Mechanical properties of the superficial biofilm layer determine the architecture of biofilms

© 2016 The Royal Society of Chemistry. Cells in biofilms sense and interact with their environment through the extracellular matrix. The physicochemical properties of the matrix, particularly at the biofilm-environment interface, determine how cells respond to changing conditions. In this study we describe the application of atomic force microscopy and confocal imaging to probe in situ the mechanical properties of these interfacial regions and to elucidate how key matrix components can contribute to the physical sensing by the cells. We describe how the Young's modulus of microcolonies differs according to the size and morphology of microcolonies, as well as the flow rate. The Young's modulus increased as a function of microcolony diameter, which was correlated with the production of the polysaccharide Psl at later stages of maturation for hemispherical or mushroom shaped microcolonies. The Young's modulus of the periphery of the biofilm colony was however independent of the hydrodynamic shear. The morphology of the microcolonies also influenced interfacial or peripheral stiffness. Microcolonies with a diffuse morphology had a lower Young's modulus than isolated, circular ones and this phenomenon was due to a deficiency of Psl. In this way, changes in the specific polysaccharide components imbue the biofilm with distinct physical properties that may modulate the way in which bacteria perceive or respond to their environment. Further, the physical properties of the polysaccharides are closely linked to the specific architectures formed by the developing biofilm

Mechanical signatures of microbial biofilms in micropillar-embedded growth chambers

Biofilms are surface-attached communities of microorganisms embedded in an extracellular matrix and are essential for the cycling of organic matter in natural and engineered environments. They are also the leading cause of many infections, for example, those associated with chronic wounds and implanted medical devices. The extracellular matrix is a key biofilm component that determines its architecture and defines its physical properties. Herein, we used growth chambers embedded with micropillars to study the net mechanical forces (differential pressure) exerted during biofilm formation in situ. Pressure from the biofilm is transferred to the micropillars via the extracellular matrix, and reduction of major matrix components decreases the magnitude of micropillar deflections. The spatial arrangement of micropillar deflections caused by pressure differences in the different biofilm strains may potentially be used as mechanical signatures for biofilm characterization. Hence, we submit that micropillar-embedded growth chambers provide insights into the mechanical properties and dynamics of the biofilm and its matrix.Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology (SMART)

Weak acids as an alternative anti-microbial therapy

Weak acids such as acetic acid and N-acetyl cysteine (NAC) at pH less than their pKa can effectively eradicate biofilms due to their ability to penetrate the biofilm matrix and the cell membrane. However, the optimum conditions for their activity against drug resistant strains, and safety, need to be understood for their application to treat infections or to inactivate biofilms on hard surfaces. Here, we investigate the efficacy and optimum conditions at which weak acids can eradicate biofilms. We compared the efficacy of various mono and triprotic weak acids such as N-acetyl cysteine (NAC), acetic acid, formic acid and citric acid, in eradicating biofilms. We found that monoprotic weak acids/acid drugs can kill mucoid P. aeruginosa mucA biofilm bacteria provided the pH is less than their pKa, demonstrating that the extracellular biofilm matrix does not protect the bacteria from the activity of the weak acids. Triprotic acids, such as citric acid, kill biofilm bacteria at pH ​< ​pKa1. However, at a pH between pKa1 and pKa2, citric acid is effective in killing the bacteria at the core of biofilm microcolonies but does not kill the bacteria on the periphery. The efficacy of a monoprotic weak acid (NAC) and triprotic weak acid (citric acid) were tested on biofilms formed by Klebsiella pneumoniae KP1, Pseudomonas putida OUS82, Staphylococcus aureus 15981, P. aeruginosa DK1-NH57388A, a mucoid cystic fibrosis isolate and P. aeruginosa PA_D25, an antibiotic resistant strain. We showed that weak acids have a broad spectrum of activity against a wide range of bacteria, including antibiotic resistant bacteria. Further, we showed that a weak acid drug, NAC, can kill bacteria without being toxic to human cells, if its pH is maintained close to its pKa. Thus weak acids/weak acid drugs target antibiotic resistant bacteria and eradicate the persister cells in biofilms which are tolerant to other conventional methods of biofilm eradication

Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA–HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ~2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.Singapore-MIT Alliance for Research and Technolog

Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn=3200 g mol-1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics simulations (MDS), Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (&#62;0.5%) inhibited biofilm formation, demonstrating a significant reduction in both biomass and biofilm height (17.8 vs. 5.5 µm; P &#60;0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of sugar residues, and extracellular (e)DNA (P &#60;0.05) with a corresponding increase in nanoparticle diffusion (P&#60;0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MDS. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections

Viscoelasticity of entangled λ -phage DNA solutions

10.1063/1.3009249Journal of Chemical Physics12918-JCPS

Mechanical signatures of microbial biofilms in micropillar-embedded growth chambers

© 2016 The Royal Society of Chemistry. Biofilms are surface-attached communities of microorganisms embedded in an extracellular matrix and are essential for the cycling of organic matter in natural and engineered environments. They are also the leading cause of many infections, for example, those associated with chronic wounds and implanted medical devices. The extracellular matrix is a key biofilm component that determines its architecture and defines its physical properties. Herein, we used growth chambers embedded with micropillars to study the net mechanical forces (differential pressure) exerted during biofilm formation in situ. Pressure from the biofilm is transferred to the micropillars via the extracellular matrix, and reduction of major matrix components decreases the magnitude of micropillar deflections. The spatial arrangement of micropillar deflections caused by pressure differences in the different biofilm strains may potentially be used as mechanical signatures for biofilm characterization. Hence, we submit that micropillar-embedded growth chambers provide insights into the mechanical properties and dynamics of the biofilm and its matrix

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

The biofilm matrix, with its diversity of extracellular polymeric substances (EPS), remains a poorly understood entity. It consists of a heterogeneous, multifunctional microenvironment that imparts a range of emergent properties to the biofilm, including social cooperation and resource sharing, adaptation to environmental changes, and resistance to harmful chemicals and antibiotics. Generally, studies of the biofilm matrix focus on the regulation of EPS gene expression and associated biofilm phenotypes (Flemming et al., 2022; Flemming & Wingender, 2010). For example, the differential regulation of exopolymers, which impart different mechanical properties, is an often-studied genetic marker for characterizing transitions between different stages of biofilm development (Chew, Kundukad, et al., 2014; Irie et al., 2012). New insights, however, suggest that the emergent properties of the matrix, which arise because of physical interactions between EPS molecules as well as those between EPS and bacterial cells, also play important roles in biofilm formation and organization (Liu et al., 2022; Rubinstein et al., 2012). For example, the secretion and accumulation of EPS components generate new physical forces, such as osmotic stresses, bridging interactions, and depletion effects within the crowded matrix (Liu et al., 2022). These forces alter the physical environment of the biofilm, affecting conformational and aggregational landscapes and dynamics, and thus functions, of matrix biopolymers. Together with the biological program, they stabilize extended structural, compositional, and morphological gradients in space and time, drive phase transitions, immobilize cells, and induce phase separation, creating spatial functional niches within the matrix (Worlitzer et al., 2022). Considering these insights, we highlight here the emerging perspective that understanding the competition and the collaboration between physical and biological factors is crucial for a more complete appreciation of biofilm formation, dynamics, organization, and function.Nanyang Technological UniversityNational Research Foundation (NRF)Published versionThe authors acknowledge funding grants fromNanyang Technological University, National Research Foundation Singapore and National University ofSingapore for conducting the research

Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides

10.1128/mBio.01536-14mBio541-No