Antimicrobial activity of ZnO-TiO₂ nanomaterials synthesized from three different precursors of ZnO: influence of ZnO/TiO₂ weight ratio

Antibiotics and antifungals that have saved so many lives during the last century become increasingly inactive because of the alarming increase in bacterial resistance (Rai et al. 2012; Mahamat et al. 2005) and fungal (Müller et al. 2000). The breadth of the issue is important and should encourage the effective participation of many multidisciplinary researchers to find other alternative composites for antimicrobial struggle, as well as the scarcity of new anti-microbial products on the market (Skrodeniene et al. 2006; Carlet et al. 2011). In recent decades, the search for novel composites such as metal and/or metal oxide nanoparticles capable to improve the antimicrobial activity has attracted growing interest from many (Liu et al. 2009; Schacht et al. 2012; Ansari et al. 2013). Zinc oxide (ZnO) and titanium dioxide (TiO₂) are very promising metal oxides in this field because of their very attractive physicochemical and antibacterial properties (Zhou et al. 2006; Tam et al. 2008; Ahn et al. 2009; Liu et al. 2009; Fu et al. 2010; Xie et al. 2011; Emami-Karvani 2012; Zegaoui et al. 2014). ZnO is known as an environmentally friendly inorganic material, that is chemically stable, nontoxic, biosafe and biocompatible (Zhou et al. 2006). That is why it is very suitable to use it as antibacterial agent. Its antibacterial properties have been largely studied in both microscale and nanoscale formulations (Jones et al. 2008; Tam et al. 2008; Karunakaran et al. 2010). In our previous work (Zegaoui et al. 2014), we reported that the antibacterial activity of ZnO nanoparticles synthesized from different precursors against Staphylococcus aureus and Escherichia coli bacteria depended on the concentration, the shape (agglomerates, hexagonal microrods and hexagonal nanoparticles) and the crystallite size of ZnO nanoparticles. The coupling of ZnO and TiO₂ seems to be one of the simplest ways to improve antibacterial and antifungal activities of these two metal oxides. Despite the various studies dealing with the antibacterial and antifungal activities of ZnO and TiO₂ the antibacterial and antifungal activity of ZnO-TiO₂ nanomaterials still remains unexplored in detail in this field. For instance, to the best of our knowledge, no study was reported so far to have undertaken the effect of nature of the ZnO precursor and the ZnO/TiO₂ weight ratio on the antibacterial and antifungal activities of the ZnO-TiO₂ nanomaterials. So, in this investigation, ZnO-TiO₂ nanomaterials were synthesized using three different precursors for ZnO (Zn(NO₃)₂.6H₂O, ZnSO₄.7H₂O and Zn(CH₃COO)₂.2H₂O) with various ZnO/TiO₂ weight ratios (R). The synthesized ZnO-TiO₂ nanomaterials were calcined at 500°C for 3 h and the effects of the nature of the ZnO precursor and ZnO/TiO₂ weight ratio on the structural and morphological properties of the ZnO-TiO₂ composites were investigated by using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy combined with energy dispersive X-ray spectroscopy (TEM-EDS). The antibacterial and antifungal activities of the synthesized nanomaterials were evaluated, in the dark, against five multi-resistant of Gram positive (Staphylococcus aureus (S. aureus)) and Gram negative (Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa) and Salmonella Paratyphi A (S. Paratyphi A)) bacteria and a fungus (Candida albicans (C. albicans)).

The reagents titanium (IV) tetraisopropoxide (TTIP) (Sigma Aldrich, 99.99%), Zn(CH₃COO)₂.2H₂O (Scharlau, 99%), Zn(NO₃)₂.6H₂O (Acros Organics, 98%), ZnSO₄.7H₂O (Fisher Scientific International Company, 99.5%), NaOH (Fisher Scientific International Company, 98%) and isopropyl alcohol (Fisher Scientific International Company, 99.99%) used in this work were of analytic grade. All chemicals were used as received without further purification.

ZnO nanoparticles were prepared by precipitation method from three different precursors (Zn(CH₃COO)₂.2H₂O, Zn(NO₃)₂.6H₂O and ZnSO₄.7H₂O) following the procedures described in our previous work (Daou et al. 2017). Titanium dioxide nanoparticles were synthesized by sol-gel following the procedure described by Daou et al. (Daou et al. 2013). The obtained nanomaterials were calcined at 500 °C for 3 h. They were denoted in the text as ZnO(A), ZnO(N), and ZnO(S) for ZnO synthesized from zinc acetate, zinc nitrate and zinc sulfate, respectively.

Two solutions – A, containing ZnO(X), and B, containing TiO₂ sol – were prepared simultaneously. The solution A was added to the solution B drop-wise under constant agitation for 2 h while respecting the desired ZnO/TiO₂ weight ratio (ZnO-TiO₂ weight ratio is specified as r = 0.5, 1 and 2). The obtained mixture was filtered under vacuum and the recovered powder was washed with distilled water, dried overnight in an oven at 100°C. The resulting materials were ground and calcined at 500°C for 3 h. The ZnO-TiO₂ nanomaterials were denoted in the text Zn(X)-Ti-r where X indicates A, N and S for nanomaterials synthesized from zinc acetate, zinc nitrate and zinc sulfate, respectively, and r is the ZnO(X)/TiO₂ weight ratio.

The crystal structures of the synthesized materials were done by powder X-ray diffraction using an X'PERT MPD_PRO diffractometer with Cu Kα radiation. The accelerating and the applied current were 45 kV and 40 mA, respectively. The determination of the crystalline phases was carried out by applying the Bragg law where λ = 1.5406 Å. FT-IR spectra were obtained as KBr pellets in the wave number range of 400–4000 cm⁻¹ using an FT-IR spectrometer (Type: JASCO 4100). The morphologies and elemental analysis of the prepared nanoparticles were determined by transmission electron microscopy (TEM, FEI Tecnai 12) combined with energy dispersive X-ray spectroscopy (EDS).

The antimicrobial activity of the prepared materials was evaluated against Gram positive (Staphylococcus aureus) and four Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella Paratyphi A). These strains were collected from private laboratories for bacteriological analysis in Meknes City, Morocco. The antifungal activity was evaluated against the Candida albicans that is the main fungus involved in human pathology (Wenzel & Gennings 2005; Whibley & Gaffen 2015).

The tests of the germ susceptibility against the synthesized nanomaterials were carried out following the procedure reported previously for ZnO nanoparticles (Liu et al. 2009; Zegaoui et al. 2014). Mueller Hinton agar medium was used for bacteria and Sabouraud chloramphenicol solid medium for Candida albicans. All tests were carried out in the dark and repeated three times.

The MIC of the bacterial strains was determined as concentration minimum of nanoparticles which inhibits the growth of 90% bacterial population after incubation time of 18–24 h at 37°C. It was determined according to the technique of the microtiter on microplate described by Eloff (Eloff 1998). The MTT [2-(4, 5-dimethyl-2-thiazolyl)-3, 5-diphenyl -2H-tetrazolium bromide] was used as an indicator of bacterial viability. 50 μL of Muller-Hinton broth were added to each well of a microtiter plate containing 96 wells, and 50 μL of the stock aqueous suspension of nanoparticles (8 mg/mL) were added to the first well of each row from which series of geometric dilution common ratio of 2 were made. 50 μL of the bacterial suspension containing 5.105 CFU(colony forming units)/mL were added to each well, and the microtiter plate was incubated at 37 °C for 24 h in the dark. After that, 20 μL of a solution of MTT (concentration: 0.4 mg/mL of a sterilized physiological saline), prepared extemporaneously, were added to each well and the microtiter plate was incubated again for 30 min at 37 °C. The violet blue color in the wells indicates a bacterial growth. A microtiter plate prepared with Mueller-Hinton broth and the bacterial inoculums without addition of nanoparticles was used as control. Each test was repeated three times.

The MIC of the Candida albicans was determined by the inoculation of 10 μL of a culture of 24 h into 1 mL of a Sabouraud's liquid medium containing various concentrations of the prepared nanomaterials. The preparations were incubated in the dark for 24 h at 37 °C. The growth and the quantification of the yeasts were made by comparing the turbidity of the cultures with control tubes starting from the lowest dilution (Djohan et al. 2012). To evaluate the yeast eradication, living cells were sought in fresh cultures by using an optical microscope. The control tubes were made of an adequate culture medium and microbial inoculum without nanoparticles. Each test was repeated three times.

The MBC was determined by reference to the results of the MIC tests. 10 μL from each well showing no bacterial viability were inoculated in Petri dishes containing Mueller-Hinton agar medium. The incubation was carried out in the dark at 37°C for 24 h. The first dilution giving the complete absence of growth of the strains has been regarded as MBC. Concerning the MFC, 10 μL of each tube exhibiting negative growth were aseptically transferred into the tubes containing 990 μL of Sabouraud-chloramphenicol broth. After incubation in the dark for 24 h at 37°C, the first dilution giving the absence of growth were considered as the MFC of the nanoparticles. Each test was repeated three times in the dark.

The X-ray patterns of the prepared Zn(A)-Ti-r, Zn(N)-Ti-r and Zn(S)-Ti-r nanomaterials are shown in Figures 1, 2 and 3, respectively. Figure 1 shows that Zn(A)-Ti-1 and Zn(A)-Ti-2 samples are amorphous, whereas Zn(A)-Ti-0.5 is crystallized because of its XRD spectrum indicates the presence of peaks attributed to TiO₂ anatase phase (JCPDS file card No. 21–1272) and two peaks at 2θ = 30.27 and 35.28° assigned to Zn₂TiO₄ phase (JCPDS file card N° 00 025 1164) (Ivanova et al. 2011). It is important to note that the absence of peaks corresponding to ZnO würtzite phase doesn't imply the absence of ZnO in the solid because it can exist in amorphous form. The XRD patterns of Figure 2 show that only the würtzite phase of ZnO is crystallized for ZnO-TiO₂ nanomaterials prepared from zinc nitrate. Nevertheless, the intensity of the peaks belonging to ZnO decreases with decreasing the ZnO weight ratio, and these peaks practically disappear for the sample prepared with r = 0.5, suggesting that the samples became more amorphous as r is decreased. XRD patterns corresponding to Zn(S)-Ti-r nanomaterials (Figure 3) indicate the presence of Zn₃O(SO₄)₂ phase (JCPDS file card N°: 00-032-1475) in addition to ZnO würtzite and TiO₂ anatase. As reported in our previous work (Daou et al. 2017), the presence of Zn₃O(SO₄)₂ phase indicates that the zinc sulfate used as a precursor was not completely converted into ZnO during the precipitation process, and a part of it was transformed into a zinc oxysulfate during the step of calcination at 500 °C. It is noteworthy that, as for Zn(N)-Ti-r materials, the intensity of the peaks attributed to ZnO decreases with decreasing the ZnO weight ratio, indicating the decrease in the degree of crystallinity. By comparing the XRD spectra of Zn(A)-Ti-r, Zn(N)-Ti-r and Zn(S)-Ti-r prepared with the same r, it is clearly seen that the ZnO precursor play a decisive role on the structure of the obtained ZnO-TiO₂ nanomaterials.

FT-IR spectra of ZnO-TiO₂ nanomaterials prepared from zinc acetate, zinc nitrate and zinc sulfate with various ZnO weight ratios are shown in Figures S1, S2 and S3, respectively (available with the online version of this paper). The FT-IR spectrum of TiO₂ shows a broad band between 420 and 750 cm⁻¹ due to the overlapping of several bands attributed to different vibration modes of Ti-O and Ti–O–Ti in TiO₂ (Daou et al. 2013). This spectrum, as well as those of ZnO and ZnO-TiO₂ materials prepared in this work, exhibit bands around 3420 and 1650 cm⁻¹ assigned to the stretching and bending vibrations of H₂O adsorbed on the surface of the solid, respectively (Daou et al. 2017; Ivanova et al. 2011). As reported in our previous work (Daou et al. 2017), the FT-IR spectra of ZnO(A) (Figure S1), ZnO(N) (Figure S2) and ZnO(S) (Figure S3) nanoparticles show a large absorption band between 425 and 510 cm⁻¹ attributed to infrared active modes of the würtzite ZnO. The bands observed at about 1425 and 1545 cm⁻¹ on the spectrum of ZnO(A) (Figure S1) are attributed to C-O and C = O stretching vibrations in acetate groups (Daou et al. 2017). The band appearing at 1389 cm⁻¹ on the spectrum of ZnO(N) (Figure S2) corresponds probably to the presence of residual nitrate (NO₃⁻) (Daou et al. 2017). The spectrum recorded for ZnO(S) (Figure S3) also shows two SO₄²⁻ characteristic peaks at 1120 cm⁻¹ and 630 cm⁻¹ (Daou et al. 2017). With the increase of ZnO weight ratio, the characteristic large band of ZnO shifts progressively to higher wave number values (about 660–700 cm⁻¹). This band shift can be associated with the formation of new Zn-O-Ti bonds in (Wang et al. 2003; Wang & Lin 2008; Kanmani & Ramachandran 2012) observation suggests that the addition of ZnO to TiO₂ causes a formation of other types of Ti-O or ZnO bonds (Ivanova et al. 2011) confirming the XRD results.

In a previous work, we have reported that ZnO nanoparticles synthesized from zinc acetate have a uniform and hexagonal shape (the particle average size is about 40 nm). Those prepared from zinc nitrate are in the form of well-defined microrod-like structures having a hexagonal shape (the particle average size is about 100 nm). The ZnO nanoparticles prepared from zinc sulfate are highly agglomerated and are in the form of clusters with an average size greater than 100 nm. In another work (Daou et al. 2013), we have reported that the synthesized TiO₂ nanoparticles have a homogeneous shape and are highly agglomerated (the average nanoparticle size is of 8–20 nm). Figures 4–6 show the observed TEM images of some samples for Zn(A)-Ti-r, Zn(N)-Ti-r and Zn(S)-Ti-r, respectively. From the images of these three figures, it can be seen that the addition of ZnO to TiO₂ leads to the formation of high agglomerated nanocomposites without any defined shape. The ZnO-TiO₂ composites are in the form of agglomerates which consist of many ZnO nanoparticles surrounded completely by TiO₂ nanoparticles. No ZnO particle was observed independently since the simultaneous EDS analyses carried out on the Zn(A)-Ti-2 sample (Figure 4(c)) and Zn(N)-Ti-2 sample (Figure 5(c)), and on Zn(S)-Ti-r samples (Figure 6) indicated the presence of Zn, Ti and O, and Zn, Ti, O and S, respectively. These results corroborate the XRD and FT-IR results and indicate that the ZnO particles were coated by TiO₂ particles to form undefined shapes of ZnO-TiO₂ agglomerates.

Tables 1–3 show the obtained results of the antibacterial and antifungal effect of TiO₂, ZnO(A) and Zn(A)-Ti-r nanoparticles oxides on the Gram positive bacteria and Gram negative as well as the yeast tested. These results indicated that pure TiO₂ showed no antibacterial or antifungal in the dark against all the strains tested in agreement with results reported in the literature (Rincón & Pulgarin 2003; Karunakaran et al. 2012). It is also clearly seen that adding ZnO(A) to TiO₂ improves the bactericidal effect of TiO₂ against K. pneumoniae (92% inhibition for r = 2, Table 2) while, for the same ratio, no more than 2.38% of S. Paratyphi A and 4.46% of S. aureus were eliminated (Tables 1 and 3). For C. albicans, the ZnO-TiO₂ materials do not show their antifungual activity until reaching a significantly higher MIC (up to 8 mg/mL for Zn (A)-Ti-0.5) (Table 3).

Tables 4–6 show the obtained results of the antibacterial and antifungal effect of the TiO₂, ZnO(N) and Zn(N)-Ti-r nanoparticles on the Gram positive and Gram negative bacteria as well as the fungus tested. The results indicate that the addition of ZnO(N) to TiO₂ is very effective against S. aureus (Gram positive bacterium) with very low MIC (0.062 mg/mL for Zn(N)-Ti-2) (Table 6). However, these oxides are active only against Gram negative bacteria and yeast at higher MIC (up to 4 mg/mL for E. coli, K. pneumoniae and C. albicans) (Tables 4–6). Gram negative bacteria seem to be more unaffected to the effect of ZnO(N) nanoparticles than Gram positive bacteria, These results are in agreement with those of Emami-Karvani (Emami-Karvani 2012). The bactericidal activity against S. aureus, which is a Gram positive bacterium, is probably due to the composition of its wall. Indeed, the cell envelope of Gram negative bacteria comprises two membranes: the inner and the outer membranes that are separated by a thin layer of peptidoglycan and the periplasm. The inner membrane is a double phospholipid lamella containing various proteins (carriers, enzymes, etc.) while the outer membrane is an asymmetric bilayer composed of lipopolysaccharide and phospholipids where many proteins are inserted (Pagès 2004; Suwanboon et al. 2010). This representation simplifies the selective barrier of the outer membrane of Gram negative bacteria for hydrophilic molecules. Indeed, the ion channels of these bacteria are too small to allow internalization of the nanoparticles which are large (Moore 2006).

Tables 7–9 show the obtained results of the antibacterial and antifungal effects of TiO₂, ZnO(S) and Zn(S)-Ti-r nanoparticles. The addition of ZnO(S) to TiO₂ has allowed the complete eradication of S. aureus, E. coli, K. pneumoniae and C. albicans with very low MICs. ZnO(S) is able to act on the Gram positive and negative bacteria (prokaryotic cells) and yeasts (eukaryotic cells). This peculiarity of efficiency of the ZnO-TiO₂ materials made from zinc sulfate heptahydrate was already demonstrated in the case of pure ZnO synthesized from the same precursor on heat-resistant prokaryotic and eukaryotic plant and animal cells (Moukrad et al. 2014; Zegaoui et al. 2014; Moukrad et al. 2015). This specific activity was linked to the high concentration of Zn²⁺ ions in ZnO(S) compared to that present in ZnO(N) or ZnO(A), since the zinc oxide prepared from the zinc sulphate contains Zn₃O(SO₄)₂ phase which releases more Zn²⁺ ions (Zegaoui et al. 2014). The influence of the solubility on the bactericidal response of the various oxides is particularly increased for certain soluble compounds (e.g. ZnO and FeO) compared to that of lower solubility nanoparticles (e.g. CeO₂ and TiO₂) (Domenech & Prieto 1986; Brayner et al. 2006). This difference in biological activity of ZnO(S) can be also explained by the difference in the shape and size of the synthesized nanoparticles (Liu et al. 2009; Raghupathi et al. 2011). The TEM observations of Zn(A)-Ti-2 (Figure 4(c)) shows that the TiO₂ nanoparticles surround zinc oxide nanoparticles which are in the form of clusters, whereas Zn(N)-Ti-2 nanoparticles appear in the form of large nanoparticles (Figure 5(c)) and Zn(S)-Ti-2 nanoparticles have irregular shapes (Figure 6(d)). On the other hand, it is noteworthy that the obtained MIC values for S. Paratyphi A, E. coli, K. pneumoniae and P. aeruginosa in the presence of Zn(S)-Ti-r increase when the ZnO/TiO₂ weight ratio increases from 0 to 1, then decrease when r increases further up to 2 (Tables 7 and 8). Similar results were reported by (Zvekić et al. 2011) after adding the ZnO to polyurethane varnish to test its antibacterial effect against E. coli. (Reddy et al. 2007) reported that the treatment of E. coli with 1 mM of ZnO nanoparticles increases in the growth of this bacterium compared to control samples, while for 5 mM ZnO becomes bactericide. (Outten & O'Halloran 2001; Reddy et al. 2007; Brayner et al. 2006) reported that low concentrations of Zn²⁺ improve growth in E. coli because this bacterium can metabolize Zn²⁺ as a trace element, and the ZnO nanoparticles are not toxic against E. coli. However, Zn²⁺ is considered as a growth inhibitor against S. aureus (Dadook et al. 2014; Zvekić et al. 2011; Bhat et al. 1994). The originality of the obtained results in this work lies in the fact that the Zn(S)-Ti-r materials have an antimicrobial efficacy more appreciable compared to Zn(A)-Ti-r and Zn(N)-Ti-r, showing the strong influence exerted by the nature of the ZnO precursor on the antibacterial and antifungal properties. It also has the particularity of being a broad spectrum antimicrobial and eradicating P. aeruginosa which is opportunistic and multiresistant bacteria.

In the dark, TiO₂ has no antibacterial or antifungal effect on the growth of all the strains tested in this work. However, the addition of ZnO to TiO₂ at increasing proportions improves the bactericidal and fungicidal activity of TiO₂. This antimicrobial activity was found to be very important for ZnO and ZnO-TiO₂ mixed oxides synthesized from zinc sulfate. These nanomaterials showed a more pronounced efficacy and a broader spectrum of activity against all strains tested for Gram positive, Gram negative bacteria and yeast C. albicans. The comparison of the obtained antimicrobial activities in the presence of the prepared ZnO-TiO₂ materials shows that for S. Paratyphi A, S. aureus and C. albicans: Zn(S)-Ti-r > Zn(N)-Ti-r > Zn(A)-Ti-r, whereas for E. coli, K. pneumoniae and P. aeruginosa: Zn(S)-Ti-r > Zn(A)-Ti-r > Zn(N)-Ti-r.