Is There a Threshold in the Antibacterial Action of Superhydrophobic

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Is there a threshold in the antibacterial action of superhydrophobic surfaces? Kosmas Ellinas, Dionysia Kefallinou, Kostas Stamatakis, Evangelos Gogolides, and Angeliki Tserepi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11402 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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ACS Applied Materials & Interfaces

Is There a Threshold in the Antibacterial Action of Superhydrophobic Surfaces? Kosmas Ellinas1,2†, Dionysia Kefallinou1,3†, Kostas Stamatakis3, Evangelos Gogolides1,2, and Angeliki Tserepi1,2* 1

Institute of Nanoscience and Nanotechnology, NCSR Demokritos, Aghia Paraskevi, Attiki, Greece, 15341 2

Nanoplasmas P.C. Technology Park “Lefkippos”, NCSR Demokritos, Aghia Paraskevi, Attiki, Greece, 15341

3

Institute of Biosciences and Applications, NCSR Demokritos, Aghia Paraskevi, Attiki, Greece, 15341 *Correspondence to: [email protected], [email protected], [email protected], [email protected] †These authors contributed equally

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ABSTRACT The realization of antibacterial surfaces is an important scientific problem, which may be addressed by the use of superhydrophobic surfaces, reducing bacterial adhesion. However, there are several limitations and contradicting reports on the antibacterial efficacy of such surfaces. Moreover, achieving antibacterial action through minimization of adhesion does not ensure complete protection against bacteria. Here, we identify the important factors affecting antibacterial action on superhydrophobic surfaces, emphasizing the role of bacterial concentration, and observing an upper concentration threshold above which antibacterial action of any surface is compromised. Finally, we propose metal enriched, superhydrophobic surfaces, as the “ultimate” “hybrid” antibacterial surfaces for in vitro applications. Keywords: Antibacterial activity, Superhydrophobicity, multifunctional surfaces, Plasma micro-nanotextured surfaces


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1. INTRODUCTION Bacteria are among the smallest and oldest living organisms and their impact in our life is important in many aspects, such as our food or health

1-2

. Pathogenic bacteria have negative

impact as they may cause serious infections and biomedical or prosthetic device failure. According to the US centers for disease control (CDC) and prevention, antimicrobial resistance is one of the most serious health threats. CDC estimates that in the United States alone, more than two million people become sick every year with antibiotic-resistant infections, with at least 23,000 dying, while globally the number of deaths is approximately 700.000 2. Bacteria have become resistant to multiple types or classes of antibiotics 3, mainly due to the extensive use of antibiotics against infections. This could be avoided if bacteria spreading was reduced. This can be achieved by using antibacterial surfaces in critical infrastructures, such as hospitals. This is the reason that the number of antibacterial surfaces proposed is rapidly growing. A recent review reports several different methods to create antibacterial surfaces 4. To date, these methods can be categorized in three main approaches: In the first approach, a bactericidal agent is used to exterminate the bacteria; such agents include metals and especially silver, metal oxides such as ZnO, and natural bactericidal substances such as chitosans and peptides or other organic antiseptics or antibiotics5. Nanotechnology, and nanomaterials such as nanoparticles or nanostructures can further improve the antibacterial and bactericidal action of surfaces

6-7

. The bactericidal nanostructured surfaces presented by

Ivanova et al. 8 are one such example. They reported a mechanical bactericidal effect with an average killing rate of 450,000 cells/ min/ cm2 simply by using a nano-scale columnar topography. This effect was also independent of the surface chemical composition. In the second approach, bacteria adhesion is eliminated using anti-adhesive surfaces (such as 3 ACS Paragon Plus Environment


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superhydrophobic surfaces, or antifouling hydrophilic surfaces)

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9-14

. Despite the extensive

investigation of these approaches, they seem insufficient to reduce bacterial infections. If these two approaches are combined, a third more effective scheme to "fight" bacteria can be realized

6, 15-18

. A recent review paper summarizes such antibacterial surfaces incorporating

combined dual functionality. However, in most of these surfaces dual functionality was achieved by combining materials with antibacterial properties or by using a stimuli-responsive antibacterial action (e.g. heating) 7. In general, the use of passive antibacterial surfaces, which do not require an external stimulation, is a simpler approach. Until now, the use of passive, superhydrophobic surfaces has resulted in some contradictory findings. Although several works report antibacterial action on such surfaces, Sousa et al.

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found that Staphylococcus aureus and Pseudomonas aeruginosa adhered on the superhydrophobic surface and even exhibited higher adhesion, when compared to the smooth hydrophobic surfaces. This is possibly observed, due to the complexity of the interaction between bacteria and surfaces. A recent review bacteria adhesion on surfaces. J. A. Lichter et al.

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reports some of the factors affecting

proposed a two stage adhesion model for

bacteria biofilm formation on surfaces, but there is yet no comprehensive study on the crucial factors to realize an antibacterial surface and this deficiency is more evident for superhydrophobic surfaces. A first effort to identify some of the critical factors in the performance of polymeric surfaces has been recently reported

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and factors such as surface

energy, roughness, wettability, and zeta potential highly affect the antibacterial properties of a surface, but important questions remain unanswered: Is there a limit in the antibacterial action of a surface? Are superhydrophobic surfaces suitable for such an application? Which is the most effective design towards the realization of a universally effective, passive antibacterial surface? Here, we investigate the most important factors influencing the antibacterial action of nanostructured surfaces focusing on the superhydrophobic ones. Additionally, we demonstrate 4 ACS Paragon Plus Environment

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the important role of concentration in the performance of an antibacterial surface and we prove that a concentration threshold exists, above which the action of any antibacterial surface (flat or superhydrophobic) is compromised. To do so, we study independently and as a combination the different approaches to realize an antibacterial surface. We also demonstrate that by tailoring the topography in the micro and nanoscale, as well as the surface chemistry and by enriching the surface with an effective antibacterial agent, a passive antibacterial surface can be realized on every substrate avoiding analogous concepts for surfaces with dual functionality (15), which require either complex synthesis methods or energy consumption 23

7,

. We present such surfaces, which we term as “hybrid” (i.e. anti-adhesive surface &

bactericidal agent) and as we will show, these surfaces induce both a short-term and a longterm, passive, antibacterial action.

2. EXPERIMENTAL 2.1 Superhydrophobic PMMA surface fabrication method 2-mm-thick poly(methyl methacrylate) (PMMA) substrates bought from IRPEN (Spain) and cleaned prior to plasma processing. The PMMA surfaces prior the plasma texturing step are smooth Figure 1 (a). The surfaces are micro-nanotextured using a high-density plasma reactor (Helicon plasma reactor, Micromachining Etching Tool, MET, from Adixen-Alcatel)

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.

Highly anisotropic, plasma etching conditions were used, i.e.1900 W, 100 sccm O2, 0.75 Pa, 15 oC, bias power 250 W and duration 10 min. The coating used for the hydrophobization was a Teflon-like film deposited in the same reactor using Octafluorocyclobutane (C4F8) gas at conditions: 900 W, 0 V, 5.33 Pa C4F8, deposition rate 30 nm min-1 25. The resulting multiscale topography is shown in Figure 1(b). The surfaces after the hydrophobic coating deposition become superhydrophobic (WSCA>155o, Hysteresis50%). The surfaces were examined every 24 h using fluorescence microscopy. Representative images were captured using Carl Zeiss Axioskop 2 and the average fluorescence intensity and standard deviation were extracted using ImagePro software. Each point is an average of 90 measurements (i.e. 30 7 ACS Paragon Plus Environment


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images per surface measurements in each surface x 3 repetitions). Additionally, the % bacteria coverage of each surface was calculated using the ImagePro software. With a view to explore possible differentiation after washing, the bacteria adhesion was also investigated after having washed the surfaces at the end of the experiment after 72 hours.

Cyanobacteria Synechococcus sp PCC 7942

1 mL

24 ώρες

Fluorescence

% Coverage

Figure 2. Schematic representation of the method followed to study the bacteria adhesion on the surfaces. 2.5 Cyanobacteria growth The unicellular cyanobacteria Synechococcus sp. PCC7942 were cultured in the BG11 medium

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with white fluorescent light (100 µE·m−2·s−1), in an orbital incubator (Galenkamp

INR-400) at 31 °C and aeration with 5% v/v CO2 in air

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. Cells were harvested during

exponential growth (after 4 days) and were resuspended in BG11 at 50 mg Chl a/ml. The Chl a concentration was determined according to Moran 30. 2.6 Cyanobacteria fluoresence measurement


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The method used in this work has been evaluated against gram-negative bacteria (E.coli) using the AATCC 100-2004 method 31 and practically exploits the ability of cyanobacteria for M = i

F0i - F00 F00

x 100

autofluorescence. More specifically, it uses the normalized fluorescence changes introduced in Mi to index the antibacterial

properties of the surface. Mi is defined as follows:

Where 𝐹!! is the value of Chl α fluorescence of cyanobacteria at zero contact time and 𝐹!! is the value of Chl α fluorescence of cyanobacteria after 1,2,..,i days . Each point is an average of 36 measurements (i.e. 3 measurements in each surface x 3 surfaces x 4 repetitions). Chl a fluorescence intensity depends on the ability of the cyanobacteria cells to proliferate

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.

Fluorescence intensity increases in the case that cells have the ability to proliferate, otherwise cells die, thus Chl a starts its degradation, fluorescence intensity is reduced and Mi takes negative values. Negative or low Mi values (when two surfaces are compared) are interpreted as enhanced antibacterial behavior. However, negative Mi values are also observed during the initial phase of the cyanobacteria cells deposition on the surfaces. This effect is equivalent to the lag phase during the bacteria growth in which bacteria are trying to adapt themselves to the new environment conditions before they start to proliferate. Nevertheless, if the surface does not have antibacterial properties Mi starts to rise after this short adaptation phase.

3. RESULTS AND DISCUSSION 3.1. Bacteria adhesion on superhydrophobic, anti-adhesive surfaces First, we studied the bacteria adhesion on three types of surfaces for a period of 72 hours which is longer than the duration proposed by existing protocols for the evaluation of antibacterial activity 34. The surfaces that were compared were: a) Untreated PMMA plates, b) Commercial, hydrophilic, non-adhesive surfaces for cells, c) Superhydrophobic plasma 9 ACS Paragon Plus Environment


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micro-nanotextured PMMA (Water Static Contact Angle (WSCA) >155o, Hysteresis72) of superhydrophobic surface immersion in bacteria solution. Thus, the incorporation of a bactericidal agent is expected to improve the surface antibacterial properties in the long-term (hybrid scheme with dual functionality). However, since the search for the most effective antibacterial agent is still going on, in order to identify the 12 ACS Paragon Plus Environment

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optimum agent, we did a comparison between two well-known antibacterial metal agents (copper and silver). Both metals are commonly used as bactericidal agents and many methods to apply them are already available. In our case, the metals were deposited through sputtering on untreated PMMA surfaces, forming a 15 nm thick coating. Their antibacterial behavior was studied according to an in-situ method of fluorescence measurement, which has recently been developed and described in previous works

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(See section 2.6 and the supporting

information file). Figure 4 shows the comparison of these two metals. Interestingly, copper (green line) outperforms silver (purple line). More specifically, the negative values of index Mi for copper, as one can see in Figure 4, indicate its antibacterial activity, which remains stable until the end of the experiment (22 days), while in silver coated surfaces this activity fades away after 7 days and a clear accelerating bacterial growth is noticed, almost reaching the bacterial growth rate on the untreated surfaces. This study clearly indicates that copper performs better than silver.

100

Antibacterial activity of metal-sputtered surfaces (1.7 x 108 cfu/cm2)

50 Mi 0 -50

0

5

10

15

Time (days)

-100

20

Untreated Ag Cu

Figure 4. Mi evolution curves of the antibacterial activity of copper and silver for a period of 22 days. The bacteria surface density is 1.7×108 cfu/cm2, which corresponds to 4.6×109 cfu/ml. Mi represents the change in normalized fluorescence intensity caused from the 13 ACS Paragon Plus Environment


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increase or the decrease in the bacteria number according to the method proposed in

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32-33

. Mi

is defined as Mi =

!! ! !!! ! !!!

x 100, where 𝐹!! is the value of Chl α fluorescence of cyanobacteria at zero

contact time and 𝐹!! is the value of Chl α fluorescence of cyanobacteria after 1,2,..,i days. The difference between the antibacterial activity of the two metals is clearly indicated by the low Mi values for the Copper coated (Cu) surfaces and the relatively high Mi values of silver coated (Ag) surfaces. One key point involved in this study and helps to explain the observed difference between copper and aluminium coated surfaces, is the release of the metal ions from the metal coated surface. Hans et al.

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have recently proposed some criteria for efficient contact killing. In

short, the criteria are summarized as follows: a) the metal should be easily oxidized, b) the produced metal oxides should have high solubility and c) metal ions should have a soft ionic character 41. The first two allow a high rate of ion release while the third one is correlated with the ability of metal ions to kill bacteria 42. Copper fulfils all these criteria and under common environmental conditions exists as metallic copper, Cu2O, Cu2+, and CuO. Previous reports suggest that Cu2O is really effective in contact killing, while CuO has a higher copper ion release rate. Additionally, Cu2O is highly soluble providing the required stability of the cooper antibacterial properties

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. On the other hand, the less effective antibacterial

performance of silver can be well explained by its physico-chemical properties. Silver is chemically stable under usual conditions and only in acidic environments can form oxides and Ag+ 43. This is the reason that most studies involving silver in antibacterial applications use silver ions or silver oxides in the form of nanoparticles 44. In our case, silver is not expected to be in oxide or ionic form and this is the main reason that the antibacterial activity of the Agcoated surface is worse than that of copper. 3.3 Realization of hybrid antibacterial surfaces 14 ACS Paragon Plus Environment

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Copper was therefore introduced on superhydrophobic surfaces to improve their antibacterial action and demonstrate that the passive “hybrid” approach can exhibit both short and longterm protection against bacteria (anti-adhesive & bactericidal properties on the same surface). To accomplish that, PMMA surfaces were first plasma micro-nanotextured, then a thin layer (15 nm) of Copper was sputtered on them, and finally a thin layer (5-10 nm) of teflon-like polymer was plasma deposited to lower the surface energy and render these surfaces also superhydrophobic. Both superhydrophobic (SH) and Cu enriched superhydrophobic surfaces (SH+Cu) exhibited the same wetting properties (WSCA>155o, Hysteresis155o, Hysteresis

Is There a Threshold in the Antibacterial Action of Superhydrophobic

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