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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Tailoring Bacteria Response by Piezoelectric Stimulation Estela O. Carvalho,†,‡ Margarida M. Fernandes,*,†,‡ Jorge Padrao,† Ana Nicolau,† Jorge Marqués-Marchán,§ Agustina Asenjo,§ Francisco M. Gama,† Clarisse Ribeiro,†,‡ and Senentxu Lanceros-Mendez∥,⊥ †
Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal Centre of Physics, University of Minho, Braga 4710-057, Portugal § Instituto de Ciencia de Materiales de Madrid, CSIC, Madrid 28049, Spain ∥ BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, Leioa 48940, Spain ⊥ Ikerbasque, Basque Foundation for Science, Bilbao 48013, Spain
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ABSTRACT: Bacteria are simple organisms with a remarkable capacity for survival by adapting to different environments, which is a result of their long evolutionary history. Taking into consideration these adapting mechanisms, this work now investigates the effect of electrically active microenvironments on bacteria and on how this stimulation may trigger bacteria growth inhibition or proliferation. Electrical microenvironments are generated via stimulation of a piezoelectric polymer with a mechanical cue, thus developing an electrical response and a variation on the surface charge of the polymeric material. Specifically, Grampositive Staphylococcus epidermidis and Gram-negative Escherichia coli were grown overnight under static and dynamic conditions on piezoelectric poly(vinylidene) fluoride (PVDF) films to further study bacteria behavior under: (i) the effect of the material surface charge in static conditions, (ii) the mechanical effect, and (iii) the piezoelectric effect, the last two performed under dynamic conditions. Bacteria viability in planktonic and biofilm forms was measured, and the microorganism morphology was characterized. Whereas E. coli responds little to any of the stimuli application, S. epidermidis growth can be regulated through the material surface charge and by the applied frequency. Positively charged PVDF induces bacterial growth inhibition in planktonic and adhered cells in static conditions, whereas antifouling properties are obtained when a mechanical or piezoelectric effect at 4 Hz stimuli is applied. By increasing the stimuli to 40 Hz, however, the adhesion of bacteria is promoted. In conclusion, the behavior of certain bacteria species is tailored through the application of piezoelectric materials, which provide sufficient mechanoelectrical stimuli for growth or inhibition of bacteria, allowing for the design of suitable anti- and promicrobial strategies. Such strategies are only found in studies related to mammalian cells, whereas in bacterial cells this type of stimuli are still unknown. Thus, this work provides one of the first insights on the effect of piezoelectric stimuli on bacterial cells. KEYWORDS: piezoelectric material, Gram-positive bacteria, Gram-negative bacteria, antimicrobial, promicrobial, mechanoelectric effect, surface charge
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INTRODUCTION
appendages on the bacterial surface, which enable them to exert physical forces at a nanoNewton range on their surroundings, the same amplitude of forces that mammalian cells exert on their own surroundings.8 The forces exerted trigger the accumulation of actin and other proteins, events critical for the colonization of the host.9 More recently, a potential mechanism of action for Escherichia coli (E. coli) mechanotransduction has also been suggested. According to this study, E. coli can sense the local mechanical environment through voltage-induced calcium flux. Mechanical contact induces the influx of calcium ions, generating an electrical pulse through them.10 Indeed, bacteria and some mammalian cells
The application of physical stimuli such as mechanical, magnetic, and electrical cues on the surrounding of mammalian cells is known to influence their biological behavior, enhancing cell target functions such as adhesion, proliferation, migration, and differentiation.1−4 Nevertheless, the potential of using these stimuli on bacterial cells of different species has been largely overlooked. The biochemical environment surrounding bacteria has been the main cue reported to affect them.5 Recent findings, however, disclosed that bacterial cells are activated by physical cues in a similar way to mammalian cells,6 being attuned to mechanical forces, which has been proven to induce an adaptive behavior.7 Swimming motility provides an elegant example of how bacteria are influenced by the mechanical nature of their surroundings. Neisseria gonorrhoeae, the bacteria causing gonorrhea, possess type IV pili, hair-like © XXXX American Chemical Society
Received: March 21, 2019 Accepted: July 3, 2019 Published: July 3, 2019 A
DOI: 10.1021/acsami.9b05013 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
ACS Applied Materials & Interfaces
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(human sensory neurons) share the electrical pathway as a common tool for sensing their environment and the voltagegated channels are considered a valuable tool for that purpose.11,12 On the basis of the knowledge that bacteria can feel the surrounding environment and change their phenotype in response to it, some reports have focused on the effect of mechanical vibration on surface adhesion, proliferation, and virulence. The application of surface acoustic waves generated from electrically activated piezo elements13 and vibration loads generated by magnetoelastic materials14 has been shown to avoid bacterial adhesion. However, the effect of electrical cues created by an electroactive material on the bacteria behavior, rather than acoustic mechanic waves, has been scarcely reported. The only report found using piezoelectric materials for bacteria inhibition was performed using ceramics,15 but these have been used exclusively under static conditions. Further, it is to be noticed that the used ceramic material possesses a piezoelectric constant |70| pC N−1 that is much higher than the one of poly(vinylidene fluoride) (PVDF) |24| pC N−1, leading to larger surface charge variations under specific mechanical stimuli.15 The direct application of strong electric fields has been widely reported as a bactericidal technology or as a mean of preventing biomaterial-associated infections, which are caused by biofilm formation or by immunomodulatory effects related to the foreign body reaction, or even to disinfect-contaminated liquids.16−20 Nevertheless, the strong electrical field constrains its applications on biological tissues. The indirect effect of mechanical stimuli inducing an electrical field on the surface of the material seems to be a valuable tool for remote effect that has never been tried. This could be valuable, for example, for the prevention of infection of orthopedic indwelling devices by external stimulation. Such an effect can be based on the potential of electroactive materials/scaffolds, mainly piezoelectric ones.21 The development of these kind of materials, namely possessing piezoelectric activity, has been widely explored in regenerative medicine, mainly for bone, muscle, and neural tissue engineering purposes.22−24 These works have proven these materials as able to effectively induce cell seeding, growth, and differentiation, taking advantage on the mechanotransduction properties of the cells,21,22 but they have been poorly investigated in microbiology. This work intends to open new perspectives on the study of bacteria behavior in contact with different physical stimuli, namely, the surface charge, the mechanical effect and, importantly, the mechanoelectrical effect. The response of Gram-negative and Gram-positive model bacteria in contact with a mechanoelectrical signal, produced through the application of mechanical stimuli on piezoelectric polymer films of PVDF, is thus studied. This polymer presents a high piezoelectric response, and may be processed in a variety of shapes and morphologies, including films and fibers, nanospheres, or three-dimensional scaffolds.25 The PVDF films used in this work bear three different surface charge states (noncharged, positively charged, and negatively charged) and were mechanically stimulated using two mechanical vibration frequencies (4 and 40 Hz), which induce surface charge variation. The effects of these stimuli on bacterial growth activation or inhibition were identified and their relevance for the development of advanced applications is discussed.
Research Article
MATERIALS AND METHODS
Materials. PVDF films in the electroactive β-phase, with different surface charges (zero “nonpoled”, positive “poled +”, and negative “poled −”) and with a thickness of 110 μm, were purchased from Measurement Specialties (USA), showing a piezoelectric constant (d33) of |24| pC N−1 in the poled samples. Gram-negative E. coli K12 and Gram-positive Staphylococcus epidermidis NCTC 11047 were purchased from American Type Culture Collection (LGC Standards S.L.U) and were used in all microbial assays. Kelvin Probe Force Microscopy. The electrostatic surface potential measurement of “nonpoled”, “poled +”, and “poled −” PVDF films was performed using Kelvin probe force microscopy (KPFM). A titanium (Ti) layer of approximately 25 nm was deposited by sputtering on the edge of the films, in order to establish a good reference for the electrostatic signal. The samples were further attached to a metallic support (∼1 cm2) with (conducting) adhesive carbon tape. The contact between the upper faces covered with Ti and the support has been made using silver paint. The KPFM measurements were then performed with a Nanotec SPM system26 and a tip BudgetSensors BS-ElectriMutli75 with an electrically conductive coating of Cr/Pt on both sides, with resonant frequency F = 63.5 kHz (the values given by the manufacturer are F = 75 kHz and force constant k = 3 N m−1). PVDF-Assisted Piezoelectric Stimulation of Bacteria. Previously to the microbiological assays, PVDF films were cut in 13 mm diameter circles. These were then sterilized by exposure to ultraviolet light for 30 min on each side and placed on 24-well nontreated tissue culture polystyrene (TCPS) plates (VWR). For the preparation of bacterial pre-inoculum, a single colony from the corresponding stock bacterium cultures was incubated overnight at 37 °C and 110 rpm in nutrient broth (NB). The optical density (OD) of both inoculated bacterial cultures was measured at 600 nm and adjusted to 0.09 ± 0.01, corresponding in both cases to approximately 1 × 108 CFU mL−1. Of the obtained bacterial suspension, 750 μL was then placed in contact with the films on the 24-well plate. Wells without any film were used as control for bacterial growth. The bacteria incubated were then allowed to grow for 2 and 12 h at a temperature of 37 °C. The bacteria were cultivated both in staticwhere no mechanical stimulus was applied but in the presence of differently surface charged environmentsas well as in dynamic conditionsunder mechanical stimuli (“nonpoled” sample) and the corresponding additional piezoelectric-related surface charge variation of the films (“poled +” and “poled −” samples). For the latter, the 24well plate was placed on a lab-made bioreactor system23 with mechanical stimulation with a vertical vibration module at a frequency of 4 and 40 Hz with an amplitude of 1 mm (Figure 1). The growth curve for each bacterium was also performed in triplicate. Flasks containing the diluted pre-inoculum with an OD at 0.09 ± 0.01 were incubated with shaking at 110 rpm at 37 °C. The
Figure 1. Schematic representation of β-PVDF films with different poled states and the corresponding effects (a) when the lab-made bioreactor system (schematic representation) is used for dynamic bacterial stimulation (b) and schematic representation of the β-PVDF chemical structure (c). B
DOI: 10.1021/acsami.9b05013 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. KPFM study of surface potential of the nonpoled PVDF film (a), positively poled PVDF film (b), negatively poled PVDF film (c), and profile of the surface potential of a randomly selected 5 μm area (blue line) on each sample (d). Note the different scales in the images.
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OD at 600 nm was recorded within defined time intervals during 12 h, which comprises the PVDF films’ incubation time. The bacterial growth was monitored spectrophotometrically without any PVDF film substrates. Planktonic Cell Viability Assay. The viability of bacterial cells in suspension after contacting the material under static and dynamic conditions, for 2 and 12 h, was evaluated using a colony-forming units (CFUs) assay. Ten-fold serial dilutions of the bacterial cultures were performed in phosphate buffer solution (PBS). For each dilution, a volume of 100 μL was plated on an NB plate agar and further incubated at 37 °C for 24 h, to determine the number of viable bacteria, allowing a quantitative assessment of the average colonyforming units per milliliter of pre-inoculum solution (CFU mL−1). Each cultivation experiment was repeated five times. Relative bacterial cell viability (%) was determined for each condition and compared with that of cells incubated only with NB, without any stimuli or material. Bacterial Adhesion and Viability on the Material. The bacterial adhesion on the PVDF surface, after 12 h of incubation with piezoelectric stimulation, was assessed using the Live/Dead kit assay (Thermo Fisher) and scanning electron microscopy (SEM). Live/Dead Kit. The samples were washed twice with PBS 1×. Then, cells were stained for 15 min in the absence of light with equal volumes of 0.1% (v/v) SYTO 9 and 0.1% (v/v) propidium iodide.27 Afterwards, the samples were observed under a fluorescence microscope (Olympus BX51 microscope). Representative fields were captured using 10× and 100× magnifications. SEM Visualization. The samples were first washed twice with PBS 1×, then fixed with 3% (v/v) glutaraldehyde, for 45 min, at 4 °C, and again washed with PBS. Then, they were dehydrated with increasing concentrations of ethanol [30, 50, 70, 80, 90, and 100% (v/v)]. The samples were coated with Au 20A and placed on aluminum pin stubs with electrically conductive carbon adhesive tape (PELCO Tabs) on a Phenom standard sample holder at 5 kV and a spot size of 3.3. Finally, the samples were characterized using a desktop SEM (Phenom-World BV, The Netherlands). All results were acquired using the ProSuite software. Data Analysis. All quantitative results obtained from each sample are exhibited as averages with the respective standard deviations. Results were analyzed by GraphPad Prism version X for windows (Graphpad Software, San Diego, CA, U.S.A.). To determine the statistical significances were used one-way analysis of variance, followed by the Dunnett post-hoc test or by the unpaired two-tailed Student’s t-test method.
RESULTS AND DISCUSSION The electrically active microenvironment provided by the piezoelectric scaffold, able to stimulate cells through electric or mechanical stimuli, has already been successfully explored in regenerative medicine, mainly for bone, muscle, and neural tissue engineering purposes,21,23,28 whereas in microbiology it has been scarcely researched. This work thus focuses on exploring the suitability of electrical and mechanical stimuli in promoting anti- and pro-microbial strategies. For that, bacteria were grown on the surface of PVDF films with three different states: “nonpoled”, “poled +”, and “poled −”. Although PVDF films possess piezoelectric properties when crystallized in the β-phase,25 because of the random orientation of the dipolar moments within the material, the samples must undergo a poling processthat is, alignment of the dipolar moments though the application of an electric fieldin order to optimize the piezoelectric response (Figure 1).29 Thus, whereas “nonpoled” samples show a zero net surface charge, “poled −” and “poled +” possess negative and positive surface charges, respectively (Figure 2), which vary with the applied mechanical solicitation after the piezoelectric effect,30 as depicted in eq 1 ij ∂D yz ij ∂x yz d33 = jjj 3 zzz = jjj 3 zzz j ∂X3 z j ∂E3 z k { k { E
X
(1)
where D is the electrical polarization, E corresponds to electrical field strength, and X and x are the mechanical stress and the strain, respectively.30 The |d33| represents the piezoelectric coefficient, whose value is ≈|24| pC N−1 for the used samples. Thus, the “nonpoled” sample has no piezoelectric response and the “poled +” and “poled −” show the corresponding surface charge variation related to the variation of the polarization with the mechanical solicitation. The microscopic piezoelectric response of the different samples has been also reported,31 showing the clear differences in the poled region distribution and size, as well as in the local piezoactivity. As the surface potential of a material plays a key role in the adhesion of microorganisms32 and is an important feature for the piezoelectric response of the materials upon mechanical stimuli,33 it was important to assess the surface charge state of the different samples. The surface potentials of “non-poled”, C
DOI: 10.1021/acsami.9b05013 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces “poled +”, and “poled −” samples were analyzed by KPFM giving rise to surface potentials of approximately 0, 6, and −4 V, respectively (Figure 2). These results confirm the zerosurface potential of the nonpoled samples, because of the random orientation of the dipolar moments and the corresponding positive and negative surface potential of the poled samples, because of the dipole orientation (Figure 1c). It is to be noticed that piezoelectric stimulation promotes the variation of the surface potential, because of mechanically induced dipolar variations, according to eq 1. The electrical cues on these electroactive materials may thus be generated using a home-made mechanical bioreactor (Figure 1) providing a mechanical stimulus with a frequency of 4 or 40 Hz. Thus, by using the different materials, it was possible to assess the effects of: (i) the material surface charge (“poled +” and “poled −”) at static conditions, (ii) the dynamic mechanical stimuli on the “nonpoled” samples, and finally (iii) the dynamic mechanoelectrical stimuli (piezoelectric effect) on poled samples on both planktonic and adhered cells’ growth. Effect of the Piezoelectric Stimuli on Planktonic Cells in Solutions. The effect of surface charge, and mechanical and piezoelectric stimuli on growing planktonic cells was studied at time points 2 and 12 h. After 2 h, there is an indication that E. coli and S. epidermidis are both in the log phase (Figure 3),
meaning that apart from the intense metabolic activity, bacteria are also experiencing cell division. Most antibiotics are active against dividing bacteria, as they typically target bacteria cell walls or the protein synthesis processes, in particular DNA transcription and RNA translation.34 The measurement of cell viability after 2 h (Figure 4) is therefore important to assess sensitivity to the applied stimuli. It is observed that E. coli shows a similar behavior regardless of the stimuli or material used during the incubation (Figure 4a), whereas S. epidermidis is clearly influenced (Figure 4b). In static conditions, when only the surface charge effect is present, as depicted in Figure 2, S. epidermidis growing on the top of “poled +” surface experience a reduction of the proliferation rate, which may indicate an inhibition effect. On the other hand, when it grows on the top of “nonpoled” and “poled −” surfaces, the cell viability is similar to the control (designated in the graph by a dash line indicating 100% cell viability), thus proliferation is unaffected. In dynamic conditions, upon application of the mechanical stimulus, a proliferation rate is observed on both poled surfaces, on which the growth of S. epidermidis increases with increasing the frequency applied. The application of piezoelectric stimuli with 40 Hz frequency promotes a significant increase in the proliferation on comparing to the static condition, indicating a fast bacterial growth when this higher frequency stimulus is applied. This indicates that the piezoelectric effect provided by mechanical stimuli through poled PVDF (piezoelectric effect) in solutions assists the proliferation of Gram-positive bacteria. However, in a different context, the mechanoelectrical effect on PVDF has also been reported to induce proliferation on mammalian cells,21 being the effect governed by the surface properties and the piezoelectricity of the material. After 12 h, it is observed that E. coli planktonic cells are not influenced by the surface charge or stimuli applied, as mostly nonsignificant differences are observed between the control and the samples (Figure 5a). Nevertheless, it is worth noticing that when the frequency of the stimuli increases from static to 40 Hz on “poled +” samples, a proliferation trend is observed, indicating a piezoelectric-mediated proliferation effect (Figure 5a), which may be associated with the fast growth induced by the 40 Hz frequency applied as previously explained for S. epidermidis at 2 h. Whereas E. coli seems to be less susceptible to the stimuli, a clear effect is observed in S. epidermidis after 12 h, both regarding the type of material and the stimuli applied (Figure
Figure 3. Growth curves of E. coli and S. epidermidis in the NB medium in planktonic form without the application of the piezoelectric material or stimuli.
Figure 4. Relative bacterial cell viability of planktonic cell E. coli (a) and S. epidermidis (b) in solution after 2 h in contact with the piezoelectric stimuli. Relative bacterial cell viability (%) was determined relatively to the control cells incubated only with NB. #P < 0.05 vs “poled +” under static conditions; *P < 0.05 vs “poled −” under static conditions. D
DOI: 10.1021/acsami.9b05013 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 5. Relative bacterial cell viability of planktonic cells E. coli (a) and S. epidermidis (b) in solution after 12 h in contact with the piezoelectric stimuli. Relative bacterial cell viability (%) was determined relatively to the control cells incubated only with NB. δP < 0.05 vs “poled +” under static conditions; #P < 0.005 vs “poled +” under static conditions; *P < 0.05 vs “poled −” under static conditions; †P < 0.05 vs “poled −” under dynamic conditions with 4 Hz frequency.
bacteria must be slowing down, making the bacteria more susceptible to the applied stimuli. Thus, lower frequencies at 4 Hz induce proliferation over both positively and negatively charged surfaces, in a similar way to what occurs to mammalian cells,21 whereas higher frequencies at 40 Hz induce an inhibition effect. This last may be explained by the rapid growth bacteria experience at 40 Hz (evidenced at 2 h in Figure 4b) that leads to a fast depletion of the nutrients and formation of inhibitory products, which is followed by a sharp decline in living bacterial cells after 12 h. Gram-positive bacteria thus seem to be more sensitive to the stimulus and type of material, which may be related with the structural composition of their cellular membrane. Whereas Gram-positive bacteria possess a thick cell wall (20−80 nm), where a peptidoglycan layer is exposed to the outside environment, the Gram-negative bacteria have a relatively thin (
