Characterization and Evaluation of Layered Bi2WO6 Nanosheets as a New Antibacterial Agent

Background: Pathogenic microorganisms are causing increasing cases of mortality and morbidity, along with alarming rates of ineffectiveness as a result of acquired antimicrobial resistance. Bi2WO6 showed good potential to be used as an antibacterial substance when exposed to visible light. This study demonstrates for the first time the dimension-dependent antibacterial activity of layered Bi2WO6 nanosheets. Materials and methods: The synthesized layered Bi2WO6 nanosheets were prepared by the hydrothermal method and characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman and Fourier transform infrared spectroscopy (FTIR). Antibacterial and antibiotic-modulation activities were performed in triplicate by the microdilution method associated with visible light irradiation (LEDs). Results: Bi2WO6 nanosheets were effective against all types of bacteria tested, with MIC values of 256 μg/mL against Escherichia coli standard and resistant strains, and 256 μg/mL and 32 μg/mL against Staphylococcus aureus standard and resistant strains, respectively. Two-dimensional (2D) Bi2WO6 nanosheets showed antibacterial efficiency against both strains studied without the presence of light. Conclusions: Layered Bi2WO6 nanosheets revealed dimension-dependent antibacterial activity of the Bi2WO6 system.


Introduction
Microbial infections triggered by resistant pathogens have raised important public health problems in the world, being currently the focus of study by multiple researchers in an intent to provide safer, effective and less harmful antimicrobials [1]. Among the Antibiotics 2021, 10 various fields under intense research, nanobiotechnology has been on high demand, with an increasing number of nanomaterials being highlighted in several scientific studies as a possible alternative to fight resistant microorganisms. Characterized by a very small size, but with a large surface area, such nanoformulations have revealed the capability of improving the delivery of drugs in specific tissues, ultimately boosting drugs' effectiveness and a reduction in adverse effects, as both dose and time of action can be thoroughly controlled [2]. After the discovery of graphene, nanomaterials with a 2D dimension are increasingly being targeted by research seeking to elucidate physical properties in relation to their bulk precursors [3]. However, in the post-graphene era, numerous inorganic materials in the form of layers have been extensively investigated, such as transition metal dichalcogenides [4] and layered metal oxides [5], among others. Monolayered bismuth tungstate (Bi 2 WO 6 ) nanosheets is a representative example; the Aurivillius oxide Bi 2 WO 6 nanosheets has a sandwich substructure of [BiO] + -[WO 4 ] 2− -[BiO] + . Layered Bi 2 WO 6 nanosheets were obtained by a cetyltrimethylammonium bromide-assisted bottom-up route [6].
Bismuth tungstate (Bi 2 WO 6 ) has received huge attention as a visible light photocatalyst [7]. Bi 2 WO 6 has shown interesting photochemical stability and reusability, being potentially useful for environmental treatment purposes, while also exhibiting photocatalytic degradation of Erichrome Black T (EBT) organic dye [8]. Furthermore, Bi 2 WO 6 exhibited photocatalytic activity for degrading norfloxacin and enrofloxacin under visible light irradiation, meaning its feasible application along with fluoroquinolone antibiotics [9]. In a study, Aurivillius oxide Bi 2 WO 6 was applied as an effective visible light-driven antibacterial photocatalyst for Escherichia coli's inactivation, where neither visible light without the photocatalyst nor Bi 2 WO 6 in the dark revealed bactericidal effects. Thus, the bactericidal effect in E. coli was certainly attributed to the photocatalytic reaction of Bi 2 WO 6 under visible light irradiation [10].
In this sense, the present study aims to assess, for the first time, the antibacterial and antibiotic modulation effects of layered Bi 2 WO 6 nanosheets against the standard and multidrug-resistant (MDR) Staphylococcus aureus and E. coli. Two-dimensional (2D) Bi 2 WO 6 nanosheets showed antibacterial efficiency in both studied strains (Gram-negative and Gram-positive bacteria) without the presence of light (in the dark), showing a dimensiondependent antibacterial activity of the Bi 2 WO 6 system and improving its properties in relation to the bulk material. Figure 1 shows the XRD powder pattern of Bi 2 WO 6 obtained by a CTAB-assisted hydrothermal method. The crystalline nature of the samples was confirmed, and all diffraction peaks can be indexed to an orthorhombic Bi 2 WO 6 phase with lattice parameters a = 5.457 Å, b = 5.436 Å and c = 16.427 Å (JCPDS Card N • 73-2020), without secondary phase. The intensity ratio of the (020)/(200) Bragg peak to the (113) peak was higher than the standard value. In addition, the full width at half maximum (FWHM) of the (200)/(020) Bragg peak was narrower than that of the (113) peak. This analysis indicates a higher grain size of the synthesized bismuth tungstate along the (100) and (010) directions compared to the (001) direction [11]. The AFM images (Figure 2a,b) and the corresponding height histograms (Figure 2c,d) of the layered Bi 2 WO 6 nanosheets show that the monolayer has a thickness of about 0.9 nm, which is in agreement with that of Bi 2 WO 6 monolayer along the (001) direction. In addition, the three-dimensional (3D) morphology in Figure 2b indicates that the layered Bi 2 WO 6 nanosheets have a dense and flat morphology. Therefore, the AFM result agrees with the XRD results. Furthermore, this result is in perfect agreement with data obtained by Zhou et al. [6], who showed by high resolution transmission electron microscopy (HRTEM) and atomic force microscopy (AFM) that layered Bi 2 WO 6 nanosheets obtained by CTAB-assisted hydrothermal synthesis exposes (001) facets. In addition, the Antibiotics 2021, 10, 1068 3 of 10 authors showed that layers had a thickness of 0.8 nm, i.e., the Bi 2 WO 6 monolayer along the (001) direction features 1/2 the size of the unit cell in the c-axis direction.

Characterization of Layered Bi 2 WO 6 Nanosheets
Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 11 transmission electron microscopy (HRTEM) and atomic force microscopy (AFM) that layered Bi2WO6 nanosheets obtained by CTAB-assisted hydrothermal synthesis exposes (001) facets. In addition, the authors showed that layers had a thickness of 0.8 nm, i.e., the Bi2WO6 monolayer along the (001) direction features 1/2 the size of the unit cell in the caxis direction.    (Figure 4), which clearly reveal Bi 2 WO 6 exhibiting thin two-dimensional structures.
As stated above and looking at Figure 3, Aurivillius Bi2WO6 is a layered material built up of [Bi2O2] layers and corner-shared WO6 octahedral layers, whereas the Bi2WO6 monolayer has a sandwich substructure configuration of [BiO] + -[WO4] 2− -[BiO] + exposing the Bi atoms on the surface. The morphology of the two-dimensional Bi2WO6 nanosheets can be shown by the SEM images ( Figure 4), which clearly reveal Bi2WO6 exhibiting thin twodimensional structures.   The room temperature Raman and Fourier transform infrared (FTIR) spectra of the synthesized layered Bi2WO6 nanosheets are presented in Figure 5 and Table 1. The Raman spectrum of Bi2WO6 monolayers in the spectra range between 100 and 900 cm −1 presents broad Raman peaks, some even overlapping forming only shoulders, making them difficult to distinguish in the unpolarized spectrum (See Figure 5a). On one hand, the Raman spectrum exhibits significant changes as the crystallite size decreases, comparing the Raman spectrum of Bi2WO6 monolayers with the spectrum of Bi2WO6 bulk structures. On the other hand, the Raman spectrum obtained is in full agreement with what is shown in the literature for nano-sized materials [12]. Looking at the FTIR spectrum in Figure 5b, we see that the broad band at 3460 cm −1 corresponds to the stretching vibration of O-H bonds, indicating water molecules adsorbed on the surface of the sample. The 2921 cm −1 and 2854 cm −1 are associated with the C-H stretching vibrations of the methyl and methylene groups of CTAB used in hydrothermal synthesis [13]. The assignment of the Raman and FTIR modes that come from the W-O or Bi-O bonds are listed in Table 1. In summary, the FTIR and Raman spectra obtained are in full agreement with what is shown in the literature and in agreement with the XRD results [12,14,15].  The room temperature Raman and Fourier transform infrared (FTIR) spectra of the synthesized layered Bi 2 WO 6 nanosheets are presented in Figure 5 and Table 1. The Raman spectrum of Bi 2 WO 6 monolayers in the spectra range between 100 and 900 cm −1 presents broad Raman peaks, some even overlapping forming only shoulders, making them difficult to distinguish in the unpolarized spectrum (See Figure 5a). On one hand, the Raman spectrum exhibits significant changes as the crystallite size decreases, comparing the Raman spectrum of Bi 2 WO 6 monolayers with the spectrum of Bi 2 WO 6 bulk structures. On the other hand, the Raman spectrum obtained is in full agreement with what is shown in the literature for nano-sized materials [12]. Looking at the FTIR spectrum in Figure 5b, we see that the broad band at 3460 cm −1 corresponds to the stretching vibration of O-H bonds, indicating water molecules adsorbed on the surface of the sample. The 2921 cm −1 and 2854 cm −1 are associated with the C-H stretching vibrations of the methyl and methylene groups of CTAB used in hydrothermal synthesis [13]. The assignment of the Raman and FTIR modes that come from the W-O or Bi-O bonds are listed in Table 1. In summary, the FTIR and Raman spectra obtained are in full agreement with what is shown in the literature and in agreement with the XRD results [12,14,15].

Antibacterial Activity of Layered Bi2WO6 Nanosheets
Bi2WO6 nanosheets revealed to be effective against all kinds of bacteria tested, with a MIC of 256 μg/mL against E. coli standard and resistant strains, and of 256 μg/mL and 32 μg/mL against S. aureus standard and resistant strains, respectively ( Table 2). Evaluating the antibacterial activity of Bi2WO6 nanosheets under LED irradiation with varying wavelengths (415, 620 and 590 nm), no photocatalytic reaction was observed, given that MIC values do not decrease under visible light irradiation. Thus, Bi2WO6 nanosheets exhibit excellent antibacterial inactivation on E. coli and S. aureus in the dark. In the study by Li et al. [16], the Bi2WO6 crystals in hierarchical flower-like morphology did not reveal antibacterial activity against E. coli in the dark, as some effect was only observed when the material was irradiated with visible light. Likewise, Hen et al. [10] using Aurivillius oxide Bi2WO6 as an effective visible light-driven antibacterial photocatalyst for E. coli inactivation, did not report any bactericidal effect under visible light without the photocatalyst nor Bi2WO6 in the dark. It is, however, worth noting that the results obtained by Hen et al. [10] and Li et al. [16] attributed the bactericidal effect to the photocatalytic reaction of Bi2WO6 (bulk) under visible light irradiation. In the present study, it was demonstrated that the antibacterial activity of the layered Bi2WO6 nanosheets was intrinsic to the material (no need for the presence of visible light).
Regarding the antibacterial effect of Bi2WO6 nanosheets, an intrinsic clinical relevance was found with the standard stated by Houghton et al. [17]. Another interesting point to underline is that this material affects Gram-positive and Gram-negative bacteria equally, which highlights the great clinical and technological potential of this material. This is especially important in view of the significant increase in resistance mechanisms developed by E.coli and S. aureus, such as the alteration of cell membrane permeability, making it difficult for antibiotics to enter and change the target drug binding sites, respectively [18,19].

Antibacterial Activity of Layered Bi 2 WO 6 Nanosheets
Bi 2 WO 6 nanosheets revealed to be effective against all kinds of bacteria tested, with a MIC of 256 µg/mL against E. coli standard and resistant strains, and of 256 µg/mL and 32 µg/mL against S. aureus standard and resistant strains, respectively (Table 2). Evaluating the antibacterial activity of Bi 2 WO 6 nanosheets under LED irradiation with varying wavelengths (415, 620 and 590 nm), no photocatalytic reaction was observed, given that MIC values do not decrease under visible light irradiation. Thus, Bi 2 WO 6 nanosheets exhibit excellent antibacterial inactivation on E. coli and S. aureus in the dark. In the study by Li et al. [16], the Bi 2 WO 6 crystals in hierarchical flower-like morphology did not reveal antibacterial activity against E. coli in the dark, as some effect was only observed when the material was irradiated with visible light. Likewise, Hen et al. [10] using Aurivillius oxide Bi 2 WO 6 as an effective visible light-driven antibacterial photocatalyst for E. coli inactivation, did not report any bactericidal effect under visible light without the photocatalyst nor Bi 2 WO 6 in the dark. It is, however, worth noting that the results obtained by Hen et al. [10] and Li et al. [16] attributed the bactericidal effect to the photocatalytic reaction of Bi 2 WO 6 (bulk) under visible light irradiation. In the present study, it was demonstrated that the antibacterial activity of the layered Bi 2 WO 6 nanosheets was intrinsic to the material (no need for the presence of visible light).
Regarding the antibacterial effect of Bi 2 WO 6 nanosheets, an intrinsic clinical relevance was found with the standard stated by Houghton et al. [17]. Another interesting point to underline is that this material affects Gram-positive and Gram-negative bacteria equally, which highlights the great clinical and technological potential of this material. This is especially important in view of the significant increase in resistance mechanisms developed by E.coli and S. aureus, such as the alteration of cell membrane permeability, making it difficult for antibiotics to enter and change the target drug binding sites, respectively [18,19].
In addition, the crystalline structure of layered Bi 2 WO 6 nanosheets has the ability to generate e − -h + pairs, while the sandwich substructure of [BiO] + -[WO 4 ] 2− -[BiO] + monolayer Bi 2 WO 6 simulates the heterojunction interface with space charge that promotes the separation of carriers generated in the interface [6]. As a result, such species interact with H 2 O, triggering the formation of OH • , H + , and O 2 •− , which can act on the cells' surface to degrade some components of bacterial cell membrane. Cell rupture can cause the bacteria to lose function and eventually lead to cell death [20]. Moreover, the high surface area/volume ratio of 2D Bi 2 WO 6 nanosheets favors the interaction between the sample and the bacterial membrane, facilitating possible adsorption processes. Therefore, based on the results discussed above, it is possible to observe the possibility that the mechanism responsible for the antibacterial activity of Bi 2 WO 6 monolayers is related to the two-dimensional property of the system.

Modulation of Antibiotic Activity by Layered Bi 2 WO 6 Nanosheets
Data obtained on the modulation of antibiotic activity demonstrated by Bi 2 WO 6 nanosheets are shown in Figure 6. The compound significantly raised the MIC of both antibiotics (amikacin and gentamicin) against Gram-negative and Gram-positive strains, show no improvements in antibiotic activity, and the associations even resulted in antagonistic effects.
The association of aminoglycosides under visible light irradiation may represent a promising strategy in the treatment of skin infections caused by resistant bacteria. Therefore, in this study the antibiotic-modulating effects of LED light exposure associated or not with Bi 2 WO 6 nanosheets was investigated (Figure 7). No potentiation of the action of aminoglycosides associated with Bi 2 WO 6 nanosheets against Gram-positive and Gramnegative strains was stated, regardless of LED wavelength. Thus, the strong antagonistic effects observed when aminoglycosides were used in combination with Bi 2 WO 6 nanosheets under visible light irradiation against Gram-positive and Gram-negative strains seem to be related to the high photocatalytic potential of bismuth tungstate in degrading organic compounds [8,9]. In addition, the crystalline structure of layered Bi2WO6 nanosheets has the ability to generate e − -h + pairs, while the sandwich substructure of [BiO] + -[WO4] 2− -[BiO] + monolayer Bi2WO6 simulates the heterojunction interface with space charge that promotes the separation of carriers generated in the interface [6]. As a result, such species interact with H2O, triggering the formation of OH • , H + , and O2 •− , which can act on the cells' surface to degrade some components of bacterial cell membrane. Cell rupture can cause the bacteria to lose function and eventually lead to cell death [20]. Moreover, the high surface area/volume ratio of 2D Bi2WO6 nanosheets favors the interaction between the sample and the bacterial membrane, facilitating possible adsorption processes. Therefore, based on the results discussed above, it is possible to observe the possibility that the mechanism responsible for the antibacterial activity of Bi2WO6 monolayers is related to the two-dimensional property of the system.

Modulation of Antibiotic Activity by Layered Bi2WO6 Nanosheets
Data obtained on the modulation of antibiotic activity demonstrated by Bi2WO6 nanosheets are shown in Figure 6. The compound significantly raised the MIC of both antibiotics (amikacin and gentamicin) against Gram-negative and Gram-positive strains, show no improvements in antibiotic activity, and the associations even resulted in antagonistic effects.  The emergence of antibiotic resistance by pathogenic bacteria occurs due to several factors and, among these, the inappropriate drugs' disposal stands out. Specifically, wastewater from health facilities, mainly hospitals, stands out as an important source of emission of antibiotics in the environment and, thus, it is important to degrade these compounds before disposal. In this sense, current research is seeking new agents capable of acting as photocatalysts [21], and Bi 2 WO 6 nanosheets showed a good potential in the treatment of effluents containing gentamicin and amikacin.

Synthesis
The synthesis of layered Bi 2 WO 6 nanosheets was performed by a hydrothermal method, as described previously [6].  3 ] were used as starting precursors. In a synthesis procedure, 1 mmol of Na 2 WO 4 ·2H 2 O, 2 mmol of Bi(NO 3 ) 3 ·5H 2 O and 0.05 g of CTAB were dissolved in 80 mL of deionized water. This aqueous solution was stirred for 30 min at an average speed of 1500 rpm. The resulting solution was transferred to a 100 mL Teflon-lined stainless autoclave and maintained at 120 • C for 24 h. The white precipitates were repeatedly washed with deionized water and dried in an air oven at 60 • C for 10 h.

Structural Characterization
Structural characterization was performed by X-ray diffraction (XRD) using a Mini-Flex Rigaku diffractometer. Morphological analysis of layered Bi 2 WO 6 nanosheets was carried out in a scanning electron microscope (SEM) model Vega3 Tescan. Atomic force microscopy images were recorded using NTMDT microscope. Fourier Transform Infrared (FT-IR) spectra were obtained using a Perkin Elmer Spectrum Two spectrophotometer. Raman measurements were performed by a Horiba LabRaman spectrometer.

Analysis of Antibacterial Activity and Antibiotic Resistance Modulation
The antibiotic-enhancing activity was assessed using the methodology of Coutinho et al. [22].

Experiments with LED Light Exposure
The Light Emitting Diodes-LED device (a light emitting diode; NEW Estética ® , Fortaleza, Brazil) was used in the experimental protocols. The LEDs with a wavelength predetermined by the device used were blue (415 nm), red (620 nm) and yellow (590 nm). To assess the effect of LED light exposure on bacterial growth in modulating antibacterial activity, cultures and treatments were performed as described above. Plates were exposed to blue, red or yellow light for 20 min and then incubated at 37 • C for 24 h. Plates without exposure to LED light were used as experimental controls. Readings were performed as described above.

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 6.0 software, with an alpha set at 0.05. One-way analysis of variance (ANOVA) and Bonferroni's posthoc tests were used to address differences between groups. More details are shown in Supplementary File S1.

Conclusions
Data obtained in this study provide significant insights into the dimension-dependent antibacterial activity of layered Bi 2 WO 6 nanosheets. The crystalline nature of the samples was confirmed, and all diffraction peaks were indexed to the orthorhombic Bi 2 WO 6 phase. Two-dimensional (2D) Bi 2 WO 6 nanosheets showed antibacterial action against