Measuring the Bacterial Adhesion

Bacterial Adhesion Determination Using a Micromanipulator in SEM

  • Fig. 1: Setup of Primary Adhesion cultivation experiment in a BIOSTAT B fermenter with sample holder (left) and E. coli (right) [8].Fig. 1: Setup of Primary Adhesion cultivation experiment in a BIOSTAT B fermenter with sample holder (left) and E. coli (right) [8].
  • Fig. 1: Setup of Primary Adhesion cultivation experiment in a BIOSTAT B fermenter with sample holder (left) and E. coli (right) [8].
  • Fig. 2: Micromanipulator with glass tip (A); spring table (B) and a polycarbonate sample colonized with bacteria (C) installed in SEM environment.
  • Fig. 3: Adhesion force measurement of a single bacteria on a polycarbonate surface.
To determine the antimicrobial efficacy, the research in the field of bioadhesion is crucial. Experiments include cultivations with E. coli K12 JM109 to reach bacteria adhesion in order to enable adhesion force measurements between bacterium and surface with a new micromanipulation system installed in SEM. To alter the surface, hydrophobic surfaces were generated with Argon/C4F8 plasma. The bacterial adhesion force for untreated polycarbonate (PC) is 96.6 ± 17.6 µN and for plasma fluorinated PC 12.9 ± 6.1 µN,  respectively.
 
 
Bacteria are ubiquitous and can accumulate on almost any biotic and abiotic surface. The milli- and micrometer thick biofilms – in the context with industry surfaces called ‘biofouling’ – are a major cause for failure in many sectors, especially in sensitive sectors such as the food industry or medicine. In order to survive, bacteria prefer to live on solid ground and to support each other. Bacteria can support each other by colonizing in a framework of secreted extrapolymeric substances (EPS). This shelter offers on the one hand protection against extrinsic factors like UV radiation, convection or chemical substances, on the other hand it serves as a supply network with nutrients. These interactions are responsible in great part for the bacteria’s resistance; hence it is more difficult to eradicate an existing surface biofilm.
 
Antimicrobial Strategies and their Efficacy Testing
To prevent the migration of bacteria and consequently the formation of biofilm, two strategies are generally pursued: killing and anti-adhesivity.
Surface-integrated disinfectants like silver particles, ammonium components and active ingredients (e.g. Triclosan) are prominent and common used examples in the food industry, medicine or water treatment. After surface contact, attached microbes will be immediately and efficiently killed. However, this strategy bears two problems: dead left bacteria could be used by other microbes as substrate, also it increases the bacterial resistance we have to face since several years [1, 2, 3, 4].
Anti-adhesive agents are used as a more environmentally friendly alternative to biocidal additives as they do not radically alter the survival strategy of bacteria.

Such surfaces primarily prevent bacteria from adhering to surfaces through the physico-chemical modifications. The anti-adhesive surfaces are equipped with nano- or microfeatures, different surface energies (ranging from super-hydrophilic like sol-gels or super-hydrophobic like PTFE) or composite coatings like carbon or hyaluronan. Nevertheless, they are insufficiently integrated to industrial usage, because they are prone to wipe disinfection or higher shear forces, and cost- and time-intensive on large-area components [5, 6, 7].

Both strategies contain agents that will have only a limited duration of action and are not generally applicable to every microorganism, though. Finally, the usage has to be weight carefully, but it remains elusive if there will be a ‘single magic bullet’ which will cover every need.
To increase the comparability, characterization and efficacy of the applied strategies, there are already numerous methods in which the cells are sucked, sheared, lifted or turn off of the surface. Noteworthy, the condition of the bacteria differs highly. Some are dried, immobilized or in wet on the surface, dead or alive, adhere with different cell appendages depending also on strain specifity. In conclusion, there is an urgent need for novel measurements that can be universally applied and that satisfy today’s industrial demands: wipe disinfection, which is used everywhere, or high-pressure radiators which exert lateral shear forces parallel to the surfaces. This gap can now be filled with a special micromanipulator device.
 
Adhesion Force Measurement Using a Micromanipulator
Micromanipulation is an effective strategy to directly determine a bacterial adhesive force. The micromanipulator interacts directly with the bacteria and yields the required force to remove the bacteria. Before the adhesive force measurements are performed by the micromanipulator, microorganism and surface have to be brought together. The University of Applied Sciences Ansbach has developed a cultivation method that allows E. coli JM109 bacteria to primarily adhere to polymer surfaces (Fig. 1). E. coli JM109 is a biofilm-forming microorganism that belongs to the safety strain K12 and is therefore an excellent candidate for pilot experiments. Polycarbonate was used as the test material due to its transparency and thermal stability.
Having the interplay of bacteria-surface, the prepared specimen (Fig. 2C) is loaded in front of the micromanipulator (Fig. 2A) on a spring table with a defined spring constant of 12.2 N/m (Fig. 2B). To orchestrate the right position to such little organism (1 – 3 µm long), the micromanipulator has three axes: X) for the rotational movement to the right and left (accuracy up to 5 nm) Y) for the height orientation from top to bottom or vice versa (3.5 nm) and Z) for the insertion and moving of the tip holder (0.5 nm) (Fig. 2A). A glass tip which tapers off from 1.2 µm to 0.8 µm at the end is attached orthogonally to the Z axis. To determine the adhesive force, the glass tip is pushed against a bacterium adhered on the surface, the spring table moves along and then jumps back to its original position when the bacterium is detached (Fig. 3). The distance from start point until detachment is a traverse path which is multiplied by the spring constant according to Hooke’s law.
 
F = s ∙ c([N] = [m] ∙ [N/m])
It results in a force - the bacterial adhesive force.
 
To have a comparison and to test different surface qualities, a plasma fluorinated polycarbonate (PC) sample and a natural polycarbonate sample were tested so far. On untreated PC, up to ten times higher adhesive forces were determined compared to fluorinated PC. The results show that the fluorination strongly influences the adhesion of the bacteria and proves the functionality of the micromanipulation system.
Regarding, for example, food processing, prone and contaminated surfaces can be checked in time series to reveal the weakest bioadhesion state to set right cleaning impulses and intervals.
In conclusion, this micromanipulation system can be easily used to test every bacterium on different surfaces and is easily incorporated to industrial applications. Therefore, this in vitro system contributes to safer and cleaner surfaces as well as to increased comparibility of different surface-bacteria combinations.

 

Authors
Philipp Häfner1, Nathalie Stefani2, Kerstin Lohbauer1, Hans-Achim Reimann1

Affiliation
1University of Applied Sciences Ansbach, Ansbach, Germany
2Leibniz Institute for Natural Product Research and Infection Biology e. V., Hans Knöll Institute (HKI), Dept. of Molecular and Applied Microbiology, Jena, Germany

 

Contact
Philipp Häfner
Prof. Dr. Hans-Achim Reimann
University of Applied Sciences Ansbach
Ansbach, Germany
philipp.haefner@hs-ansabch.de
a.reimann@hs-ansbach.de
www.hs-ansbach.de

 

More information on force measurements

Read more about  the microscopy of bacteria

References
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[2] C. Romão, CA. Miranda, J. Silva, M. Mandetta Clementino, I. de Filippis, M. Asensi: Presence of qacEΔ1 Gene and Susceptibility to a Hospital Biocide in Clinical Isolates of Pseudomonas aeruginosa Resistant to Antibiotics: Curr. Microbiol., 63 (1) 16–21 (2011) doi: 10.1007/s00284-011-9934-0
[3] S. Buffet-Bataillon, B. Branger, M. Cormier, M. Bonnaure-Mallet, and A. Jolivet-Gougeon: Effect of higher minimum inhibitory concentrations of quaternary ammonium compounds in clinical E. coli isolates on antibiotic susceptibilities and clinical outcomes, J. Hosp. Infect. 79 (2) 141–146 (2011) doi: 10.1016/j.jhin.2011.06.008
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, JAMA 298 (15) 1763–1771 (2007) doi: 10.1001/jama.298.15.1763
[5]  F. Siedenbiedel and J. C. Tiller: Antimicrobial Polymers in Solution and on Surfaces: Overview and Functional Principles, Polymers 4 (1) 46–71 (2012) doi: 10.3390/polym4010046
[6] J. C. Tiller, “Antimicrobial Surfaces,” in Bioactive Surfaces, H. G. Börner and J.-F. Lutz, Eds. Springer Berlin Heidelberg, 2011, pp. 193–217.
[7] J. C. Tiller, C.-J. Liao, K. Lewis, and A. M. Klibanov: Designing surfaces that kill
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, Proc. Natl. Acad. Sci. 98 (11) 5981–5985 (2001) doi: 10.1073/pnas.111143098
[8] N. Stefani, K. Bogendörfer, P. Häfner, H.-A. Reimann, Applied Research Conference 2015; Primary Adhesion of E.coli JM109 and Adhesive Behaviour on Plasma Modified Polycarbonate.

 

Contact

University of Applied Sciences Ansbach


University of Applied Sciences Ansbach


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