scholarly journals Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

2019 ◽  
Author(s):  
Georgina Benn ◽  
Alice L. B. Pyne ◽  
Maxim G. Ryadnov ◽  
Bart W Hoogenboom
Keyword(s):  
Cell Envelope ◽  
Bacterial Cells ◽  
Label Free ◽  
Bacterial Surface ◽  
Force Microscopy ◽  
Correlative Imaging ◽  
Atomic Force ◽  
Live Bacteria ◽  
Surface Functionalisation ◽  
Bacterial Adsorption

AbstractAtomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-L-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane.

scholarly journals Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy

2008 ◽  
Vol 190 (12) ◽  
pp. 4225-4232 ◽  
Author(s):  
Virginia Vadillo-Rodriguez ◽  
Terry J. Beveridge ◽  
John R. Dutcher
Keyword(s):  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Cell Surface ◽  
Cell Envelope ◽  
Compressive Force ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cell Surface Structure

ABSTRACT The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium, Pseudomonas aeruginosa PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant, k 1, and an effective time constant, τ, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in k 1 together with a significant decrease in τ. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.

Atomic Force Microscopy Studies of the Interaction of Antimicrobial Peptides with Bacterial Cells

2017 ◽  
Vol 70 (2) ◽  
pp. 130 ◽  
Author(s):  
Anna Mularski ◽  
Frances Separovic
Keyword(s):  
Atomic Force Microscopy ◽  
Antimicrobial Peptides ◽  
Turgor Pressure ◽  
Living Cells ◽  
Bacterial Cells ◽  
Diverse Range ◽  
Bacterial Surface ◽  
Force Microscopy ◽  
Atomic Force ◽  
Conventional Antibiotics

Antimicrobial peptides (AMPs) are promising therapeutic alternatives to conventional antibiotics. Many AMPs are membrane-active but their mode of action in killing bacteria or in inhibiting their growth remains elusive. Recent studies indicate the mechanism of action depends on peptide structure and lipid components of the bacterial cell membrane. Owing to the complexity of working with living cells, most of these studies have been conducted with synthetic membrane systems, which neglect the possible role of bacterial surface structures in these interactions. In recent years, atomic force microscopy has been utilized to study a diverse range of biological systems under non-destructive, physiologically relevant conditions that yield in situ biophysical measurements of living cells. This approach has been applied to the study of AMP interaction with bacterial cells, generating data that describe how the peptides modulate various biophysical behaviours of individual bacteria, including the turgor pressure, cell wall elasticity, bacterial capsule thickness, and organization of bacterial adhesins.

scholarly journals Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

2019 ◽  
Author(s):  
Dennis J Doorduijn ◽  
Bart W Bardoel ◽  
Dani AC Heesterbeek ◽  
Maartje Ruyken ◽  
Georgina Benn ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Cell Envelope ◽  
Membrane Attack Complex ◽  
Bacterial Killing ◽  
Bacterial Surface ◽  
Force Microscopy ◽  
Bacterial Cell Envelope ◽  
Atomic Force ◽  
Multiple Copies ◽  
Local Assembly

AbstractAn important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

scholarly journals Nano- and Macroscale Imaging of Cholesterol Linoleate and Human Beta Defensin 2-Induced Changes in Pseudomonas aeruginosa Biofilms

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1279
Author(s):  
Brent A. Beadell ◽  
Andy Chieng ◽  
Kevin R. Parducho ◽  
Zhipeng Dai ◽  
Sam On Ho ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Cells ◽  
Biofilm Inhibition ◽  
Bacterial Surface ◽  
Force Microscopy ◽  
Novel Treatments ◽  
Atomic Force ◽  
Biofilm Production ◽  
Extracellular Structures ◽  
Induced Changes

The biofilm production of Pseudomonas aeruginosa (PA) is central to establishing chronic infection in the airways in cystic fibrosis. Epithelial cells secrete an array of innate immune factors, including antimicrobial proteins and lipids, such as human beta defensin 2 (HBD2) and cholesteryl lineolate (CL), respectively, to combat colonization by pathogens. We have recently shown that HBD2 inhibits biofilm production by PA, possibly linked to interference with the transport of biofilm precursors. Considering that both HBD2 and CL are increased in airway fluids during infection, we hypothesized that CL synergizes with HBD2 in biofilm inhibition. CL was formulated in phospholipid-based liposomes (CL-PL). As measured by atomic force microscopy of single bacteria, CL-PL alone and in combination with HBD2 significantly increased bacterial surface roughness. Additionally, extracellular structures emanated from untreated bacterial cells, but not from cells treated with CL-PL and HBD2 alone and in combination. Crystal violet staining of the biofilm revealed that CL-PL combined with HBD2 effected a significant decrease of biofilm mass and increased the number of larger biofilm particles consistent with altered cohesion of formed biofilms. These data suggest that CL and HBD2 affect PA biofilm formation at the single cell and community-wide level and that the community-wide effects of CL are enhanced by HBD2. This research may inform future novel treatments for recalcitrant infections in the airways of CF patients.

scholarly journals 3D phonon microscopy with sub-micron axial-resolution

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Richard J. Smith ◽  
Fernando Pérez-Cota ◽  
Leonel Marques ◽  
Matt Clark
Keyword(s):  
Signal To Noise Ratio ◽  
Single Cells ◽  
Optical Resolution ◽  
Acquisition Time ◽  
Label Free ◽  
Axial Resolution ◽  
Force Microscopy ◽  
Atomic Force ◽  
Accuracy And Precision ◽  
Good Agreement

AbstractBrillouin light scattering (BLS) is an emerging method for cell imaging and characterisation. It allows elasticity-related contrast, optical resolution and label-free operation. Phonon microscopy detects BLS from laser generated coherent phonon fields to offer an attractive route for imaging since, at GHz frequencies, the phonon wavelength is sub-optical. Using phonon fields to image single cells is challenging as the signal to noise ratio and acquisition time are often poor. However, recent advances in the instrumentation have enabled imaging of fixed and living cells. This work presents the first experimental characterisation of phonon-based axial resolution provided by the response to a sharp edge. The obtained axial resolution is up to 10 times higher than that of the optical system used to take the measurements. Validation of the results are obtained with various polymer objects, which are in good agreement with those obtained using atomic force microscopy. Edge localisation, and hence profilometry, of a phantom boundary is measured with accuracy and precision of approximately 60 nm and 100 nm respectively. Finally, 3D imaging of fixed cells in culture medium is demonstrated.

scholarly journals Effect of glutaraldehyde fixation on bacterial cells observed by atomic force microscopy

Scanning ◽  
2011 ◽  
Vol 34 (1) ◽  
pp. 6-11 ◽  
Author(s):  
Bao You Liu ◽  
Guang Min Zhang ◽  
Xue Ling Li ◽  
Heng Chen
Keyword(s):  
Atomic Force Microscopy ◽  
Bacterial Cells ◽  
Glutaraldehyde Fixation ◽  
Force Microscopy ◽  
Atomic Force

Atomic Force Microscopy Measurement of Heterogeneity in Bacterial Surface Hydrophobicity

Langmuir ◽  
2008 ◽  
Vol 24 (9) ◽  
pp. 4944-4951 ◽  
Author(s):  
Loredana S. Dorobantu ◽  
Subir Bhattacharjee ◽  
Julia M. Foght ◽  
Murray R. Gray
Keyword(s):  
Atomic Force Microscopy ◽  
Surface Hydrophobicity ◽  
Atomic Force Microscopy Measurement ◽  
Bacterial Surface ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Measurement

scholarly journals Intragenic Antimicrobial Peptide Hs02 Hampers the Proliferation of Single- and Dual-Species Biofilms of P. aeruginosa and S. aureus: A Promising Agent for Mitigation of Biofilm-Associated Infections

2019 ◽  
Vol 20 (14) ◽  
pp. 3604 ◽  
Author(s):  
Lucinda J. Bessa ◽  
Julia R. Manickchand ◽  
Peter Eaton ◽  
José Roberto S. A. Leite ◽  
Guilherme D. Brand ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Laser Scanning ◽  
Multidrug Resistant ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Bacterial Cells ◽  
Generalized Polarization ◽  
Force Microscopy ◽  
Atomic Force

Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in a large variety of infections. Their co-occurrence in the same site of infection has been frequently reported and is linked to enhanced virulence and difficulty of treatment. Herein, the antimicrobial and antibiofilm activities of an intragenic antimicrobial peptide (IAP), named Hs02, which was uncovered from the human unconventional myosin 1H protein, were investigated against several P. aeruginosa and S. aureus strains, including multidrug-resistant (MDR) isolates. The antibiofilm activity was evaluated on single- and dual-species biofilms of P. aeruginosa and S. aureus. Moreover, the effect of peptide Hs02 on the membrane fluidity of the strains was assessed through Laurdan generalized polarization (GP). Minimum inhibitory concentration (MIC) values of peptide Hs02 ranged from 2 to 16 μg/mL against all strains and MDR isolates. Though Hs02 was not able to hamper biofilm formation by some strains at sub-MIC values, it clearly affected 24 h preformed biofilms, especially by reducing the viability of the bacterial cells within the single- and dual-species biofilms, as shown by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) images. Laurdan GP values showed that Hs02 induces membrane rigidification in both P. aeruginosa and S. aureus. Peptide Hs02 can potentially be a lead for further improvement as an antibiofilm agent.

scholarly journals Atomic force microscopy recognition of protein A onStaphylococcus aureuscell surfaces by labelling with IgG–Au conjugates

2013 ◽  
Vol 4 ◽  
pp. 743-749 ◽  
Author(s):  
Elena B Tatlybaeva ◽  
Hike N Nikiyan ◽  
Alexey S Vasilchenko ◽  
Dmitri G Deryabin
Keyword(s):  
Atomic Force Microscopy ◽  
Protein A ◽  
Bacterial Cells ◽  
Force Microscopy ◽  
Selective Labelling ◽  
Atomic Force ◽  
Novel Approach ◽  
Preferential Binding ◽  
Igg Receptors ◽  
Labelling Method

The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces ofStaphylococcus aureusby using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM). The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG) allowed the visualization, localization and distribution of protein A–IgG complexes on the surface ofS. aureus. The selectivity of the labelling method was confirmed in mixtures ofS. aureuswithBacillus licheniformiscells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG–Au conjugates toS. aureuswas obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations.

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