New Insights into Antibiofilm Effect of a Nanosized ZnO Coating

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New insights into antibiofilm effect of a nano-sized ZnO coating against the pathogenic methicillin resistant Staphylococcus aureus Marta M. Alves, Ons Bouchami, Ana Tavares, Laura Córdoba, Catarina Ferreira Santos, Maria Miragaia, and Maria de Fatima Montemor ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02320 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 7, 2017

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ACS Applied Materials & Interfaces

New insights into antibiofilm effect of a nano-sized ZnO coating against the pathogenic methicillin resistant Staphylococcus aureus Marta M. Alvesa,*, Ons Bouchami b, Ana Tavaresb, Laura Córdobaa, Catarina F. Santosa,c, Maria Miragaiab, Maria de Fátima Montemora a

CQE, DEQ Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1049-001, Lisboa, Portugal

b

Laboratory of Bacterial Evolution and Molecular Epidemiology, Instituto de Tecnologia Química e Biológica

António Xavier, Nova University (ITQB-NOVA), 2780, Oeiras, Portugal c

EST Setúbal, DEM, Instituto Politécnico de Setúbal, Campus IPS, 2910, Setúbal, Portugal

Corresponding author Marta M. Alves [email protected] [email protected]

Phone: +351 21 841 77 64

Address: Instituto Superior Técnico Av. Rovisco Pais 1049-001 Lisboa Portugal

1 ACS Paragon Plus Environment


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Abstract ZnO nanoparticles (NPs) are arising as promising novel antibiotics towards device-related infections. The surface functionalization of Zn, a novel resorbable biomaterial, with ZnO NPs could present an effective solution to overcome such threat. In this sense, the antibacterial and antibiofilm activity of nano and micro-sized ZnO coatings was studied against clinically relevant bacteria - methicillin resistant Staphylococcus aureus (MRSA). The bacterial viability of planktonic and biofilm cells together with the corresponding biofilms structures revealed that only the nano-sized ZnO coating had an antibiofilm effect. To elucidate this effect, a novel approach was taken: preconditioning of bacteria with this ZnO coating followed by exposure to sub-inhibitory concentrations of antibiotics with well-known modes of actions. This approached revealed i) a decreased biofilm formation in combination with gentamycin, targeting protein synthesis and ii) an increased biofilm formation in the presence of rifampicin and vancomycin, acting on RNA and cell wall biosynthesis, respectively. The increased bacteria resistance to these two antibiotics gave new insights into the antibiofilm effect of this nanosized ZnO coating. The synergistic effect observed for gentamycin opened new perspectives for the design of effective solutions against implant-related infections. During the in vitro degradation of this nano-sized ZnO coated Zn a specific corrosion product, hopeite [Zn3(PO4)2], was depicted. Interestingly, the increased deposition of hopeite-derived compounds on MRSA cells’ surface seemed to be related with unhealthy and dead bacterial cells. This observation suggested that hopeite may be as well playing a key role in this antibiofilm activity. The results obtained herein shed light on the possible antibacterial effect of a nano-sized ZnO coating, and strengthened its antimicrobial (antibacterial and antifungal) potential, therefore providing a potential effective material to overcome the growing trend of implant-related infections.

Keywords: Antibiotics; Biofilm; MRSA; Nanoparticles; ZnO coating.

1. Introduction ZnO has been known for a long time for its antibacterial properties 1-3. The technological progresses have led to the synthesis of ZnO crystals down to the nanoscale size further enhancing its antibacterial properties 4, 5. In face of that ZnO nanoparticles (NPs) are being now considered as novel potent antibiotics with several advantages over the conventional organic counterparts, namely enhanced stability at high temperatures 3. This is of especial importance in an era where the microbial resistance to conventional antibiotics has been declared an increasing threat worldwide 6. Medical implanted devices, from a simple catheter to complex prostheses, are especially prone to develop infections particularly due to their increasing use in clinical practices. Currently, medical-device related infections account for more than 60 % of all of the hospital-acquired associated infections in the USA 7. In addition, the rising number of immunocompromised patients and aged people in hospitals further aggravate these


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numbers. Once acquired, infections can lead to implant failure and in more severe cases to patient's death 3. Upon developing these device-related infections result in the production of bacterial biofilms

8

composed by

bacterial cells, polysaccharides, proteins and extracellular DNA. In an initial and decisive stage cells attachment to the abiotic surface or to the host matrix proteins that covers the surface of the medical device. Only then is followed by the aggregation of cells and further maturation. In the form of biofilm bacterial cells are particularly protected against antibiotics and the host immune system what makes the treatment of these infections quite challenging. Within this context, antibiofilm surfaces represent a step forward in the prevention of implantrelated infections with ZnO NPs-based coatings being presented as a promising alternative 9. In fact, the antibacterial properties of ZnO in the free form of NPs have been reported effective against a broad spectrum of clinically relevant microorganisms. Among those microbes are the multidrug resistant strains, methicillin resistant Staphylococcus aureus (MRSA), which are prevailing bacteria typically associated to these type of infections

4, 10, 11

. However some caution should be taken when translating the already known

antibacterial properties of ZnO NPs. The majority of these conclusions are reported from free ZnO NPs acting against planktonic bacterial cells 1-3. When envisaging the development of ZnO-derived antibiofilm surfaces one must take into consideration the immobilization of the particles and the formation of bacteria biofilms. In fact the immobilization per se may impact the exposed surfaces (e. g. preferential exposure of a ZnO crystal face 12) and the formation of a bacteria biofilm that represents a much more resistant form than the bacteria planktonic counterparts. Up to now, few studies have addressed the antibacterial effect of ZnO NPs functionalized surfaces against staphylocococci

13-15

. Applerot et al.

15

reported an antibiofilm activity for a ZnO NPs-derived coating

with no decrease in cells’ viability. On the other hand, Jansson et al. 13 and McGuffie et al. 14 reported that ZnO NPs-derived coatings allowed the formation of bacterial biofilms, however with less viable cells. The antibacterial activity of these coatings was suggested to occur upon cells contact with ZnO NPs

13, 14

, probably

2+

trough the production of reactive oxygen species (ROS), releasing of toxic Zn doses and membrane damaging caused by the direct or electrostatic interactions of the NPs with cells membrane 1-3, 10. Despite the high number of studies related to the antimicrobial effect of ZnO NPs, the effect lying behind the ZnO NPs antibiofilm activity is still not fully understood 1-3. In this context, Zn a promising resorbable material 16-18, coated with nano and micro-sized ZnO-derived coatings was evaluated and tested against MRSA. In an attempt to disclose the antibiofilm effect of ZnO NPs–derived coatings, a non-conventional approach was used: preconditioning of cells with ZnO NPs followed by exposure to sub-inhibitory concentration of five different antibiotics with well-known mechanisms of action. Additionally, the evaluation of the degradation behavior of the ZnO-derived coatings opened a new perspective towards the antimicrobial action of nano-sized ZnO against MRSA.



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2. Materials and Methods 2.1. Biomaterial fabrication The ZnO coatings were electrodeposited on Zn according to the procedure previously described by Alves et al. 12

. Briefly, in a conventional three-electrodes electrochemical cell, Zn coupons (99%) were used as working

electrode, a platinum foil as counter electrode and saturated calomel electrode (SCE) as reference electrode. The electrolyte solution was composed of 50 mM Zn(NO3)2 and 50 mM H3BO3 at pH 6. The electrodeposition process was carried out in a Voltalab PGZ 100 potentiostat (Radiometer Analytical) by applying a constant cathodic potential of −1.9 V for 20, 100 and 300 s to produce nanostructured flower, rod-like and hexagon ZnO films morphologies, respectively 12.

2.2. Methicillin resistant Staphylococcus aureus (MRSA) cells colonization on bare or ZnO-coated Zn 2.2.1. Assessment of cells viability The hospital-associated MRSA strain HAR22, a proficient biofilm forming strain which belonging to EMRSA15 clone, which is a pandemic MRSA clone disseminated worldwide

19

, was initially streaked from -70°C

glycerol stock on a tryptic soy agar (TSA) plate and a fresh single colony was then inoculated in 5 ml tryptic soy broth (TSB) and cultured at 37 oC with shaking. Overnight culture of HAR22 strain was adjusted to the turbidity of a 0.5 McFarland standard at 600 nm and bacterial suspensions (500 µl) were incubated with both bare Zn (used as a control) or ZnO-coated Zn samples (nanostructured flower, rod-like and hexagon) for 24 h without shaking at 37 oC. For that purpose mini cuvettes were glued on the surface of the coupons previously mounted in cold curing epoxy resin [13] (Scheme 1). The exposed area was of 1 cm2. For the assessment of the planktonic cells viability, the cultures supernatants were gently removed by aspiration and the planktonic cell suspensions were sequentially diluted 10 to 1016 fold. For the assessment of the biofilm forming cells viability, after the supernatant removal, 500 µl of TSB was added to the biofilm fixed at the surfaces of bare or ZnO-coated Zn samples. The biofilms-grown bacteria (adherent layer of bacterial cells) were then suspended by scraping the samples’ surfaces with a sterile pipette tip. The solution was pumped up and down until cell aggregates were destroyed and a homogenous suspension was obtained. To assess cell viability, the biofilm cell suspensions were sequentially diluted 10 to 1016 fold and 0.1 ml aliquots were spread on TSA agar plates with sterile glass beads (Scheme 1). The numbers of colony forming units (CFU) from both the supernatant and biofilm were determined after 48 h growth at 37 oC. All the experiments were performed at least with three independent assays with internal replicates and averaged.


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Scheme 1: Diagram of the experimental setup for the antibacterial and antibiofilm assays of bare or ZnO-coated Zn samples against Staphyloccocus aureus (MRSA).

2.2.2. Morphological characterization of the biofilms For the morphological characterization of the biofilms the cells were fixed on the samples’ surfaces. For that purpose, the biofilms washed with distilled water were fixed overnight with glutaraldehyde at 4 oC, followed by 70 % (v/v) ethanol for 10 min, 95 % (v/v) ethanol for 10 min and finally by absolute ethanol for 20 min. After complete air-drying, the samples analyzed by SEM (JEOL- JSM7001F) were previously coated with a thin layer of conductive chromium (Polaron E-5100). For the atomic force microscope (AFM) analyses a Nanosurf Easyscan 2 was used to characterize the biofilms’ morphology. Topographical and amplitude images were obtained for all conditions in non-contact mode at different magnifications and random locations. The measurements were carried out in air at room temperature with NanoAndMore GMBH® silicon probes. Images were acquired at a resolution of 512 × 512 pixels. 2.3. Methicillin resistant Staphylococcus aureus (MRSA) biofilm formation in the presence of antibiotics on ZnO nanostructured flower coating 2.3.1. Antibiotic susceptibility testing Antibiotic susceptibility patterns of S. aureus HAR22 strain to various antibiotics (S1) were performed by disk diffusion (Kirby-Bauer) and minimum inhibitory concentration (MIC) was determined by both dilution and Etest (AB BioMérieux, Solna, Sweden) methods according to the CLSI guidelines

20

. The sub-inhibitory

concentrations values used were half the MIC (S1).

2.3.2. ZnO preconditioning and antibiotic susceptibility Overnight cultured HAR22 (500 µl) were incubated on ZnO nanostructured flower-coated Zn samples for 24 h without shaking at 37 oC. The culture supernatants were gently removed by aspiration. Then, 500 µl of five different antimicrobial drugs including rifampicin, gentamicin, ciprofloxacin, trimethoprim and vancomycin (Sigma Aldrich, St. Louis, MO, USA) at sub-inhibitory concentrations (half the MIC) (S1) were added to the adherent biofilm cells and incubated for 18 h at 37 oC. A volume of 500 µl of fresh TSB was then added and the biofilms were scraped from the samples’ surfaces as above described. Biofilm cell suspensions were serially diluted in TSB and plated on TSA with glass beads for CFUs determination (Scheme 2a). All the experiments



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were performed at least with three independent assays with internal replicates and averaged. To assess the eventual emergence of antimicrobial resistant mutants, the biofilm cell suspensions previously in contact with sub-inhibitory concentrations of antibiotics were also spread on TSA agar plates containing rifampicin, ciprofloxacin, trimethoprim and vancomycin at concentrations above the resistance MIC breakpoints (Scheme 2 b; see S1).

Scheme 2. Representation of the assays used to assess the preconditioning effect of the ZnO-nanostructured flower coating on Staphyloccocus aureus (MRSA) cells challenged with a) distinct antibiotics and b) mutants selection.

2.3.3. Morphological characterization of the biofilms To characterize the morphology of the biofilms cells grown on the ZnO - nanostructured flower coated Zn, cells were fixed on the samples’ surfaces as above described and analyzed by SEM (JEOL- JSM7001F) and the elemental chemical composition was analyzed by the respective energy dispersive X-ray (EDS). Prior to these analyses the samples were coated with a thin layer of conductive chromium (Polaron E-5100) to increase the conductivity. Topographical images of the biofilms were acquired by AFM (Nanosurf Easyscan 2) in the noncontact mode. The AFM measurements were carried out in air at room temperature with NanoAndMore GMBH® silicon probes. Images were acquired at a resolution of 512 × 512 pixels. 2.4. In vitro corrosion evaluation 2.4.1. Potentiodynamic polarization measurements The in vitro degradation behavior of bare and ZnO-coated Zn was studied by immersing the samples in simulated body fluid (SBF) solution (NaCl 137.5 mM, NaHCO3 4.2 mM, KCl 3.0 mM, K2HPO4 1.0 mM, MgCl2·1.5 mM, CaCl2 2.6 mM, Na2SO4 0.5 mM, (HOCH2)3CNH2 50.5 mM at pH 7.4) at 37 oC

21

.

Potentiodynamic measurements were carried out in a three-electrodes electrochemical cell with the sample as the working electrode, a platinum coil as counter electrode and a SCE as reference electrode. These measurements were carried out in a Voltalab PGZ 100 potentiostat (Radiometer Analytical) within the range of ± 10 mV from OCP until ± 1 V vs SCE at a scan rate of 1 mV/s. The Tafel extrapolation method used to determine the


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corrosion current density (icorr) and the corrosion potential (Ecorr) was performed by using OriginPro 8.5.0 software.

2.4.2. Physico-chemical characterization of the surfaces The microstructure of the products formed on the surface of bare or ZnO-coated samples upon anodic polarization in SBF at 37 oC was analyzed by SEM using a JEOL-JSM7001F or Hitachi S2400 apparatus and the elemental chemical composition by the respective EDS. Raman spectra were collected using the radiation source with a solid-state laser operating at 532 nm with an output power of 20 mW (Horiba LabRAM HR800 Evolution). A spectrograph with a 600 lines/mm grating and a 50 objective lens were used. Spectra were obtained with acquisition time of 10 s and 10 accumulations.

2.5. Statistical analysis One way ANOVA with a multiple Comparisons versus Control Group using the Holm-Sidak method (p ≤ 0.05) was used with SigmaStat v3.10 (Systat Software, Inc., Erkrath, Germany) software.

3. Results and Discussion

3.1. Biofilm formation on bare and ZnO-coated Zn samples The fabrication of distinct and reproducible ZnO coatings morphologies on Zn was successfully attained, with the detailed electrodeposition process already discussed elsewhere

12

. As depicted in Fig. 1 a) the ZnO-

nanostructured flower coating was heterogeneously distributed on the Zn surface with the ZnO units (either from the flowers or from the non-flowered coating) composed by nano-sized-hexagon crystals. Both the rod-like and hexagon coatings covered uniformly Zn surface, each one with micro sized rod-like and hexagon ZnO crystals units, respectively. When envisaging biomedical applications the formulation of the material per se is just the first step on a long process. A number of essential assays have to be performed to assess the feasibility of the novel materials before their clinical application. In this sense, to understand how the structure of ZnO coatings on Zn samples influenced the formation of bacterial biofilm, MRSA were inoculated on bare or ZnO-coated Zn samples for 24 h. When compared to bare Zn, the planktonic cells in contact with ZnO coated samples had a decrease of one log in the colony forming units (CFUs), whereas the cells composing the biofilm presented a decrease of two log CFUs solely when in contact with the ZnO-nanostructured flower coating (Fig. 1 b). No change in cells viability was observed in the rod-like and hexagon coated samples (Fig. 1 b). While there is no apparent relation between the amount of electrodeposited ZnO (S2) and the antibacterial effect depicted in Fig. 1 b), ZnO morphology whatsoever seems to. Although no differential effect was depicted on the planktonic cells’ viability, the same



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was not true for biofilm formation where the size of ZnO crystals was influencing the antibiofilm properties of the nanostructured flower coating against MRSA. This result is in agreement with the recent finding of Cai et al. 22

that reported a differential antibacterial activity for distinct ZnO NPs morphologies. Jansson et al.

McGuffie et al.

14

13

and

that had also reported an antibiofilm ability for different shaped ZnO NPs-derived coatings,

refereed that although these coatings allowed cell adhesion, bacterial killing occurred upon contact with ZnO NPs afterwards. In the same line of thought, the decreased cell viability depicted in the biofilm caused by the ZnO-nanostructured flower coating seemed to result from the direct contact of cells with ZnO NPs as similar planktonic cells viabilities were depicted for all ZnO-coatings (Fig. 1) .

Fig.1. Bare or ZnO-coated Zn a) micrographs with the insets revealing the ZnO crystals subunits composing the coatings and b) viability of the methicillin resistant Staphylococcus aureus (MRSA) planktonic cells (grey) or composing the biofilm (black) incubated on bare or ZnO-coated Zn surfaces for 24 h. The results shown are means of, at least, three independent experiments with bars representing average errors and different letters p-values below 0.05.


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In addition to the antibiofilm effect of the here studied nanostructured flower ZnO coating against MRSA, the same effect was already proven against clinically relevant Candida spp. 23. Together, these results suggest that this ZnO nanostructured flower coating has an antibiofilm effect against a wide variety of microbes (both pathogenic bacterial and fungi) which suggests that using this coating for the treatment of implant-related infections is a promising strategy. In an attempt to further elucidate the antibiofilm effect of nano-sized ZnO, the biofilms of MRSA formed on the top of nano and micro-sized ZnO-derived coatings were investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM).



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Fig. 2. Biofilms of methicillin resistant Staphylococcus aureus (MRSA) formed on the top of bare or ZnO-coated Zn samples a) scanning electron microscopy (SEM) images, b) surface parameters calculated by the Nanosurf Easyscan 2 software using three random areas per sample scanned across 25 µm2 areas and, c) topographical and d) amplitude atomic force microscopy (AFM) images. Bars represent average errors and different letters p-values below 0.05.

As depicted in Fig. 2, SEM and AFM images revealed that mature biofilms were grown on bare and ZnO-coated samples, as illustrated by the high level of cell-to-cell aggregation, by the existence of channels within the biofilm matrix, by the visualization of polysaccharide in localized areas of the biofilm (smooth structures on the top of several cells in Fig. 2 a) and by the presence of cells grouped into mushroom-like structures (Fig. 2 a). However, different morphological features can be highlighted on the different biofilms. The presence of fissures in the biofilms was observed for bare, rod-like and hexagon coatings but the frequency of those cracks over the surface was distinct depending on the sample (Fig. 2 a): the highest number of fissures was found on the biofilm formed on bare Zn, followed by the one formed on the rod-like coating and hexagon coating, both cases with almost no fissures. On the other hand, the biofilm grown on the nanostructured flower coating was more heterogeneous, with a few number of polysaccharide islets and prominent mushroom-like structures separated by large distances, suggesting the covering of the flower-like structures (Fig. 1 a). The presence of tube-like structures revealed a type of arrangement that is not usually seen in a healthy biofilm matrices (Fig. 2 a). Although some fissures in the biofilm were also observed in the nanostructured flowers, those were predominantly located on the tube-like structures. All these morphological features contributed for an increased roughness of this biofilm as corroborated by the maximum peak height (Sp) and the maximum valley depth (Sv)


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parameters measured by AFM. The values of these roughness parameters on the flower-like coating statistically differed from those of the biofilms grown on bare, rod-like and hexagon coatings (Fig. 2 b). For a more detailed characterization of the morphology of the biofilms, topographical (Fig. 2 c) and amplitude (Fig. 2 d) AFM images were taken. Overall, the biofilms showed high surface covering with cell sizes of 1 µm in average diameter. However, differences were detected in the cells shape within the distinct samples. Whereas on bare Zn cells exhibited a smooth surface, the ZnO-coated samples cells presented an irregular shape, showing multiple bumps at the surface in several bacterial cells (Fig 2 c and d). Since the cell-surface roughness and cell shape are indicators of cell healthiness (a healthy cell does not show signs of pores, holes, grooves or breakages in the cell envelope) the occurrence of bumps at the cells’ surface suggested that ZnO coatings may be impacting cell wall synthesis and growth. Among the biofilms grown on the top of ZnO-coated samples, there were other relevant morphological features that distinguished the biofilm formed on the top of the nanostructured flower coating from those grown over rod-like or hexagon coated Zn samples, e.g. looser cell-to-cell connections and increased number of irregular shaped, wrinkled and lyzed cells (Fig 2 c and d). Although there were distinct morphological features distinguishing the biofilm formed on the top of the nano-sized ZnO coating, these were not enough to explain its antibiofilm activity (Fig. 1 b). In an attempt to elucidate the antibiofilm activity lying behind ZnOnanostructured flower coating, further assays were performed.

3.2. Effect of ZnO-nanostructured flower coating against methicillin resistant Staphylococcus aureus (MRSA) 3.2.1. Antibiofilm effect – from a cell point of view Bacterial cells, as part of the biofilm formed on the ZnO-nanostructured flower coating, were individually analyzed by AFM and SEM. Fig. 3 a) and b) depict representative AFM images of three cells, one of them with some visible protuberances on the cell surface. In Fig. 3 c) the increased number of these protuberances observed in the cell surface can be related with cells lysis and death, since the highest number of protuberances was observed on busted cells. To the best of our knowledge, this work is pioneer in reporting such a detailed characterization of bacterial cells grown on a ZnO NPs-derived coating

13-15

. On Fungi cells, similar

protuberances were reported for Candida parapsilosis when in contact with the ZnO-nanostructured flower coating. The number of protuberances were also related with cell death 23. The EDS results for the protuberances found in MRSA cells revealed that these were mainly composed of zinc and phosphorous (Fig. 3 d i), which clearly contrasts with the low zinc and phosphorous content in the bacteria cell wall (Fig. 3 d ii). This data agrees with chemical elements detected on C. parapsilosis, which in addition to zinc and phosphorous had also calcium 23

. With this data, no direct correlation can be withdrawn from the precipitation of these zinc-phosphate-derived

compounds and ZnO antimicrobial effect. If this precipitation is a consequence of ZnO cell wall damage or if it is itself responsible for the observed cell wall damage is still a mystery to be solved.



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Fig. 3. Detailed analysis of the methicillin resistant Staphylococcus aureus (MRSA) biofilm cells grown on the top of the ZnO-nanostructured flower coating; representative atomic force microscopy (AFM) a) topography image and b) corresponding 3D projection, representative scanning electron microscopy (SEM) images of c) biofilm and d) a single busted cell with the corresponding EDS analyses.

3.2.2 Preconditioning of MRSA with ZnO nanostructured flower coating A myriad of studies have explored the antibacterial mechanisms of ZnO NPs 3. This antibacterial effect has been described as resulting from the i) generation of ROS (reactive oxygen species), ii) release of Zn2+ from ZnO and iii) penetration and disorganization of the bacterial membrane upon contact with nano sized ZnO crystals 3. The approach used in the majority of these studies is focused on exploring similar metabolic pathways (e. g. ROS responsive genes, activity of antioxidant enzymes or ROS detection) 3. In an attempt to bring a novel insight into the antibiofilm activity of ZnO, a novel approach was here tested: cell preconditioning, which is a phenomenon where a stressful stimulus to the cell confers it the ability to protect itself from a subsequent stimulus caused by the same type of stress 24. An example of this is the preconditioning of cells in BHI containing dipyridyl, an iron chelator to reduce intracellular iron stores, and prepare cells to survive in environments with iron-deficiency 25. In this context, when a particular kind of stress (e. g. antibiotics) is applied to MRSA cells that were previously exposed to ZnO, would prepare the cell to respond better when compared with a cell that had no preconditioning. To evaluate this, the viability of the biofilm cells was examined after sequential exposure to ZnO nanostructured flowers coating and to five antibiotics, at sub-inhibitory concentration, that disrupt distinct bacterial metabolic pathways (Scheme 2, Fig. 4).


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Fig. 4. Preconditioning of methicillin resistant Staphylococcus aureus (MRSA) inoculated in bare or ZnO-nanostructured flower coated Zn sample. After forming a biofilm (24 h) the MRSA cells were further inoculated (12 h) with or without the addition of an antibiotic (Scheme 2). The results shown are means of, at least, three independent experiments. Bars represent average errors and * p-values below 0.05; Dihydrofolic acid, DHF and tetrahydrofolic acid, THF.

As presented in Fig. 4, the viability of cells composing the biofilm formed on the ZnO-nanostructured flower coating presented different response that may be related to the underlying antibacterial effect of each antibiotic. While without adding antibiotics the viability of cells decreased in the presence of ZnO-nanostructured coating (Fig. 1 and 4), when combining antibiotics, different responses were attained when comparingthe bare Zn control samples with the ZnO-nanostructured flower coating (Fig. 4). An evident increase in cells viability was observed when combining vancomycin, a glycopeptide antibiotic that inhibits cell wall peptidoglycan at a late stage 26, rifampicin, a potent antibiotic that inhibits bacterial RNA polymerase antibiotic that rapidly blocks DNA replication

28

27

or ciprofloxin, a fluoroquinolone

. Both vancomycin and rifampicin resulted in a statistically

significant increase of the biofilm cells viability. A decrease in viability of the biofilm cells with gentamicin was observed, although with no statistical significance. Gentamicin is an aminoglycoside antibiotic that compromise protein synthesis

29

. No difference in the biofilm cells’ viability was observed when using trimethoprim, an

antibiotic that exerts its antimicrobial activity by blocking the formation of folic acid 30.



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Exposure of MRSA to sub-inhibitory concentrations of antibiotics was previously reported as promoting the emergence of mutants resistant to the applied antibiotic 6. A possible emergency of mutants could result from the exposure of cells to nano-sized ZnO combined with sub-inhibitory concentration of antibiotics. To confirm that hypothesis cells were grown at concentrations above the MIC breakpoint (S1) after the preconditioning assay (Scheme 2 b). A proportion of cells treated with ZnO NPs, ciprofloxacin and trimethoprim became resistant to these antibiotics. The generation of these resistant phenotypes was possibly due to stress-induced mutagenesis 28, 30

either by the antibiotics themselves or possibly by the ROS generated by the presence of ZnO NPs

1-3

. The

generation of hydroxyl radicals originated from a ZnO NPs coating, has been reported to play a key role in its antibiofilm activity 15. The generation of ciprofloxacin and trimethoprim resistant phenotypes did not allow us to go any further in the inference of the underlying antibiofilm effect of ZnO NPs. However, a very pertinent question in terms of health-care and veterinary systems was raised, as the broad use of ZnO NPs as an active ingredient, that goes from dermatological applications to food additives 2, may be incompatible with the use of such antibiotics. As the emergency of mutations was not observed for neither rifampicin nor vancomycin, one can infer that ZnO NPs preconditioning induced stress-responses that allowed cells to cope better with the damage caused by these two antibiotics. The protection response elicited by ZnO NPs on the MRSA cells can thus be related to the mode of action of both rifampicin and vancomycin 26, 27. Rifampicin strongly binds to the β-subunit of DNA-dependent RNA polymerase inhibiting RNA synthesis

27

. Vancomycin, inhibits a late stage of cell wall peptidoglycan

biosynthesis by binding to the terminal peptide chain avoiding cell wall cross-linking and promoting a deficient cell wall and bacterial death

26

. Indeed, cell wall damage due to the contact with ZnO NPs has been already

addressed 1-3 and our own results clearly evidence the bacterial cell wall deficiencies after exposure to ZnO (Fig. 2 and 3). While rifampicin and vancomycin induced biofilm viability was proposed to be due to similar stress responses induced by ZnO NPs, the synergistic effect with gentamicin although much speculative could somehow result from a ZnO overloading or blocking efflux pumps leading to higher effective doses of the antibiotics. It is noteworthy to mention that only few publications reported an integrated treatment of free ZnO NPs with antibiotics (e.g. 31, 32) and, to the best of our knowledge, only the work published by Applerot et al. 15 addressed it in the form of a ZnO NPs-derived coating. These authors

15

reported a moderate susceptibility of planktonic S.

aureus cells, but not to the biofilm forming cells, in the presence of chloramphenicol, an antibiotic that impairs protein synthesis through the inhibition of peptidyl transferase activity of ribosome 50S subunit. Overall, cell wall and RNA damage have been suggested to be related with ROS generation and/or Zn2+ release, with the ROS generation being widely proven to be related with ZnO

1-3

. Whatsoever, Zn2+release has been

disregarded in the sense that Zn2+ release does not occur as an isolated event. In the presence of the most typical inorganic molecules, e.g. phosphates, Zn can readily precipitate in the form of Zn-derived phosphates 23. To have


Page 15 of 35

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a better understanding of Zn/ZnO dissolution, the degradation behavior of ZnO-coated Zn samples was further evaluated.

3.2.3 In vitro degradation of ZnO-coated Zn When a resorbable Zn biomaterial gets in contact with a physiological medium, electrochemical reactions leading to the metal reduction and consequently to Zn2+ release take place. These events that may occur even before cells attach to the surface will influence the material-cell interplay. To simulate the degradation behavior of ZnO-coated Zn, potentiodynamic polarization experiments were conducted in SBF. As depicted in Fig. 5 a) and b) the electrochemical responses of all ZnO-coated Zn samples were similar. A slightly higher corrosion potential (Ecorr) was observed for the rod-like coating, followed by the nanostructured flower coating. The hexagon coating presented the lowest Ecorr among all tested conditions. A different trend was observed for the corrosion current density (icorr). The Tafel extrapolation revealed that the rod-like coating presented the lowest icorr value. Higher values resulted for the nanostructured flower and the hexagon coatings. These observations suggest that upon in vitro degradation important physico-chemical alterations occurred on these materials’ surfaces. As depicted in Fig. 5 c) the corroded surfaces in simulated physiological conditions presented distinct morphological features. In all ZnO-coated samples degradation resulted in the formation of lamina-like structures; however distinct morphological features were revealed according to ZnO crystals morphology. The degradation of the nanostructured flower coated samples resulted in the agglomeration of corrosion products on the top of the flowers forming mushroom-like structures. On the rod-like and hexagon coated samples precipitation of small cubic-like structures occurred on what seems to be the remaining of micro sized ZnO crystals. To characterize these products, EDS and Raman analyses were performed to identify the species that participated in the built-up of these distinct corrosion layers. EDS analyses in Fig. 5 c) revealed the accumulation of chloride on the corrosion layer of micro-sized Zn coatings - the rod-like and the hexagon ZnO coatings.



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Page 16 of 35

Fig. 5. Assessment of the in vitro degradation of ZnO-coated Zn samples by a) potentiodynamic polarization curves and b) corresponding electrochemical values. Anodic corroded surfaces of ZnO-coated Zn samples in SBF solution at 37oC c) micrographs with the corresponding the EDS quantifications, d) Raman spectra and e) EDS maps. Raman spectra were compared with the patterns deposited RRUFF database (http://rruff.info) and the peaks matched those for: hydrozincite, HDZ (ID: R050635); hopeite, HPT (ID: R050254); simonkolleite, SMK (ID: R130616); smithsonite, SMT (ID: R040035) and zincite, ZnO (ID: R050635).

On the ZnO nanostructured flower coating the accumulation of phosphorous was detected instead. Then, the corrosion products consisted of simonkolleite [Zn5Cl2(OH)8] and hopeite [Zn3(PO4)2]

23, 33-35

, respectively. The

presence of carbon can be also related with the precipitation of smithsonite [ZnCO3] and hydrozincite [Zn5(CO3)2(OH)6] 23, 33-35. The formation of these corrosion products was confirmed by Raman spectroscopy. As shown in Fig. 5 d), the presence of peaks assigned to simonkolleite was consistent with the presence of chloride on the corroded surfaces of rod-like and hexagon ZnO coatings. Despite the peaks overlap, the presence of hopeite was suggested by the distorted peak at 1047 cm-1 detected on the corroded surface on ZnO nanostructured flower coating. This observation is in close agreement with the detection of phosphorous on the degraded surface of the material. The presence on zincite can be related either to the ZnO that composes the


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coatings or to the formation of ZnO as corrosion product. Lastly, the presence of carbonated-derived compounds in all samples was confirmed as hydrozincite as identified by Raman technique; smithsonite was only detected on the microstructured ZnO coatings. In order to determine the spatial distribution of the corrosion products over the degraded surfaces of ZnO-coated Zn samples, EDS maps were drawn. As shown in Fig. 5 e) chloride and carbon were predominantly detected on the lamina-like structures area of the corroded surfaces of ZnO rod-like coating; the EDS oxygen mapping is more intense on what seems to be the remaining of the ZnO coating. On the degraded surface of ZnO hexagon coated Zn samples, there was a preferential formation of simonkolleite over the carbon-derived compounds, as revealed by the low carbon percentage (Fig. 5 c) and the low intensity carbon map. On the corroded surfaces of both rod-like and hexagon coated samples, overlapping of chloride and oxygen in the maps suggested the preferential deposition of simonkolleite on top of the ZnO crystals. Finally, the ZnO nanostructured flower coated samples presented no evidence of simonkolleite, but hopeite and hydrozincite instead. The precipitation of this phosphate and carbon-derived compounds, coincident with the EDS oxygen mapping, support the idea that the corrosion products preferentially precipitate on the top of the nanostructured flowers. The favored deposition of Zn-derived corrosion products on the ZnO flower structures was already reported by other authors

23, 35

. These new data depicted in this work suggests that distinct morphological ZnO

features promoted the precipitation of specific Zn-derived corrosion products: on nano-sized ZnO crystals there was a preferential deposition of hopeite and hydrozincite while on micro sized ZnO crystals the preferentially deposition of simonkolleite occurred along with hydrozincite and smithsonite. Similar results to the micro sized ZnO-coated Zn samples were obtained for bare Zn samples (S3). Despite the similarity of the polarization curves (Fig. 5 a), the degradation of ZnO-coated Zn samples resulted in the built-up of a different corrosion layers. The basis for these differences seems to lay on the unique surface properties of nano-sized ZnO crystals that may be as well contributing for the different MRSA colonization observed on these ZnO-coatings (Fig. 1 b).

3.2.4 An integrated overview In summary, a previous work have shown that the mechanism of antibacterial activity of ZnO could be related with damaged caused by ROS generation and/or toxicity of Zn2+ released from ZnO 3. It is well-known that ROS can have a detrimental effect on cells viability since it readily reacts with almost every type of organic biomolecules: nucleic acids, lipids, carbohydrates, proteins, DNA and amino acids. Our results contribute to the state of the art by suggesting that similar ROS effects may be contributing for the observed antibiofilm activity of ZnO NPs-derived coatings. It is possible that the generation of mutants resistant to ciprofloxacin and trimethoprim, the RNA and the cell wall damage may be in some way related with the proposed generation of ROS by ZnO NPs (Fig. 6 a). In this intricate antibiofilm effect of the ZnO-nanostructured flower coating, toxicity from Zn2+ could play a preponderant role. The simplistic view of Zn2+ as free moving ion (Fig. 6) is far from representing the antibacterial effect on MRSA cells. There are much more complex reactions underlying



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this effect since Zn2+ dissolution mandatorily entails the formation of Zn-derived corrosion products

Page 18 of 35

23, 35

. The

differentiated presence of hopeite on the corroded ZnO-nanostructured flower coating, may be somehow related with the detection of ZnP-derived protuberances on the bacteria cell wall (Fig. 3 d). Interestingly, in both MRSA (Fig. 3 d) and C. parapsilosis 23 the composition of the protuberances matched the composition of the corrosion products found on the ZnO nanostructured flower coating (Fig. 5 d and e) 23. Hopeite precipitation seems to have a strong correlation with microbial death

36

trough cell wall damage (Fig. 6). Now if this is a direct or indirect

consequence of the exposure of cells to ZnO NPs, and its possible relation with the corrosion products detected, is still as a brand new and unexplored pathway opened by our work.

Fig. 6. Schematic representation of the a) antibiofilm effect of ZnO nanostructured coating on a methicillin resistant Staphylococcus aureus (MRSA) cell and b) corrosion products formation in the presence of MRSA cells. ROS (reactive oxygen species) damage, Zn2+toxicity and cell wall damage are effects already described in the literature 3. The fine-tuned metabolic effects inferred from our work are marked with circles.

4. Conclusions Bacterial proliferation on materials’ surfaces is a critical step in the development of implant related infections. Once a bacterial biofilm is formed, the cells can become much more resistant to antibacterial agents and to the human immune system than their planktonic counterparts. Therefore, research efforts towards the modification of the biomaterial surfaces towards inhibiting biofilm proliferation are of utmost importance. In this sense, the well- known antibacterial agent ZnO was used to functionalize the surface of metallic Zn, a new material emerging in in the field of resorbable implants. In terms of the antibiofilm effect, only the nano-sized ZnOderived coating was effective against MRSA biofilm formation, an important pathogenic strain in medical implant related infections. In an attempt to explain the effect laying behind this antibiofilm action, an innovative approach using the preconditioning response to ZnO NPs, revealed distinct MRSA cells responses when challenged with different antibiotics. The combined response with gentamicin suggested that the ZnO NPs


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antibiofilm action was probably not related to protein synthesis damage. The production of mutants with increased resistance to ciprofloxacin and trimethoprim suggested that a stress-induced mutagenesis conferring resistance to these antibiotics was probably triggered by this ZnO NPs-derived coating on MRSA. The promoted growth of MRSA in the presence of sub-inhibitory concentrations of rifampicin and vancomycin, which are antibiotics that inhibit cell wall and RNA synthesis, respectively, suggested that the preconditioning of bacteria with ZnO elicited envelope and ribosomal stress responses that helped bacteria to persist to these two antibiotics. It can be suggested that ZnO, rifampicin and vancomycin were sharing common mechanism of action, putatively related to inhibition of RNA and/or cell wall synthesis. Besides the attempt to elucidate the ZnO NPs antibiofilm effects, relevant health issues were also raised with this study. While the higher efficacy of ZnO-nanostructured coating together with gentamicin might be useful for the treatment of MRSA infections, the use of trimethoprim may be quite inefficient. On the other hand the use of ciprofloxacin, rifampicin and vancomycin might even aggravate these infections. As surface modifications of a material leads to variations on its degradation behavior, the evaluation of Zn coated with different ZnO coatings morphologies revealed minor alterations in the corrosion rate of micro-sized (rod-like or hexagon) or nano-sized (nanostructured flower) coated samples. Whatsoever, different corrosion products occurred on these materials surfaces with their composition depending upon ZnO crystals morphology. On the micro-sized ZnO crystals a preferential deposition of simonkolleite was observed while on the nano-sized ZnO crystals hopeite was the main corrosion product. The presence of hopeite was also speculated to influence the antibiofilm role of the nanostructured flower coating through bacteria cell wall damage. Overall, our results of MRSA antibiofilm activity together with our previous published data on antifungal properties 23, depicts this ZnO nanostructured coating as a promising material for further in vivo and clinical trial studies in the field of implanted-related materials.

Acknowledgements The author Marta Alves would like to thank FCT (SFRH/BPD/76646/2011) for providing financial support. UID/QUI/00100/2013 (Funded by FCT). This work was financially supported by: Project LISBOA-01-0145FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT - Fundação para a Ciência e a Tecnologia; Project PTDC/FIS-NAN/0117/2014 from Fundação para a Ciência e a Tecnologia. Ons Bouchami was supported by fellowship SFRH/BPD/98317/2013 from Fundação para a Ciência e a Tecnologia.

Supporting information: S1. Antimicrobial susceptibility of MRSA strain HAR22.



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Page 20 of 35

S2. Weight of ZnO-coatings electrodeposited on Zn using a constant cathodic current of -1.9V s for 20 s, 100 s and 300 s. These conditions resulted in the electrodeposition of ZnO coatings with nanostructured flower, rod-like or hexagon morphologies, respectively. The results shown are means of, at least, five independent experiments with bars representing average errors and different letters p-values below 0.05. S3. Assessment of the in vitro degradation of the bare Zn sample by a) potentiodynamic polarization curves and b) corresponding electrochemical values. Anodic corroded surfaces of bare Zn samples in SBF solution at 37oC c) micrograph with the corresponding the EDS quantification, d) Raman spectrum and e) EDS maps. Raman spectrum was compared with the patterns deposited RRUFF database (http://rruff.info) and the peaks matched those for: hydrozincite, HDZ (ID: R050635); simonkolleite, SMK (ID: R130616); smithsonite, SMT (ID: R040035) and zincite, ZnO (ID: R050635).

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Graphical Abstract



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Infection

ZnO crystal Corrosion products (hopeite and hydrozincite) Methicillin resistant Staphylococcus aureus (MRSA)

Graphical abstract ACS Paragon Plus Environment

Page 24 of 35

Page 25 of 35

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Bacterial suspension Planktonic cells

Mini cuvette

Bare or ZnO-coated Zn Samples

Biofilm forming cells

Epoxy resin Methicillin resistant Staphylococcus aureus (MRSA)

Scheme 1

ACS Paragon Plus Environment


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Bacterial suspension

Page 26 of 35

Antibiotic a)

b) Antibiotic

ZnO crystal Methicillin resistant Staphylococcus aureus (MRSA)

Scheme 2


Page 27 of 35

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ZnO COATED Zn

BARE Zn

Nanostructured flower

a) 1 µm

100 µm

100 µm

ZnO COATED Zn Rod-like

Hexagon

1 µm

100 µm

Fig. 1 ACS Paragon Plus Environment

1 µm

100 µm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Log (CFUs)


14 12 10 8 6 4 2 0

a' a

BARE Zn

Page 28 of 35

a'

a' b

b'

b

b


Rod-like

ZnO COATED Zn


Hexagon

b)

Page 29 of 35

ZnO COATED Zn

BARE Zn


1 µm

1 µm

1 µm


b)

3 Peak height (Sp)

50 µm

50 µm

1 µm

a)

Hexagon

Valley depth (Sv)

b Distance (µm)

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2 b' a

1

a'

a a' 50 µm

1 µm

a'

50 µm

0 1 µm

a

1 µm

1 µm


BARE Zn


Rod-like

ZnO COATED Zn

Hexagon


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

BARE Zn

ZnO COATED Zn


Page 30 of 35

BARE Zn c)


ZnO COATED Zn


ZnO COATED Zn Hexagon

Rod-like


Hexagon

d)

b)

Y*

1 µm

a)

0 µm

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Line ft 326 nm

Page 31 of 35

0 µm

X*

1 µm

c)

Atomic % Element

i

ii

C

51.5

70.6

O

36.3

26.8

P

4.0

1.7

Cl

0.6

--

Zn

7.7

0.9

i ii

1 µm

500 nm


d)


Page 32 of 35

6

Plasmid

5

Cell membrane

4 3 2

*

1 0

Bare Zn

Ribosome

Capsule

Cytoplasm

Cell wall

ZnO

Nucleoid

NO ANTIBIOTIC

METABOLITE SYNTHESIS



DHF

CFU s(x 107)

CFUs (x 109)

3 2 1

10

5

8

4 3 2

Bare Zn

ZnO

0

Bare Zn

ZnO

TRIMETHOPRIM

Cell wall synthesis

DNA synthesis 3

8

4

0



10

6

2

1

GENTAMICIN

Fig. 4

6

STRUCTURAL INTEGRITY



RNA synthesis

CFUs (x 1010)

4

DNA



THF

Folic acid synthesis

Ribosome

0



NUCLEIC ACID SYNTHESIS

CFUs (x 107)

PROTEIN SYNTHESIS

*

CFUs (x 107)

CFU s(x 1013)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6 4

2

1

*

2

Bare Zn

ZnO

RIFAMPICIN ACS Paragon Plus Environment

0

Bare Zn

ZnO

CIPROFLOXIN

0

Bare Zn

ZnO

VANCOMYCIN

Page 33 of 35

-0.2

V (V vs. SCE)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nanostructured Flower Rod-like Hexagons

-0.4 -0.6

a)

Ecorr

Icorr

(V vs. SCE)

(μA cm−2)

Nanostructured Flower

-0.999

1.786

Rod-like

-0.961

0.335

Hexagon

-1.025

3.090

Material

-0.8 -1.0 -1.2 -1.4 -1.6 -1.8

-8

-6

-4

Log |i|

-2

0


b)

Fig. 5

// 400 600 1000 1200 Wavenumber (cm-1)


Page 34 of 35

c)

200

2 µm

20 µm

HDZ 1083.8 cm-1 SMT 1127.6 cm-1

At. % 17.7 36.4 1.1 44.8

HDZ 431.8 cm-1 ZnO 476.0 cm-1 ZnO 561.9 cm-1

20 µm

Element C O Cl Zn

SMK 269.0 cm-1 ZnO 324.9 cm-1

2 µm

// 400 600 1000 1200 Wavenumber (cm-1)

SMK Zn5Cl2(OH)8

200

Hexagon

HDZ 1105.3 cm-1 SMT 1159.8 cm-1

At. % 23.0 32.5 0.7 43.8

SMK 254.7 cm-1 ZnO 323.0 cm-1 HDZ 392.8 -1cm-1 SMK 422.9 cm ZnO 476.0 cm-1 ZnO 551.4 cm-1

20 µm

ZnO

200

Element C O Cl Zn

HPT 1047.0 cm-1 HDZ 1068.5 cm-1

ZnO, HPT 338.7 cm-1 HDZ 398.1 cm-1 ZnO 484.8 cm-1 ZnO 553.2 cm-1

At. % 15.5 35.7 0.8 47.9

SMT ZnCO3 HDZ Zn5(CO3)2(OH)6

ZnO

HPT Zn3(PO4)2

Relative intensity (a. u.)

Element C O P Zn

HDZ Zn5(CO3)2(OH)6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Rod-like ACS Applied Materials & Interfaces Nanostructured flower Rod-like Flowers

d)

// 400 600 1000 1200 Wavenumber (cm-1)

e)

Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a)

b)

500 nm



ZnO crystal Corrosion products (hopeite and hydrozincite) Methicillin resistant Staphylococcus aureus (MRSA)


New Insights into Antibiofilm Effect of a Nanosized ZnO Coating

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