Background

Spread of antibiotic-resistant bacteria is a major concern worldwide due to infections leading to high morbidity and mortality rates [1, 2]. The impact of antibiotic resistance on health and disease has been further underlined by recent developments in understanding the microbiome [3,4,5].To combat this issue, alternative compounds such as natural products and nanoparticles have been explored [6]. Metallic nanoparticles (NPs) such as zinc, cerium, aluminum, nickel, zirconium, magnesium, and titanium dioxide have the potential to act as antimicrobial agents due to their small size and high surface-to-volume ratio [7,8,9,10,11]. In particular, titanium dioxide has been shown to have enhanced photoelectrochemical performance and effective photocatalytic properties [12, 13] These properties allow them to generate reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals, and superoxide anions, that can penetrate bacterial cell walls and disrupt cell functions, leading to cell death [14,15,16]. Additionally, NPs can be modified with organic compounds to enhance their antimicrobial activity, making them promising candidates for treating infections caused by antibiotic-resistant bacterial strains [17, 18]. One of the NPs that has gained attention for its antimicrobial properties is titanium dioxide (TiO2) [14].

Titanium dioxide (TiO2) is a unique material that displays various physical and chemical characteristics, including the ability to produce ROS when exposed to light, creating pairs of holes. These electron-hole pairs can react with water or oxygen and, as a result, generate ROS [19, 20] Multiple studies have demonstrated that TiO2 exhibits good antibacterial and antifungal activity against a broad range of Gram-positive and Gram-negative bacteria [21]. TiO2 has three different crystalline phases: rutile, anatase, and brookite; each can exhibit metastable behavior as various factors affect its crystal shape and cause a possible conversion among the three phases [22, 23]. It was shown that anatase and rutile phases are stable and have great photocatalytic activity [24,25,26]. The cytotoxicity of TiO2 varies depending on the crystal shape, as results of several studies showed that the toxicity of TiO2 was dependent on the concentration and the crystal phase; therefore, the response of bacteria to the crystals can be different [16, 27,28,29].

Using natural compounds can greatly enhance the inherent physical and chemical properties of NPs [30, 31]. For instance, TiO2 has been used as an antimicrobial additive in polyamide 12 powder, and nitrogen-doped titanium dioxide nanoparticles have demonstrated photoinduced antimicrobial properties under visible-light irradiation [32, 33] The small size, high surface energy, and strong hydrophilicity for surface hydroxylation may increase TiO2 NPs compatibility with polymer materials [34, 35]. Incorporating inorganic NPs into organic polymeric nanocomposites is a promising strategy to enhance their antimicrobial properties [36,37,38]. For example, monoterpenes incorporated with TiO2 had a greater antimicrobial effect [39]. Monoterpenes are natural products containing active compounds such as phenols, flavonoids, and terpenoids. Geraniol is a mono-terpenoid with low toxicity and high solubility, and when combined with TiO2 NPs, it may exhibit enhanced antimicrobial properties [40, 41].

The objectives of this study were to: (a) synthesize TiO2 NPs and combine them with geraniol; (b) assess the antimicrobial activity of this material against planktonic and biofilm-forming bacterial cells. This is a novel study focusing on the combined effect of geraniol and TiO2 NPs and their synergistic activity against E. coli, Staphylococcus aureus, and MRSA. This distinguishes it from the previous studies that have mainly focused on the effects of individual agents.

Results

Characterization of the TiO2 NPs

Synthesized TiO2 NPs and their combination with GER were characterized through elemental analysis, and GER was detected on the surface of TiO2. Scanning electron microscopy revealed the size, shape, and surface morphology of the anatase and rutile TiO2 material and showed that NPs were successfully formed. The anatase form of the TiO2 had clusters of particles with an average size of 300 ± 100 nm. High-magnification imaging revealed dense nanocrystalline domains on the surface. The anatase form of the TiO2 with GER resembled unmodified TiO2 NPs, did not vary in size after surface modification with GER, and showed a strong tendency for aggregate. However, imaging revealed nanocrystalline domains and a relatively smooth surface (Fig. 1A, B), compared to the unmodified form of TiO2 indicating the presence of GER on the surface. In the rutile form of TiO2 without surface modification (Fig. 1A, B), the nanoparticles (NPs) had irregular rock shapes with an average size of 100 ± 10 nm and a tendency to aggregate. The same properties were observed for the rutile form of the TiO2 NPs modified with GER. However, in the case of the rutile form, the surface was not smooth as it did in the case of the anatase form after modification with GER. To further confirm the presence of GER on the surface of TiO2 NPs, we used the energy-dispersive X-ray spectroscopy (EDS) for an elemental mapping analysis. The elemental dispersion on the TiO2 surface in both forms showed the presence of carbon, which confirms the successful modification with GER. Both non-modified forms also showed signs of carbon; however, in trace amounts (contamination for ambiance) only (Fig. 1).

X-ray crystallography (XRD) patterns confirmed the crystallinity of the TiO2 (anatase and rutile) observed under SEM. These patterns showed that both forms of TiO2 NPs were polycrystalline with well-defined peaks indicating a high degree of crystallinity. The XRD pattern exhibited diffraction peaks at 27.26°, 36.14°, 41.16°, 43.79°, 54.23°, 56.30°, 62.92°, and 68.67°, which belongs to the anatase and tetragonal rutile form of TiO2 NPs [42, 43] (Fig. 2). These results indicate that increasing the temperature without using any additive changed the biphasic anatase/rutile to rutile TiO2 NPs.

To confirm the presence of GER on the surface of TiO2 NPs, we also performed FTIR analysis (Fig. 3). The data revealed the presence of vibrational peaks that belong to organic functional groups, indicating the presence of GER. In particular, the broadband in the range of 3.500–3.000 cm − 1 was observed in the anatase and rutile TiO2 NPs, which is related to the stretching of hydroxyl groups (O–H), indicating the presence of surface water as moisture (Fig. 3A and B). The FTIR spectrum of anatase TiO2 NPs (Fig. 3A) showed a typical band on 1630 cm − 1 that is attributed to the H–O–H vibrations from physisorbed water [19, 20] and an absorption band detected at 438 cm − 1 that belongs to the Ti–O–Ti vibrations related to the anatase phase [21, 22]. Similar absorption band peaks were observed in the case of rutile TiO2 NPs (Fig. 3B), with the exception of the Ti–O–Ti vibration, which is shifted and detected at 512 cm − 1 as a result of the temperature increase (900 °C) during rutile preparation [18, 21]. Investigating the molecular vibration features of the GER-modified TiO2 NPs, we detected additional peaks indicating successful surface modification. In the case of anatase TiO2 NPs (Fig. 3A), the vibrational peaks detected at 3406 cm − 1 and 1639 cm − 1 are related to the O–H groups coming from surface and physisorbed water, similar to the case of unmodified TiO2 NPs. The peaks detected at 2970 cm− 1, 2922 cm− 1, and 2871 cm− 1 belong to the CH stretching vibrations of GER [19, 23], while the stretching vibrations at 1710 cm− 1, corresponding to the carbonyl group (C = O), confirm the consumption of hydroxyl groups from GER during surface modification of TiO2 [24, 25]. The stretching of other peaks at 493 cm− 1 and 573 cm− 1 belongs to TiO2 [18]. A similar position of detected peaks can be observed in the case of rutile TiO2 NPs modified with GER (Fig. 3B). The only exception is the deformed C–H bending vibrations detected in two peaks at 1377 cm− 1 and 1439 cm− 1, which belong to the methylene (–CH2–) and methyl (–CH3) groups [24], confirming the interaction of GER with TiO2 NPs.

In addition to the analyses described above, the FTIR spectra of free GER and GER-modified TiO2 NPs were examined, and the results showed notable alterations in peak locations indicating GER-TiO2 interaction (Figure S1). For instance, in the rutile and anatase GER-modified TiO2 NPs, the O-H stretching vibration peak changed from 3.330 cm− 1 in free GER to 3.406 cm− 1 and 3.143 cm− 1, respectively. Similar considerable changes in the C-H bending and stretching vibrations of the -CH2 and -CH3 groups were seen after surface modification. These peak shifts show the impact of GER-TiO2 interactions on the vibrational behavior of these functional groups and confirm the effective surface modification of TiO2 NPs with GER. Additional characteristics of our synthesized TiO2 nanoparticles, including particle size distribution and zeta potential, are further explored in the supplementary materials (Figure S2 and S3).

Fig. 1
figure 1

SEM images of unmodified and modified TiO2 NPs. A1-A4 unmodified anatase TiO2 NPs and their corresponding surface elements (Ti, C, and O, respectively). B1-B4 unmodified rutile TiO2 NPs and their corresponding surface elements (Ti, C, and O, respectively). C1-C4 and D1-D4. Modified anatase and rutile TiO2 NPs and their corresponding surface elements (Ti, C, and O, respectively).

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Fig. 2
figure 2

X-ray powder diffraction patterns of anatase and rutile form of TiO2.

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Fig. 3
figure 3

Baseline-corrected FTIR spectra of the TiO2/anatase and TiO2/anatase/geraniol (A) and TiO2/rutile and TiO2/rutile/geraniol (B) in the 4000 to 400 cm–1 range

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Effect of TiO2/GER treatments on bacteria

It was observed that both TiO2/anatase/GER and TiO2/rutile/GER exhibit activity against S. aureus, MRSA, and E. coli, with MIC values ranging from 0.25 to 1.0 mg/ml (Table 1). In contrast, anatase and rutile forms alone did not display any strong bactericidal properties, and GER displayed indifferent activity with MIC values of 4.25 mg/ml The interaction between GER and TiO2 inhibitory was confirmed by the FIC index values, which showed additive effects in the case of MRSA with a value of 0.735294 and synergism for S. aureus and E. coli with values of 0.284314 and 0.22549 respectively (Table 1). Furthermore, the supporting data can be found in (Figure S4, and Tables S1 and S2). Live and dead assays showed that the viability of S. aureus, MRSA, and E. coli was significantly reduced after treatment with both TiO2/anatase/GER and TiO2/rutile/GER. At concentrations of 0.25 mg/ml and 0.5 mg/ml, dead cells (stained in red) and live cells (stained in green) were barely visible, indicating that the treatment had a bactericidal effect on all strains, including the MRSA (Fig. 4). Images of control samples showed an increase of live cells (stained in green) and a decrease in dead cells (stained in red).

Table 1 Minimum inhibitory concentration (MIC) and FIC index of GER, anatase, and rutile and their combination against various bacterial strains
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Fig. 4
figure 4

The LIVE/DEAD cell images of S. aureus, MRSA, and E. coli treated with TiO2 anatase, TiO2 rutile, TiO2 /anatase/GER, and TiO2 /rutile/GER—magnification 100×. The scale bar is 50 μm. The saturation was processed equally across all micrographs

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Biofilm assay

It was observed that TiO2/anatase/GER treatment was highly effective against the MRSA biofilm, reducing its growth by 84.6% (p < 0.0001). Similarly, the TiO2/rutile/GER treatment reduced the biofilm growth by 80% (p < 0.0001). We also measured the effect of UV light (c 280 nm) under the same conditions and found that after 60 min, the results were similar to the non-UV light treatment, with the exception of a significant reduction in viability after geraniol treatment compared to that of the negative control (bacteria alone) (Fig. 5A and B).

Confocal laser microscopy was employed for a more qualitative, in-depth evaluation of the mixed TiO2 nanoparticles (NPs) with GER on MRSA biofilm. By analyzing fluorescence intensity distribution, it was found that both TiO2/anatase/GER and TiO2/rutile/GER significantly reduced biofilm thickness to values less than 30,000 nm compared to that of the control (p < 0.0001) (Fig. 6A). Interestingly, the treatment with anatase and rutile forms alone also decreased the biofilm thickness (Fig. 6B).

Fig. 5
figure 5

Fluorescence intensity (a.u.) of bacterial biofilm: Efficiency of modified and non-modified TiO2 against MRSA biofilm with UV-irradiation A) and without UV irradiation B). 

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Fig. 6
figure 6

A) 3D images from confocal laser scanning microscopy of cells in biofilm with intact membranes (green) and disturbed bacterial biofilm (black areas). B) Biofilm thickness after different treatments.

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Discussion

Titanium dioxide NPs have the potential to function as antimicrobial agents with several chemical modifications to optimize their biochemical properties. In this work, we report the synthesis and characterization of TiO2 NPs in anatase and rutile forms together with surface modification by GER for antibacterial effect enhancement. The antibacterial and antifungal properties of TiO2 against a broad range of pathogens were demonstrated in several previous studies [21] and the use of natural compounds to enhance physical and chemical properties of NPs has also been documented [30, 31, 39]. In our study, GER was chosen as a potential candidate for coupling with TiO2 to enhance its antimicrobial properties due to its high solubility and controlled toxicity [40, 41].

The morphology of the TiO2 NPs can be tuned by calcination temperature [44]. In line with previous reports [45,46,47], we successfully synthesized TiO2 with two different calcination temperatures, 500 °C confirming the creation of TiO2 in the anatase form and 900 °C to generate TiO2 in the rutile form. The success of TiO2/GER modification was confirmed by the XRD results showing the impact of homogenization on the crystalline structure of the combination [48, 49]. Another verification of the successful modification was done by elemental composition with EDS. Carbon was a definite indicator of response and coverage in the sample. Several earlier studies have detected the presence of carbon, which is attributed to carbon-containing functional groups of natural products [50,51,52].

Several investigations employing modification of TiO2 demonstrated the antibacterial activity of this material [53]. These studies attributed the increase in antimicrobial susceptibility to modifications of TiO2 with natural elements and non-metallic compounds [45, 46, 54, 55]. Our MIC results are consistent with previous findings reporting MIC against various G-positive and G-negative bacteria in the similar range [45, 56,57,58,59] while other studies reported the MIC against MRSA to be much higher (7.0–13.0 mg/ml) [60, 61]. Our study showed that the UV light on TiO2 had no significant changes in the reduction rate. This finding contradicts a previous report that suggested that UV light irradiation of TiO2 nanorod arrays exhibited excellent antibacterial properties against both S. aureus and E. coli biofilm [40].

Although the confocal microscopy results supported the quantitative Alamar blue results, some variation was noticed. In the Alamar blue analysis, both TiO2 modifications exhibited greater effectiveness against MRSA biofilms, and neither treatment alone had a statistically significant effect on the biofilms. In contrast, the confocal microscopy indicated that the different TiO2 treatments influenced the architecture of the biofilms, as they showed a reduction in biofilm thickness. These findings are supported by previous studies emphasizing the importance of examining biofilms using quantitative and qualitative methods [45]. Therefore, we have demonstrated that TiO2 has the potential to act as an anti-biofilm agent on its own without the need for any modification.

The increased antimicrobial activity and reduction in bacterial cell viability due to the exposure of TiO2/anatase/GER and TiO2/rutile/GER showed that both treatments had a high adsorption capacity on the cell surface. The increased antimicrobial activity of modified TiO2 can be explained by several mechanisms. First, geraniol ability to adhere to cell membrane lipids of bacteria can mediate antimicrobial activity by increasing the permeabilization of the membrane [62, 63]. In addition, the presence of TiO2, TiO2/anatase/GER, and TiO2/rutile/GER can lead to loss of cell wall integrity and direct interaction with DNA leading to cell death [64, 65]. Secondly, modifications of NPs are commonly used to prevent aggregation, allowing them to disperse in aqueous environments or other hydrophilic media, thereby enhancing their antimicrobial activity [47]. Lastly, modifications are also one of the most effective methods to regulate and control NPs and bacteria interactions and increase ROS production [31, 32].

Based on these findings, combining TiO2 with geraniol, offers an opportunity for the antibacterial use. Low cytotoxic effects, described in Figure S5., highlight the usefulness of these compounds. Nevertheless, further research is needed to find out the full range of potential applications and any additional potential hazards.

Conclusions

In conclusion, TiO2 nanoparticles modified with GER in the form of anatase and rutile were successfully synthesized and tested for their antimicrobial activity. The modified TiO2 nanoparticles had the elevated antimicrobial activity and also inhibited MRSA biofilm formation. TiO2 alone also proved to be effective against biofilms.

Methods

Preparation of TiO2 NPs

Titanium isopropoxide (1.48 ml) (Sigma Aldrich in St. Louis, MO, USA) was mixed with 229.0 µl of isopropyl alcohol (Lach-Ner in Neratovice, Czech Republic) and 18.0 µl of MilliQ water (pH 8) (Thermofisher, USA) and stirred for 10 min. Then, the solution was washed three times with MilliQ water and dried overnight in the Memmert UM 400 drying oven (Memmert, Schwabach, Germany) at 60 °C. Finally, TiO2 NPs were produced by calcining the dried solution at the temperature of 500 °C for the anatase form and 900 °C for the rutile form for 2 h in the laboratory chamber furnace (LAC in Rajhrad, Czech Republic).

Modification of TiO2 NPs by GER

The TiO2 NPs were redispersed in 50 ml of MilliQ water. Then, 10 ml of the dispersion with 161.1 mg of GER (Sigma Aldrich, Saint Louis, USA) was mixed in 200 µl of GER solvent with MilliQ water and stirred overnight. Afterward, the solution was washed three times with MilliQ water.

Characterization of TiO2 NPs

The absorbance wavelength was determined using the UV/Vis spectrophotometer (Perkin-Elmer, USA) with the quartz cuvette with acetonitrile/tetrahydrofuran as a reference. The X-ray powder diffraction patterns of the TiO2 NPs were recorded on the Bruker 8D advanced X-ray diffractometer (Bruker, AXS GmbH, Karlsruhe, Germany) with CuKα radiation of wavelength = 1.54056 Å. The morphology of the TiO2 NPs was studied by SEM using the JEOL JSM-6330 LA operated at 20.0 kV and 1.0000 nA. The elemental analysis used SEM on a Tescan MIRA 2 equipped with a FEG (Tescan Ltd., Brno, Czech Republic) and EDX detector MAX 50 (Oxford Instruments plc, Abingdon, UK). The images were obtained using the E-T SE detector at a working distance of 15 mm and 15 kV acceleration voltages, with 50 000-fold magnification. Fourier transform infrared spectroscopy (FTIR) analysis was used on a film of each freeze-dried sample using a Jasco FT/IR-4700 (Jasco, MD, USA) spectrophotometer with a wavenumber range of 400 to 4000 cm − 1 with 4 cm − 1 resolutions. The zeta potential and size of particles were measured using the dynamic light scattering technique on the Zeta sizer Nano ZS instrument (Malvern Instrument Ltd, UK). The particle size distribution and ζ-potential were studied using dynamic light scattering (DLS) on the Malvern Zetasizer (NANO-ZS, Malvern Instruments Ltd., Worcestershire, U.K.) at a detector angle of 173° and the refractive index of 1.33 at 25 °C. The size distribution and ζ-potential measurements were performed in MilliQ water at pH 6.8. Measurements were taken for each sample in triplicates.

Determination of minimum inhibitory concentration (MIC)

Bacterial cultures were obtained from the Czech Collection of Microorganisms (Brno, Czech Republic), including Staphylococcus aureus CCM 4223, MRSA CCM 7110, and Escherichia coli CCM 3954. All bacterial strains were cultured on Columbia agar with 5% sheep blood (LMS, Czech Republic) at 37 °C for 24 h. The antibacterial activity of TiO2/anatase/GER, TiO2/rutile/GER, along with control treatments anatase, rutile, and GER, was evaluated against bacterial strains cultured in Muller Hinton broth (company). Bacteria were incubated in the 96 well plate at 37 °C, with 150 rpm shaking; the optical density (OD) at 600 nm was measured after 24 h.

Determination of fractional inhibitory concentration (FIC)

The effect of the GER and TiO2 combination was assessed against bacterial strains using the checkerboard technique [16]. We then calculated the FIC index values using the formula: FICI = FIC (A) + FIC (B). The Σ FICI values were interpreted as follows:

≤ 0.5 = synergistic; >0.5–1.0 = additive; > 1.0–4.0 = indifferent (non-interactive); > 4.0 = antagonistic.

LIVE/DEAD® Cell Assay

Live/dead assay was carried out by following the manufacturer instructions of the LIVE/DEAD™ BacLight™ bacterial viability kit for microscopy (Invitrogen, USA). Modified nanomaterials TiO2/anatase/GER, TiO2/rutile/GER, and non-modified anatase and rutile samples were examined at the defined MIC after 24 h and these bacterial samples were observed by an inverted fluorescent microscope Olympus IX71 (Olympus C&S Ltd., Czech Republic).

MRSA biofilm assay

The efficacy of TiO2 with and without GER agent. MRSA biofilm was evaluated using qualitative and quantitative methods. MRSA was incubated in BHI with 1% glucose for 72 h at 37 °C. Biofilms were treated based on the MIC values, washed twice with PBS, and stained with the alamarBlue™ cell viability reagent (Invitrogen, USA) for 20 min. The 96 well plate was used to carry out the experiment, and biofilm viability was measured using fluorescence measurements (Tecan, Switzerland) (590/570 nm, excitation/emission). Qualitative assessment of the biofilm thicknesses and high-quality visualization images were done using confocal microscopy (LSM 880, USA) with the incubation and washing conditions, with the exception that IBIDI dishes were used and stained using the LIVE/DEAD™ BacLight™ bacterial viability kit for microscopy (Invitrogen, USA).