Quantification of Adhesion Force of Bacteria on the Surface of

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Quantification of Adhesion Force of Bacteria on the Surface of Biomaterials: Techniques and Assays Fahad Alam, Shanmugam Kumar, and Kartik M Varadarajan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00213 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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ACS Biomaterials Science & Engineering

Quantification of Adhesion Force of Bacteria on the Surface of Biomaterials: Techniques and Assays Fahad Alama,b, Shanmugam Kumarb*, Kartik M. Varadarajanc,d aBiomaterials

Processing and Characterization Laboratory, Materials Science and

Engineering, Indian Institute of Technology Kanpur, UP, India bDepartment

of Mechanical and Materials Engineering, Khalifa University of Science and

Technology, Masdar Institute, Masdar City, P.O. Box 54224, Abu Dhabi, UAE. cDepartment

of Orthopaedic Surgery, Harvard Medical School, A-111, 25 Shattuck Street, Boston, USA

dDepartment

of Orthopaedic Surgery, Harris Orthopaedics Laboratory, Massachusetts General Hospital, 55 Fruit St, Boston, USA

*Corresponding Author. Tel: +971 28109239. Email: [email protected] (S. Kumar)

Abstract Biomaterials associated infection (BAI) has been identified to be one of the leading causes of failure of bio-implants. Failed implant requires revision surgery which is about twenty times costlier and more painful than primary surgery. Infection starts from initial attachment of bacteria onto the surface of biomaterials followed by colonization and biofilm formation. Once a biofilm is developed the bacteria become resistant towards antibiotics. On account of microbial cell development, their metabolic activity and viability get strongly affected by the adhesion. Hence a thorough investigation warrants an in-depth understanding of the interfacial adhesion. Several methods such as plate-and-wash assay, spinning–disc assay, centrifugation assay, step-pressure technique, optical tweezers, atomic force microscopy (AFM) and nanoindentation are used for the measurement of the bacterial adhesion. Most of the aforementioned techniques are non-quantitative and only provide approximate values of adhesion forces. Techniques such as AFM and nanoindentation can quantify a wide range of force 10 pN-1µN and 1nN–10µN respectively, and hence they are particularly used for exact quantification of the adhesion force as well as adhesion strength of bacterial cells on various surfaces of biomaterials. In this review, we present a comparative study of the techniques

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Prior to initial attachment, the bacteria stay in the planktonic state and are sensitive towards antibiotics and can easily be killed by antibiotics23. Soon after the initial attachment, the bacteria enter the interaction state where they become less sensitive to antibiotics and gradually develop resistance towards the same17. Prolonged adhesion of bacteria on the surfaces results in the formation of stable and mature biofilm18-19. The biofilm provides protection for bacterium from antibiotics, disinfectants, and dynamic environments20. Within the biofilm, the bacterium starts intercellular communications and rapidly stimulates the up- and downregulation of gene expression enabling temporal adaption such as phenotypic variation and the ability to survive in low nutrient conditions21-22. Almost all population of bacteria are reported to form a biofilm at various stages of growth, and they differ in composition and properties depending on the type of bacteria19, 23. Various techniques, as listed in Table 1, have been used to measure the adhesion force of cells (bacterial as well as mammalian cells) such as, plate-and-wash assay24, spinning–disc assay25, centrifugation assay26-28, buoyancy force technique

29,

micropipette suction (step

pressure technique)30-32, reflectance interference contrast microscopy 33, optical tweezers34-35, atomic force microscopy (AFM) and scratch based techniques such as nanoindentation and cyto-detacher36-41. Most of the aforementioned techniques are qualitative, with the exception of optical tweezers, AFM, cyto-detacher and nanoindentation, which provide a quantitative measure of interfacial forces between cell and biomaterial surface. In this review, we discuss the techniques that can be used to measure adhesion force between bacteria and surface of the biomaterial. Some of the highlighted methods have largely been used for the assessment of adhesion forces of mammalian cells, but they are included herein with the thought that these methods can be utilized for adhesion studies of bacterial cells as well. Among the different techniques available, few are quantitative whereas others are qualitative. Here, we have tried to categorize different techniques as quantitative and non-



quantitative based on their primary output metric. This review is expected to help the researchers newly entering into the area of bacterial adhesion and infection studies to select appropriate technique for the measurement of bacterial adhesion to various substrates. 2. Why is quantification of bacterial adhesion important? Bacterial infections represent one of the most significant challenges in modern medicine. For example, according to study conducted by Bozic K.J. et al., “total direct medical cost associated with revision total hip arthroplasty due to an infection are 2.8 times higher than that due to aseptic loosening and 4.8 times higher than the primary total hip arthroplasty” 42. Busscher and van der Mei (2012), hypothesized that bacterial growth on the surface of an artificial implant can cause infection and failure of the implant at different stages 17 (figure 2). Colonization that can occur on the surface of biomaterials can be classified into following three types depending on the stage of infection: I. II. III.

Pre-prosthetic infection Infection during operation Post-prosthetic infection

very early failure of the implant early failure of the implant Implant can withstand for longer time

Bacterial adhesion to the substrate has three different force regimes:


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Figure 2: Different regimes of bacterial adhesion on surfaces of the substrate that dictate the bacterial response towards the surface, adapted from Busscher and van der Mei 201217. Planktonic regime: It is free floating stage of the bacterial cells where the protective phenotype changes remains disabled and biofilm formation does not takes place leaving themselves susceptible to antimicrobials agents. It happens generally when bacterial cells don’t realize they are on the surface for examples the surface with polymer-brush coatings and hydrogels. Interaction regime: Bacterial cell in this stage realized the mating surface and thus enabling phenotypes changes, resulting in biofilm formation. Progressive phenotype change leads to increasing adhesion force and thus becoming more resistant to antimicrobial agents. Lethal regime De-activated adhesion: Very strong adhesion force prevails between the bacteria and the substrate. It happens when the surface of the substrate is positively charged, that leads to strong interaction and bacterial cells become sessile in nature. A positive charge



density of ~ 8.1015 per cm2 can kill 108 bacterial cells adhering per cm2. The positive charge density required to kill bacterial cell may be different for different bacterial specie. The series of events that take place when bacteria encounter the surface of the biomaterial are adhesion due to van der Waals forces, hydrophobicity, and electrostatic interaction, eventually followed by biofilm formation. Due to strong adhesion forces caused by opposite charges a mechanical stress is generated on the bacteria leading to de-activation state and hence they are called as de-activated adhesion. Most of the bacterial strains are negatively charged and therefore a very strong adhesion can be observed on the surfaces which are positively charged. Coating on surfaces which are known to kill the bacteria upon contact, such as quaternary ammonium coating, induces positive charges. Strong adhesion hinders the growth and causes death, usually termed as lethal. 3.1. Adhesion mechanism of bacteria to the surface of the substrate The physio-chemical interaction of bacteria to the surface of substrate has been explained by different models including Derjaguin–Landau–Verwey–Overbeek (DLVO) (Figure 3) theory43-45 , extended DLVO theory and thermodynamic theory. Lifshitz-Van der Waals forces and electrostatic (repulsive or attractive) forces in combination can be utilized to predict the overall interaction between bacterium and other mating surface (another bacterium or surface of a substrate) such as shown in the fig. 3. This theory, also called as DLVO theory. has been used as a qualitative model to calculate the free energy change involved in bacterial adhesion 46-47. At a very small bacteria-surface separation, there is a very low (deep) primary potential energy minimum which comes from the van der Waals attraction. An energy barrier created by electrostatic repulsion (electrostatic stabilization) prevents the bacteria from approaching this primary minimum. There is a secondary minimum before the energy barrier, which is responsible for the reversible adhesion due electrostatic attraction. If the energy barrier


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is strong enough, a bacterium trapped in the secondary minimum will remain there only and will not fall into the primary minimum (irreversible adhesion).

Figure 3: Schematic representing the DLVO theory of bacterial adhesion in different states, adapted from Faraudo et al., 2013 48. Plot shows potential energy as a function of distance from surface of the substrate. Initial attachment is an instantaneous process and bacterial cells are held weakly to a mating surface by physical attractive forces (van der Waals forces) of mass attraction and electrostatic forces (caused by ionic groups) interacting on or around the mating surfaces. The van der Waals forces involved in this instantaneous process are different from the strong van der Waals forces responsible for the primary minimum. The net force acting on a single bacterial cell on the flat surface is the sum of coulomb interaction and van der Waals interaction. In case of bacteria on a surface, using present thermodynamic model, the contact angle between the substrate and bacteria is measured and from Dupré equation49, adhesion state is measured with known interfacial energies. If free energy per unit area

) is negative as a result of

adhesion, i.e., when the interfacial energy of the bacterium and substrate ( liquid is smaller than the sum of bacteria and liquid (

), bacteria and

) and substrate and liquid interfaces (



) the adhesion will be favored. The adhesion process can be described in two different phases (phase I and phase II). Phase I of the adhesion process is time-dependent. Phase II starts after bacterial cells get attached to a surface resulting in irreversible adhesion. In phase II the bacteria get firmly attached to the surface due to synthesis of extracellular adhesive materials. DLVO theory does not account for the bacterial cell surface nature (bacterial cell surface charges and extracellular matrix) in the determination of bacterial interaction because the substrate is being considered as flat, however this is not the case all the time50-51. The DLVO theory does not explain the change in the bacterial adhesion when there is a variation on the substrate surface (e.g. surface roughness or surface wettability) or in the solution (e.g. electrolyte concentration)52. Moreover this theory is not adequate to explain the molecular interactions involved in the bacterial adhesion53-54. The second theoretical model that has been used to describe the bacterial adhesion to the surface is the thermodynamic model in which the overall attractive and repulsive forces are represented in terms of free energy50, 55. In this model the thermodynamic parameters such as free energies of bacterial surface, substratum surface and surface tension of solution are estimated to calculate the Gibbs energy of bacterial adhesion. According to this model if the bacterial adhesion results in negative free energy per unit surface then adhesion will be favored and spontaneous attachment will take place45, 49. The thermodynamic model also has certain limitations since it is an equilibrium model and it does not allow interpretation of bacterial kinetics56. Furthermore, the calculation of correct values of surface free energies are very difficult as bacterial surface possesses a complex chemistry and changes with in-vitro hydration. Considering the limitations of these DLVO and thermodynamic theories, an extended DLVO (XDLVO) theory has been suggested

46.

Along with the Lifshitz-Van der

Waals forces and electrostatic forces the acid-base interaction forces are also included in the XDLVO theory.


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Although the development of XDLVO theory helped to explain the bacterial adhesion better it still does not account for the biological nature of the bacterial surface in the adhesion. 53.

Therefore, experimental techniques for study of bacterial cell adhesion remain of critical

importance. 4. Techniques to study cell adhesion Numerous techniques have thus far been in use for the determination of cell (bacterial and mammalian) adhesion force and are summarized in Table 1. On the basis of output metric of these techniques, we have classified them as non-quantitative (plate-and-wash technique, spinning–disc assay, buoyancy force technique, reflectance interference contrast microscopy and centrifugation), semi-quantitative (RICM, Micropipette assay) and quantitative techniques (optical tweezers, step pressure technique, AFM, nanoindentation and cyto detacher). The techniques which measure cell adhesion in qualitative terms (percentage of cells that remain attached), are considered non-quantitative while those measuring true magnitude of adhesion force have been classified as quantitative. Table1: Techniques for measurement of bacterial adhesion forces Technique Plate-andwash assay Buoyancy force Centrifugatio n assay Spinning disk assay Flow chamber Well plate RICM Micropipette assay Membrane force probe Optical tweezers

Applied Force type Shear force

Force/press ure range -

-

-

Shear force

1-2000 pN

Shear force

1-350 Pa

Shear force

Output

Remarks

Ref.

Ratio of attached/detached cells

Non-quant.

57-58

Ratio of attached/ or detached cells Number of attached cells

Non-quant.

29, 59

Non-quant.

26, 28, 60

Non-quant.

25

1-30 Pa

Force at which 50% cell detached Ratio of attached/detached cells

Non-quant.

61

Gravitationa l force adhesion patches Pulling force

-

Number of attached cells

Non-quant.

62

~200 nN

adhesion patch analysis

33, 63

~1 nN

the hydrodynamic lifting force

Pulling force

(0.1–1 nN)

Detachment force

Pulling force

0.1 pN-100 pN

Detachment force

Semiquantitative Semiquantitative Semiquantitative Quantitative


31, 64

65

34, 66-67


AFM-SCFS Nanoindenter

Pulling force Lateral force

Cytodetacher

Shear force

10 pN-1µN 1 nNt-100 µN ~100 µN

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Detachment force Detachment force

Quantitative Quantitative

38, 68-71

Detachment force

Quantitative

72

41

4.1. Non-quantitative Techniques 4.1.1. Well-plate assay This technique has been mostly employed in study of mammalian cell. However, this method can be used for estimation of adhesion force of bacterial cell as well. Jason L et al., 201173 used this method to study the adhesion of Escherichia coli bacteria (a human pathogenic strain) to epithelial cells (HEp-2 human epithelial cell line) to see the bacterial interactions to the host mammalian cells. Similar study was done by Qiuyan et al., 201174, where they observed the adhesion behavior of E. coli to uroepithilial cell line and concluded that cranberry extract can help to reduce the adhesion and thus the extent of infection. This assay is based on the quantification of number of cells that lose the adhesion from a layer of adherent cells by utilizing the gravitational force. In this method the cells of the desired type (either primary or adherent cell line) are first allowed to grow on cell culture plate or onto the substrate followed by, seeding of different cell line, for which the adhesion force is being measured, to grow on the plate as a co-culture. The non-adherent cells are collected by putting well plate upside down and vital unbonded cells are quantified calorimetrically as described schematically in figure 4. The ratio of total cells seeded initially to the number of cells that remain attached after washing is counted 75. For better quantification of adhesion, the substrates can be coated with a protein such as collagens, which can provide better attachment of cells

76.

The adherent cells remain fixed and are stained with dye. Finally, the stain is

extracted and quantified calorimetrically77. The advantages of this method is low cost and easy to perform as compared to other adhesion assays. The quantification of bacterial cell count that lose the adhesion is performed by calorimetric methods so the contribution from the dead cells


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or debris can be avoided. The limitation of the current assay is that it is only applicable to initial adhesion because the force applied in the plate-and-wash assay is typically

Quantification of Adhesion Force of Bacteria on the Surface of

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