enterococcal , Sy |
Akane Shojima,Jiro Nakayama |
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Abstract
A thiolactone/lactone peptide-mediated quorum sensing (QS) system is commonly employed in gram-positive bacteria to control the expression of a variety of phenotypes, including the production of virulence factors and biofilm formation. Here, we describe assay protocols for the well-studied QS systems (. and .) of two representative gram-positive pathogens, . and .. These convenient assay systems are useful for the screening of QS inhibitors as well as for basic research to address the mechanism of these QS systems.
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Advanced Techniques for In Situ Analysis of the Biofilm Matrix (Structure, Composition, Dynamics) by |
Thomas R. Neu,John R. Lawrence |
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Abstract
The extracellular constituents in bioaggregates and biofilms can be imaged four dimensionally by using laser scanning microscopy. In this protocol we provide guidance on how to examine the various extracellular compartments in between microbial cells and communities associated with interfaces. The current options for fluorescence staining of matrix compounds and extracellular microhabitats are presented. Furthermore, practical aspects are discussed and useful notes are added. The chapter ends with a brief introduction to other approaches for EPS analysis and an outlook for future needs.
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Multiplex Fluorescence In Situ Hybridization (M-FISH) and Confocal Laser Scanning Microscopy (CLSM) |
Lamprini Karygianni,Elmar Hellwig,Ali Al-Ahmad |
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Abstract
Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (. spp., .) and late colonizers (., . spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images.
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Field Emission Scanning Electron Microscopy of Biofilm-Growing Bacteria Involved in Nosocomial Infec |
Claudia Vuotto,Gianfranco Donelli |
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Abstract
Scanning electron microscopy (SEM) provides useful information on the shape, size, and localization within the biofilm of single bacteria as well as on the steps of biofilm formation process, on bacterial interactions, and on production of extracellular polymeric substances..When biofilms are constituted by microbial species involved in health care-associated infections, information provided by SEM can be fruitfully used not only for basic researches but also for diagnostic purposes..The protocols currently used in our laboratory for biofilm investigation by SEM are reported here. Particularly, the procedures to fix, dehydrate, and metalize in vitro-developed biofilms or ex vivo clinical specimens colonized by biofilm-growing microorganisms are described as well as the advantages of the observation of these samples by field emission scanning electron microscopy.
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Experimental Approaches to Investigating the Vaginal Biofilm Microbiome |
Marc M. Baum,Manjula Gunawardana,Paul Webster |
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Abstract
Unraveling the complex ecology of the vaginal biofilm microbiome relies on a number of complementary techniques. Here, we describe the experimental approaches for studying vaginal microbial biofilm samples with a focus on specimen preparation for subsequent analysis. The techniques include fluorescence microscopy, fluorescence in situ hybridization, and scanning and transmission electron microscopy. Isolation of microbial DNA and RNA from these samples is covered along with a brief discussion of chemical analysis methods.
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Imaging Bacteria and Biofilms on Hardware and Periprosthetic Tissue in Orthopedic Infections |
Laura Nistico,Luanne Hall-Stoodley,Paul Stoodley |
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Abstract
Infection is a major complication of total joint arthroplasty (TJA) surgery, and even though it is now as low as 1 % in some hospitals, the increasing number of primary surgeries translates to tens of thousands of revisions due to prosthetic joint infection (PJI). In many cases the only solution is revision surgery in which the hardware is removed. This process is extremely long and painful for patients and is a considerable financial burden for the health-care system. A significant proportion of the difficulties in diagnosis and treatment of PJI are associated with biofilm formation where bacteria attach to the surface of the prosthesis and periprosthetic tissue and build a 3-D biofilm community encased in an extracellular polymeric slime (EPS) matrix. Bacteria in biofilms have a low metabolic rate which is thought to be a major contributor to their recalcitrance to antibiotic treatment. The diagnosis of biofilm infections is difficult due to the fact that bacteria in biofilms are not readily cultured with standard clinical microbiology techniques. To identify and visualize in situ biofilm bacteria in orthopedic samples, we have developed protocols for the collection of samples in
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Animal Models to Evaluate Bacterial Biofilm Development |
Kim Thomsen,Hannah Trøstrup,Claus Moser |
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Abstract
Medical biofilms have attracted substantial attention especially in the past decade. Animal models are contributing significantly to understand the pathogenesis of medical biofilms. In addition, animal models are an essential tool in testing the hypothesis generated from clinical observations in patients and preclinical testing of agents showing in vitro antibiofilm effect. Here, we describe three animal models — two non-foreign body . biofilm models and a foreign body . model.
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Animal Models to Investigate Fungal Biofilm Formation |
Jyotsna Chandra,Eric Pearlman,Mahmoud A. Ghannoum |
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Abstract
Microbial biofilms play an essential role in several infectious diseases and are defined as extensive communities of sessile organisms irreversibly associated with a surface, encased within a polysaccharide-rich extracellular matrix (ECM), and exhibiting enhanced resistance to antimicrobial drugs. Forming a biofilm provides the microbes protection from environmental stresses due to contaminants, nutritional depletion, or imbalances, but is dangerous to human health due to their inherent robustness and elevated resistance..The use of indwelling medical devices (e.g., central venous catheters, CVCs) in current therapeutic practice is associated with 80–90 % of hospital-acquired bloodstream and deep tissue infections. Most cases of catheter-related bloodstream infections (CRBSIs) involve colonization of microorganisms on catheter surfaces where they form a biofilm. Additionally, . and . were the causative organisms of the 2005/2006 outbreak of contact lens-associated fungal keratitis in the United States, Europe, the UK, and Singapore, and these infections involved formation of biofilms on contact lens. Fungal biofilm formation is studied using a number of techniques, involving the us
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Nonmammalian Model Systems to Investigate Fungal Biofilms |
Marios Arvanitis,Beth Burgwyn Fuchs,Eleftherios Mylonakis |
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Abstract
Medical advances have resulted in an increase in the number of patients in immunocompromised states, vulnerable to infection, or individuals fitted with medical devices that form niches for microbial infections. These infections are difficult to treat and have significant morbidity and mortality rates. An important factor in the pathogenesis of fungal diseases is the development of biofilm-forming communities, enabling the invasion of host tissues and resistance to antimicrobial compounds. To investigate the genetic requirements for filamentation and seek compounds that inhibit the process, invertebrate hosts are employed as models of in vivo infection. The purpose of our review is to highlight methods that can be utilized to investigate fungal filamentation, an important step in the development of biofilms, in the invertebrate hosts ., and ..
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Abstract
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Microbiological Methods for Target-Oriented Screening of Biofilm Inhibitors |
Livia Leoni,Paolo Landini |
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Abstract
The ability of many pathogenic bacteria to grow as a biofilm results in lower susceptibility to antibiotic treatments and to the host immune response, thus leading to the development of chronic infections. The understanding that biofilms can play an important role in bacterial virulence has prompted the search for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we present two examples of target-based microbiological screenings for antimicrobials endowed with anti-biofilm activity, aimed respectively at the inhibition of the signal molecule cyclic di-GMP and of quorum sensing.
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In Vitro Screening of Antifungal Compounds Able to Counteract Biofilm Development |
Marion Girardot,Christine Imbert |
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Abstract
Fungi are able to grow as a single-species or a more complex biofilm attached to inert surfaces (catheters…) or tissues (lung…). This last form is a microbial niche which must be considered as a major risk factor of developing a human fungal infection. Nowadays, only a few therapeutic agents have been shown to be active against fungal biofilms in vitro and/or in vivo. So there is a real need to find new anti-biofilm molecules. Here we describe in detail some rapid, 96-well microtiter plate-based methods, for the screening of compounds with anti-biofilm activity against . spp. yeasts. Two approaches will be considered: prophylactic or curative effects of the tested compounds by producing biofilms on two supports – polystyrene well surfaces and catheter sections.
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Biofilm Matrix-Degrading Enzymes |
Jeffrey B. Kaplan |
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Abstract
Polymeric substances such as proteins, polysaccharides, and DNA constitute a major component of the biofilm matrix. Enzymes that depolymerize and degrade these components are useful tools for investigating the composition and function of the biofilm matrix. This chapter provides a brief overview of the most commonly used biofilm matrix-degrading enzymes and presents examples of their applications in biofilm research.
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Efficacy Evaluation of Antimicrobial Drug-Releasing Polymer Matrices |
Iolanda Francolini,Antonella Piozzi,Gianfranco Donelli |
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Abstract
To assay in vitro antimicrobial activity of substances such as antibiotics or antiseptics, standard methods both in liquid and on solid media are available. These procedures cannot be adequate for testing antimicrobial-releasing or biocidal polymer systems..This chapter is focused on the description of methods that the authors have developed to evaluate the antimicrobial activity of either antimicrobial agent-releasing polymers or biocidal polymers. These assays can be applied to different types of water-soluble or insoluble polymer matrices.
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Antibiotic Polymeric Nanoparticles for Biofilm-Associated Infection Therapy |
Wean Sin Cheow,Kunn Hadinoto |
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Abstract
Polymeric nanoparticles are highly attractive as drug delivery vehicles due to their high structural integrity, stability during storage, ease of preparation and functionalization, and controlled release capability. Similarly, lipid–polymer hybrid nanoparticles, which retain the benefits of polymeric nanoparticles plus the enhanced biocompatibility and prolonged circulation time owed to the lipids, have recently emerged as a superior alternative to polymeric nanoparticles. Drug nanoparticle complex prepared by electrostatic interaction of oppositely charged drug and polyelectrolytes represents another type of polymeric nanoparticle. This chapter details the preparation, characterization, and antibiofilm efficacy testing of antibiotic-loaded polymeric and hybrid nanoparticles and antibiotic nanoparticle complex.
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Pharmacokinetics and Pharmacodynamics of Antibiotics in Biofilm Infections of , In Vitro and In Vivo |
Wang Hengzhuang,Niels Høiby,Oana Ciofu |
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Abstract
Although progress on biofilm research has been obtained during the past decades, the treatment of biofilm infections with antibiotics remains a riddle. The pharmacokinetic (PK) and pharmacodynamic (PD) profiles of an antimicrobial agent provide important information helping to establish an efficient dosing regimen and to minimize the development of antimicrobial tolerance and resistance in biofilm infections. Unfortunately, most previous PK/PD studies of antibiotics have been done on planktonic cells, and extrapolation of the results on biofilms is problematic as bacterial biofilms differ from planktonic grown cells in the growth rate, gene expression, and metabolism. Here, we set up several protocols for the studies of PK/PD of antibiotics in biofilm infections of . in vitro and in vivo. It should be underlined that none of the protocols in biofilms have yet been certificated for clinical use or proved useful for guidance of antibiotic therapy.
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发表于 2025-3-21 19:11:07
