Showing papers on "Biofilm matrix published in 2017"

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria

Abstract: Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Understanding the mechanism of bacterial biofilms resistance to antimicrobial agents

Abstract: A biofilm is a group of microorganisms, that causes health problems for the patients with indwelling medical devices via attachment of cells to the surface matrix. It increases the resistance of a microorganism for antimicrobial agents and developed the human infection. Current strategies are removed or prevent the microbial colonies from the medical devices, which are attached to the surfaces. This will improve the clinical outcomes in favor of the patients suffering from serious infectious diseases. Moreover, the identification and inhibition of genes, which have the major role in biofilm formation, could be the effective approach for health care systems. In a current review article, we are highlighting the biofilm matrix and molecular mechanism of antimicrobial resistance in bacterial biofilms.

Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment

Abstract: Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. A limited number of antifungal agents from only a few drug classes are available to treat patients with these serious infections. Resistance can be either intrinsic or acquired. Resistance mechanisms are not exchanged between Candida; thus, acquired resistance either emerges in response to an antifungal selection pressure in the individual patient or, more rarely, occur due to horizontal transmission of resistant strains between patients. Although multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins, and polyenes have occurred in several Candida species, most notably Candida glabrata and more recently Candida auris. Drivers are overall antifungal use, subtherapeutic drug levels at sites of infection/colonization, drug sequestration in the biofilm matrix, and, in the setting of outbreaks, suboptimal infection control. Moreover, recent research suggests that DNA mismatch repair gene mutations may facilitate acquisition of resistance mutations in C. glabrata specifically. Diagnosis of antifungal-resistant Candida infections is critical to the successful management of patients with these infections. Reduction of unnecessary use of antifungals via antifungal stewardship is critical to limit multidrug resistance emergence.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process.

Abstract: Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

Candida Species Biofilms’ Antifungal Resistance

Abstract: Candida infections (candidiasis) are the most prevalent opportunistic fungal infection on humans and, as such, a major public health problem. In recent decades, candidiasis has been associated to Candida species other than Candida albicans. Moreover, biofilms have been considered the most prevalent growth form of Candida cells and a strong causative agent of the intensification of antifungal resistance. As yet, no specific resistance factor has been identified as the sole responsible for the increased recalcitrance to antifungal agents exhibited by biofilms. Instead, biofilm antifungal resistance is a complex multifactorial phenomenon, which still remains to be fully elucidated and understood. The different mechanisms, which may be responsible for the intrinsic resistance of Candida species biofilms, include the high density of cells within the biofilm, the growth and nutrient limitation, the effects of the biofilm matrix, the presence of persister cells, the antifungal resistance gene expression and the increase of sterols on the membrane of biofilm cells. Thus, this review intends to provide information on the recent advances about Candida species biofilm antifungal resistance and its implication on intensification of the candidiasis.

Advances in the microbial etiology and pathogenesis of early childhood caries.

Abstract: Early childhood caries (ECC) is one of the most prevalent infectious diseases affecting children worldwide. ECC is an aggressive form of dental caries, which, left untreated, can result in rapid and extensive cavitation in teeth (rampant caries) that is painful and costly to treat. Furthermore, it affects mostly children from impoverished backgrounds, and so constitutes a major challenge in public health. The disease is a prime example of the consequences arising from complex, dynamic interactions between microorganisms, host, and diet, leading to the establishment of highly pathogenic (cariogenic) biofilms. To date, there are no effective methods to identify those at risk of developing ECC or to control the disease in affected children. Recent advances in deep-sequencing technologies, novel imaging methods, and (meta)proteomics-metabolomics approaches provide an unparalleled potential to reveal new insights to illuminate our current understanding about the etiology and pathogenesis of the disease. In this concise review, we provide a broader perspective about the etiology and pathogenesis of ECC based on previous and current knowledge on biofilm matrix, microbial diversity, and host-microbe interactions, which could have direct implications for developing new approaches for improved risk assessment and prevention of this devastating and costly childhood health condition.

More than Enzymes That Make or Break Cyclic Di-GMP-Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli.

Abstract: The bacterial second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases (DGCs, with GGDEF domains) and specific phosphodiesterases (PDEs, with EAL or HD-GYP domains). Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is "local" c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of Escherichia coli Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or "supermodule" of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems.IMPORTANCE c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of E. coli, together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as "lonely players" without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of "local" c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.

Cleaning and Disinfection of Biofilms Composed of Listeria monocytogenes and Background Microbiota from Meat Processing Surfaces

Abstract: Surfaces of food processing premises are exposed to regular cleaning and disinfection (C&D) regimes, using biocides that are highly effective against bacteria growing as planktonic cells. However, bacteria growing in surface-associated communities (biofilms) are typically more tolerant toward C&D than their individual free-cell counterparts, and survival of pathogens such as Listeria monocytogenes may be affected by interspecies interactions within biofilms. In this study, Pseudomonas and Acinetobacter were the most frequently isolated genera surviving on conveyor belts subjected to C&D in meat processing plants. In the laboratory, Pseudomonas, Acinetobacter, and L. monocytogenes dominated the community, both in suspensions and in biofilms formed on conveyor belts, when cultures were inoculated with eleven-genus cocktails of representative bacterial strains from the identified background flora. When biofilms were exposed to daily C&D cycles mimicking treatments used in food industry, the levels of Acinetobacter and Pseudomonas mandelii diminished, and biofilms were instead dominated by Pseudomonas putida (65 to 76%), Pseudomonas fluorescens (11 to 15%) and L. monocytogenes (3 to 11%). The dominance of certain species after daily C&D correlated with high planktonic growth rates at 12°C and tolerance to C&D. In single-species biofilms, L. monocytogenes developed higher tolerance to C&D over time, for both the peracetic acid and quaternary ammonium disinfectants, indicating that a broad-spectrum mechanism was involved. Survival after C&D appeared to be a common property of L. monocytogenes strains, as persistent and sporadic subtypes showed equal survival rates in complex biofilms. Biofilms established preferentially in surface irregularities of conveyor belts, potentially constituting harborage sites for persistent contamination.IMPORTANCE In the food industry, efficient production hygiene is a key measure to avoid the accumulation of spoilage bacteria and eliminate pathogens. However, the persistence of bacteria is an enduring problem in food processing environments. This study demonstrated that environmental bacteria can survive foam cleaning and disinfection (C&D) at concentrations used in the industrial environment. The phenomenon was replicated in laboratory experiments. Important characteristics of persisting bacteria were a high growth rate at low temperature, a tolerance to the cleaning agent, and the ability to form biofilms. This study also supports other recent research suggesting that strain-to-strain variation cannot explain why certain subtypes of Listeria monocytogenes persist in food processing environments while others are found only sporadically. The present investigation highlights the failure of regular C&D and a need for research on improved agents that efficiently detach the biofilm matrix.

The Candida albicans Biofilm Matrix: Composition, Structure and Function

Abstract: A majority of infections caused by Candida albicans-the most frequent fungal pathogen-are associated with biofilm formation. A salient feature of C. albicans biofilms is the presence of the biofilm matrix. This matrix is composed of exopolymeric materials secreted by sessile cells within the biofilm, in which all classes of macromolecules are represented, and provides protection against environmental challenges. In this review, we summarize the knowledge accumulated during the last two decades on the composition, structure, and function of the C. albicans biofilm matrix. Knowledge of the matrix components, its structure, and function will help pave the way to novel strategies to combat C. albicans biofilm infections.

Hyaluronic Acid and Its Composites as a Local Antimicrobial/Antiadhesive Barrier.

Abstract: Living in biofilms is probably the most common condition for bacteria and fungi and biofilm-related infections account for the majority of bacterial infectious diseases worldwide. Among others biofilm-related infections, those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma, cardio-surgery and several other surgical disciplines. Given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms, new strategies are needed to protect implants and host tissues, overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix. Adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach. Among various polymers, tested as antibacterial coatings, hyaluronic acid and some of its composites do offer a well-established long-term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation. Aim of the present review is to summarize the available evidence concerning the antiadhesion/antibiofilm activity of hyaluronic acid and some of its derivatives to reduce/prevent bacterial adhesion and biofilm formation in various experimental and clinical settings.

Targeting microbial biofilms using Ficin, a nonspecific plant protease.

Abstract: Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24 hours treatment with Ficin at 10 μg/ml and six-fold at 1000 μg/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000 μg/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications.

Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production

Abstract: Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances, largely polysaccharides. Multiple types of extracellular polymeric substances can be produced by a single bacterial strain. The distinct polymer components of biofilms are known to provide chemical protection, but little is known about how distinct extracellular polysaccharides may also protect biofilms against mechanical stresses such as shear or phagocytic engulfment. Decades-long infections of Pseudomonas. aeruginosa biofilms in the lungs of cystic fibrosis patients are natural models for studies of biofilm fitness under pressure from antibiotics and the immune system. In cystic fibrosis infections, production of the extracellular polysaccharide alginate has long been known to increase with time and to chemically protect biofilms. More recently, it is being recognized that chronic cystic fibrosis infections also evolve to increase production of another extracellular polysaccharide, Psl; much less is known about Psl’s protective benefits to biofilms. We use oscillatory bulk rheology, on biofilms grown from longitudinal clinical isolates and from genetically-manipulated lab strains, to show that increased Psl stiffens biofilms and increases biofilm toughness, which is the energy cost to cause the biofilm to yield mechanically. Further, atomic force microscopy measurements reveal greater intercellular cohesion for higher Psl expression. Of the three types of extracellular polysaccharides produced by P. aeruginosa, only Psl increases the stiffness. Stiffening by Psl requires CdrA, a protein that binds to mannose groups on Psl and is a likely cross-linker for the Psl components of the biofilm matrix. We compare the elastic moduli of biofilms to the estimated stresses exerted by neutrophils during phagocytosis, and infer that increased Psl could confer a mechanical protection against phagocytic clearance. Bacteria in lungs of people with cystic fibrosis can evolve through decades to build a tough biofilm that resists the body’s defences. Vernita Gordon and colleagues at the University of Texas, with co-workers in Europe, examined biofilms cultured from lung samples taken from patients at intervals over many years. The infecting bacterial populations had steadily evolved to increase production of specific carbohydrate components of the biofilms. The researchers found that increasing production of one carbohydrate component strengthens the biofilms, most likely due to the carbohydrate being crosslinked by protein molecules. The investigation suggests that the mechanics of the carbohydrate-protein network protects the biofilms from being broken into smaller pieces that can be engulfed by defensive cells called phagocytes. This new insight into the evolution of mechanical toughness complements the previously observed evolution of increasing chemical protection. Understanding these processes will assist efforts to combat them.

Pathogenic factors of Pseudomonas aeruginosa - the role of biofilm in pathogenicity and as a target for phage therapy.

Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that can cause several acute and chronic infections in humans, and it has become an important cause of nosocomial infections and antibiotic resistance. Biofilm represents an important virulence factor for these bacteria, plays a role in P. aeruginosa infections and avoidance of immune defence mechanisms, and has the ability to protect the bacteria from antibiotics. Alginate, Psl and Pel, three exopolysaccharides, are the main components in biofilm matrix, with many biological functions attributed to them, especially with respect to the protection of the bacterial cell from antibiotics and the immune system. Pseudomonas infections, biofilm formation and development of resistance to antibiotics all require better understanding to achieve the best results using alternative treatment with phage therapy. This review describes the P. aeruginosa pathogenicity and virulence factors with a special focus on the biofilm and its role in infection and resistance to antibiotics and summarizes phage therapy as an alternative approach in treatment of P. aeruginosa infections.

Boronic Acid Functionalized Photosensitizers: A Strategy To Target the Surface of Bacteria and Implement Active Agents in Polymer Coatings.

Abstract: Advanced methods for preventing and controlling hospital-acquired infections via eradication of free-floating bacteria and bacterial biofilms are of great interest. In this regard, the attractiveness of unconventional treatment modalities such as antimicrobial photodynamic therapy (aPDT) continues to grow. This study investigated a new and innovative strategy for targeting polysaccharides found on the bacterial cell envelope and the biofilm matrix using the boronic acid functionalized and highly effective photosensitizer (PS) silicon(IV) phthalocyanine. This strategy has been found to be successful in treating planktonic cultures and biofilms of Gram-negative E. coli. An additional advantage of boronic acid functionality is a possibility to anchor the tailor made PS to poly(vinyl alcohol) and to fabricate a self-disinfecting coating.

Modulation of the mechanical properties of bacterial biofilms in response to environmental challenges

Abstract: Bacterial communities form biofilms on a wide range of surfaces by synthesizing a cohesive and protective extracellular matrix. The morphology, internal structure and mechanical stability of a biofilm are largely determined by its constituent polymers. In addition to mediating adhesion to surfaces, biofilms control the uptake of molecules and regulate the permeability of the matrix to gases and chemicals. Since biofilms can cause significant problems in both industrial and healthcare settings, there is great interest in developing strategies that either inhibit their formation or facilitate their elimination. However, although important in this context, the material properties of bacterial biofilms are poorly understood. In particular, little is known about how the different components of a biofilm matrix contribute to its various physical characteristics, or how these are modified in response to environmental cues. In this review, we present an overview of the molecular composition of different bacterial biofilms and describe techniques for the characterization of their viscoelastic properties. Finally, we summarize our current understanding of how the mechanical properties of bacterial biofilms are altered by different environmental challenges, and we discuss initial insights into the relationship between these responses and the composition of the matrix.

Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates

Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that can infect the lungs of cystic fibrosis (CF) patients and persist in the form of antibiotic-tolerant aggregates in the mucus. It has recently been suggested that such aggregates are formed due to restricted bacterial motility independent of the production of extracellular matrix components, and that they do not rely on an extracellular matrix for antimicrobial tolerance. However, we show here that biofilm matrix overexpression, as displayed by various clinical isolates, significantly protects P. aeruginosa aggregates against antimicrobial treatment. Alginate-overproducing mucA mutant bacteria growing in aggregates showed highly increased antibiotic tolerance compared to wild-type bacteria in aggregates. Deletion of algD in the mucA mutant strain abrogated alginate production and reversed the antibiotic tolerance displayed by the aggregates to a level similar to that observed for aggregates formed by the wild type. The P. aeruginosa ΔwspF and ΔyfiR mutant strains both overproduce Pel and Psl exopolysaccharide, and when these bacteria grew in aggregates, they showed highly increased antibiotic tolerance compared to wild-type bacteria growing in aggregates. However, the ΔwspF and ΔyfiR mutant strains, deficient in Pel/Psl production due to additional ΔpelA ΔpslBCD deletions, formed aggregates that displayed antibiotic tolerance levels close to those of wild-type aggregates. These results suggest that biofilm matrix components, such as alginate, Pel, and Psl, do play a role in the tolerance toward antimicrobials when bacteria grow as aggregates.

Phage “delay” towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages

Abstract: In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their "vigor" when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such "less-robust" mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically "running away" to more phage-free locations. Delay mechanisms-to the extent that they are less specific in terms of what phages are targeted-collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.

Curcumin Quantum Dots Mediated Degradation of Bacterial Biofilms.

Abstract: Bacterial biofilm has been reported to be associated with more than 80 percent of bacterial infections. Curcumin, a hydrophobic polyphenol compound, has anti-quorum sensing activity apart from having anti-microbial action. However, its use is limited by its poor aqueous solubility and rapid degradation. In this study, we attempted to prepare quantum dots of the drug curcumin in order to achieve enhanced solubility and stability and investigated for its antimicrobial and antibiofilm activity. We utilized a newer two-step bottom up wet milling approach to prepare Curcumin Quantum Dots (CurQDs) using acetone as a primary solvent. Minimum inhibitory concentration against select gram-positive and gram-negative bacteria was performed. The antibiofilm assay was performed at first using 96-well tissue culture plate and subsequently validated by Confocal Laser Scanning Microscopy. Further, biofilm matrix protein was isolated using formaldehyde sludge and TCA/Acetone precipitation method. Protein extracted was incubated with varying concentration of Curcumin quantum dots for 4 hrs and was subjected to SDS-PAGE. Molecular docking study was performed to observe interaction between curcumin and phenol soluble modulins as well as curli proteins. The biophysical evidences obtained from TEM, SEM, UV-VIS, fluorescence, Raman spectroscopy and zeta potential analysis confirmed the formation of curcumin quantum dots with increased stability and solubility. The MICs of curcumin quantum dots, as observed against both select gram positive and negative bacterial isolates, was observed to be significantly lower than native curcumin particles. On TCP assay, Curcumin observed to be having antibiofilm as well as biofilm degrading activity. Results of SDS-PAGE and molecular docking have shown interaction between biofilm matrix proteins and curcumin. The results indicate that aqueous solubility and stability of Curcumin can be achieved by preparing its quantum dots. The study also demonstrates that by sizing down the particle size has not only enhanced its antimicrobial properties but it has also shown its antibiofilm activities. Further, study is needed to elucidate the exact nature of interaction between curcumin and biofilm matrix proteins.

Flow environment and matrix structure interact to determine spatial competition in Pseudomonas aeruginosa biofilms.

Abstract: Bacteria often live in biofilms, which are microbial communities surrounded by a secreted extracellular matrix. Here, we demonstrate that hydrodynamic flow and matrix organization interact to shape competitive dynamics in Pseudomonas aeruginosa biofilms. Irrespective of initial frequency, in competition with matrix mutants, wild-type cells always increase in relative abundance in planar microfluidic devices under simple flow regimes. By contrast, in microenvironments with complex, irregular flow profiles - which are common in natural environments - wild-type matrix-producing and isogenic non-producing strains can coexist. This result stems from local obstruction of flow by wild-type matrix producers, which generates regions of near-zero shear that allow matrix mutants to locally accumulate. Our findings connect the evolutionary stability of matrix production with the hydrodynamics and spatial structure of the surrounding environment, providing a potential explanation for the variation in biofilm matrix secretion observed among bacteria in natural environments.

Raman microspectroscopy, surface-enhanced Raman scattering microspectroscopy, and stable-isotope Raman microspectroscopy for biofilm characterization

Abstract: Biofilms represent the predominant form of microbial life on our planet. These aggregates of microorganisms, which are embedded in a matrix formed by extracellular polymeric substances, may colonize nearly all interfaces. Detailed knowledge of microorganisms enclosed in biofilms as well as of the chemical composition, structure, and functions of the complex biofilm matrix and their changes at different stages of the biofilm formation and under various physical and chemical conditions is relevant in different fields. Important research topics include the development and improvement of antibiotics and medical devices and the optimization of biocides, antifouling strategies, and biological wastewater treatment. Raman microspectroscopy is a capable and nondestructive tool that can provide detailed two-dimensional and three-dimensional chemical information about biofilm constituents with the spatial resolution of an optical microscope and without interference from water. However, the sensitivity of Raman microspectroscopy is rather limited, which hampers the applicability of Raman microspectroscopy especially at low biomass concentrations. Fortunately, the resonance Raman effect as well as surface-enhanced Raman scattering can help to overcome this drawback. Furthermore, the combination of Raman microspectroscopy with other microscopic techniques, mass spectrometry techniques, or particularly with stable-isotope techniques can provide comprehensive information on monospecies and multispecies biofilms. Here, an overview of different Raman microspectroscopic techniques, including resonance Raman microspectroscopy and surface-enhanced Raman scattering microspectroscopy, for in situ detection, visualization, identification, and chemical characterization of biofilms is given, and the main feasibilities and limitations of these techniques in biofilm research are presented. Future possibilities of and challenges for Raman microspectroscopy alone and in combination with other analytical techniques for characterization of complex biofilm matrices are discussed in a critical review. Graphical Abstract Applicability of Raman microspectroscopy for biofilm analysis.

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

Abstract: Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

Bifunctionality of a biofilm matrix protein controlled by redox state.

Abstract: Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is critical to the success of biofilms in diverse habitats; however, many details of the composition, structure, and function remain enigmatic. Biofilms formed by the Gram-positive bacterium Bacillus subtilis depend on the production of the secreted film-forming protein BslA. Here, we show that a gradient of electron acceptor availability through the depth of the biofilm gives rise to two distinct functional roles for BslA and that these roles can be genetically separated through targeted amino acid substitutions. We establish that monomeric BslA is necessary and sufficient to give rise to complex biofilm architecture, whereas dimerization of BslA is required to render the community hydrophobic. Dimerization of BslA, mediated by disulfide bond formation, depends on two conserved cysteine residues located in the C-terminal region. Our findings demonstrate that bacteria have evolved multiple uses for limited elements in the matrix, allowing for alternative responses in a complex, changing environment.

Differences in internalization and growth of Escherichia coli O157:H7 within the apoplast of edible plants, spinach and lettuce, compared with the model species Nicotiana benthamiana

Abstract: Summary Internalization of food-borne bacteria into edible parts of fresh produce plants represents a serious health risk. Therefore, internalization of verocytotoxigenic E. coli O157:H7 isolate Sakai was assessed in two species associated with outbreaks, spinach (Spinacia oleracea) and lettuce (Lactuca sativa) and compared to the model species Nicotiana benthamiana. Internalization occurred in the leaves and roots of spinach and lettuce throughout a 10 day time-course. The plant species, tissue type and inoculum dose all impacted the outcome. A combination of low inoculum dose (~102 CFU) together with light microscopy imaging highlighted marked differences in the fate of endophytic E. coli O157:H7 Sakai. In the fresh produce species, bacterial growth was restricted but viable cells persisted over 20 days, whereas there was > 400-fold (~2.5 Log10) increase in growth in N. benthamiana. Colony formation occurred adjacent to epidermal cells and mesophyll cells or close to vascular bundles of N. benthamiana and contained components of a biofilm matrix, including curli expression and elicitation, extracellular DNA and a limited presence of cellulose. Together the data show that internalization is a relevant issue in crop production and that crop species and tissue need to be considered as food safety risk parameters.

A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis

Abstract: Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences. Gram-negative bacteria assemble biofilms from amyloid fibres, which translocate across the outer membrane as unfolded amyloid precursors through a secretion system. Here, the authors characterise the structural details of the amyloid transporter FapF in Pseudomonas.

Boronic Acid-Functionalized Photosensitizers: a Straightforward Strategy to Target the Sweet Site of Bacteria and Implement Active Agents in Polymer Coating

Abstract: Advanced methods for preventing and controlling hospital-acquired infections via eradication of free-floating bacteria and bacterial biofilms are of great interest. In this regard, the attractiveness of unconventional treatment modalities such as antimicrobial photodynamic therapy (aPDT) continues to grow. This study investigated a new and innovative strategy for targeting polysaccharides found on the bacterial cell envelope and the biofilm matrix using the boronic acid functionalized and highly effective photosensitizer (PS) silicon(IV) phthalocyanine. This strategy has been found to be successful in treating planktonic cultures and biofilms of Gram-negative E. coli. An additional advantage of boronic acid functionality is a possibility to anchor the tailor made PS to poly(vinyl alcohol) and to fabricate a self-disinfecting coating.

Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

Abstract: Background & objectives: Among cell surface proteins, biofilm-associated protein (Bap) promotes biofilm development in Staphylococcus aureus strains. The aim of this study was to investigate proteinase-mediated biofilm dispersion in different isolates of S. aureus. Methods: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA), before and after the proteinase K treatment. Results: Biofilm assay showed that 2 μg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352), but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain). Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 μg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. Interpretation & conclusions: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies againstS. aureus biofilms.