# Antibacterial Activity of Bismuth Oxide Nanoparticles Compared to Amikacin againstAcinetobacter baumannii and Staphylococcus aureus

Authors: Kareem H. Jawad; Thorria R. Marzoog; Buthenia A. Hasoon; Ghassan M. Sulaiman; Majid S. Jabir; Elsadig M. Ahmed; Khalil A. A. Khalil
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

In this work, bismuth oxide nanoparticles (Bi2O3 NP) were prepared by laser ablation of a solid target in distilled water. The optical and structural properties of Bi2O3 NPs were studied using UV–VIS spectroscopy, energy-dispersive X-ray spectroscopy (EDS), transmission electron microscope (TEM), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR). The results showed that the average particle size is less than 20 nm. The antibacterial activity of the Bi2O3 NPs was determined and compared with amikacin against Acinetobacter baumannii and Staphylococcus aureus. The activity of Bi2O3 NPs was higher than the amikacin. Furthermore, the antibacterial activity of Bi2O3 NPs against S. aureus bacteria was higher than Acinetobacter baumannii; this result was confirmed by studying the effect of Bi2O3 NPs in bacterial biofilm formation in eyes contact lenses using atomic force microscopy (AFM) technique. Taken together, Bi2O3 NPs could be used as a preservative for contact lenses as eye drops to prevent the formation of microbial biofilms in contact lenses.


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## Body

## 1. Introduction

Antibiotics are the primary foundation for the treatment of bacterial diseases. The medical community was convinced that the discovery of these antibiotics and their usage as chemotherapeutic agents would lead to the eradication of infectious diseases in the future. Antibiotic misuse, on the other hand, has become a major role in the development and spread of multidrug-resistant strains of various bacteria [1]. Microorganism keratitis, which is commonly associated with contact lenses, is a significant risk factor for the progression of microbial keratitis [2].Many patients have bad clinical results unless aggressive and adequate treatments are initiated quickly [3]. Even if effectively handled, microbial keratitis may lead to corneal scarring and neovascularization, leading to a loss of core corneal clarity, requiring a corneal transplant to restore sight. Successful leadership can lead to a continuous loss of visual function and possibly even eye loss [4]. This situation forced researchers to look for new antimicrobial and anti-inflammatory substances from different sources, such as Bi2O3 NPs [5–7]. The growth of drug resistance, allergic reactions, and side effects has resulted in exploring alternative drugs for nosocomial infection treatment [8, 9]. Different researches have confirmed that the transitions in Bi2O3 are of the direct permissible type, and the width of the optical energy gap ranges between (2.80-2.95 eV). Bi2O3 is a crucial derivative in the fabrication of glass and ceramics, as also hydrocarbon oxidation catalysts. Sensors, microelectronics, and optical engineering are all frequent applications [7, 8]. Thus, it has a broad range of applications and may be used as a surface disinfectant in hospitals and the food, pharmaceutical, glass, and ceramic manufacturing industries [9].The pulsed laser ablation technique is a simple, novel, nonpolluting, eco-friendly, low-cost, one-step, and quick method for producing pure metal nanoparticles with regular and minimum buildup [10–13]. The aim of this study was to synthesize Bi2O3 NPs by laser ablation technique and study their effect as a preservative of contact lenses against eye infections. The results showed the ability of Bi2O3 NPs as an antibacterial agent to prevent bacterial biofilm formation.


## 2. Materials and Methods

A pure plate of bismuth (Sigma Aldrich), contact lenses (Alcon-china), and DMSO was purchased from Sigma.


### 2.1. Preparation of Bi2O3 NPs

The Bi2O3-NPs were prepared via laser ablation of solids submerged in deionized distilled water [12, 14] as shown in the experiment illustrated in Figure 1. Then, the NPs were passed through a (0.22) m size filter for size uniformity by applying an Nd: YAG laser with a fundamental wavelength of (1064) nm., the target was irradiated for (15) min., using (150) pulses, a pulse length of (6) ns., a repetition rate of (2) Hz, spot size (10) mm, the focal length of the lens (8) cm, and the maximum output energy of (500) mJ.Figure 1
Setup for nanoparticle synthesis.


### 2.2. Characterizations of Bi2O3 NPs

The formation of Bi2O3 NPs was achieved by measuring the solution’s absorption spectrum with a UV-vis (UV/Vis, Seoul, Korea) in the 200-800 nm range [12, 15]. The microstructure and particle size of the nanocrystals were determined from (SEM) images (INSPECTS50-USA) and analyzed using Image J software [16]. FTIR was measured on the functional groups present on the surface of Bi2O3 NPs prepared from the laser ablation method were sections made by KBr pellet method (Bruker Equinox55, Germany) in the wavenumber scope of (400–4000)cm-1 on (FTLA 2000-100) version [17]. TEM (Philips EM 208S) was used to evaluate the morphology of the nanoparticles and size distribution [18]. AFM was used to determine the beneficial groups and position of each atom on a three-dimensional diagram of the surface topography of a substance at the atomic level (CSPM-AA3000) [19]. EDS tests were done by (Inspect S 50/FEI Company/Netherlands). These assays were achieved at the University of Tehran.


### 2.3. Evaluation of MIC of Amikacin and Bi2O3 NPs

The microdilution assay determined the minimal inhibitory concentration (MIC) of amikacin and bismuth oxide NPs [20, 21].


### 2.4. Antibacterial Activity

The well diffusion technique was utilized to identify the antibacterial effects of amikacin and bismuth oxide NPs againstStaphylococcus aureus and Acinetobacter baumannii isolates at the concentration for sub-MIC (32) μg/ml according to [22, 23].


### 2.5. Fluorescence-Activated Cell Sorting Assay

Bacterial strains were colored by applying the LIVE/DEAD BacLight Bacterial Viability kit corresponding to the production command (Invitrogen AG, Basel, Switzerland). The dilution of SYTO9 was (1 : 60) allowing for prior titrations to get to the best dilution of pigments to be utilized for stain, propidium iodide (PI) (equipment instructions). The samples remained kept for (30) min. at (4°C) and was analyzed by applying a flow cytometer assay (FCA). One unit cell was confined on the up-scatter (FSC-A) versus up-scatter height (FSC-H) plots. The cell remains were rejected. Consequently, 20000 incidents were established in the entry, particularly for single incidents. Unblemished and particular-color controllers were utilized to set a door for PI (+ve cells). FCA was achieved on a BD Verse method, Biosciences, U.S.A., and statistics were examined by applying FACS DIVA computer software (BD), San Jose, CA, U.S.A.


### 2.6. Effect of Sub-MIC of Amikacin and Bi2O3 NPs in Biofilm Formation in Contact Lenses Visualized by AFM

This assay was performed according to previous work, with a few changes to produce biofilm on contact lenses. Briefly, lenses were put in (10) ml of nutrient broth containing bacterial cell growth in a concentration of1.5×108cfu/ml, which exhibited the highest reduction in biofilm formation capacity after treatment with NPs, and amikacin at sub-MIC was chosen for this experiment. For 24 hours, containers were incubated aerobically at (37°C). Decantation was used to remove the media and planktonic cells, followed by two washes with distilled water (DW). The adhering cells were allowed to dry for 30 minutes after being washed twice by DW. The lenses were then stained and prepared for examination under an atomic force microscope. Control positive was created by cultivating lenses with a bacterial culture free of NPs. At the same time, the control negative was prepared by cultivating lenses in nutrient broth without any treatment or bacteria.


### 2.7. Statistical Analysis

At a significance level ofP0.05, an unpaired t-test was used to evaluate the significance level in the observed values across the treatment groups [24].


## 2.1. Preparation of Bi2O3 NPs

The Bi2O3-NPs were prepared via laser ablation of solids submerged in deionized distilled water [12, 14] as shown in the experiment illustrated in Figure 1. Then, the NPs were passed through a (0.22) m size filter for size uniformity by applying an Nd: YAG laser with a fundamental wavelength of (1064) nm., the target was irradiated for (15) min., using (150) pulses, a pulse length of (6) ns., a repetition rate of (2) Hz, spot size (10) mm, the focal length of the lens (8) cm, and the maximum output energy of (500) mJ.Figure 1
Setup for nanoparticle synthesis.


## 2.2. Characterizations of Bi2O3 NPs

The formation of Bi2O3 NPs was achieved by measuring the solution’s absorption spectrum with a UV-vis (UV/Vis, Seoul, Korea) in the 200-800 nm range [12, 15]. The microstructure and particle size of the nanocrystals were determined from (SEM) images (INSPECTS50-USA) and analyzed using Image J software [16]. FTIR was measured on the functional groups present on the surface of Bi2O3 NPs prepared from the laser ablation method were sections made by KBr pellet method (Bruker Equinox55, Germany) in the wavenumber scope of (400–4000)cm-1 on (FTLA 2000-100) version [17]. TEM (Philips EM 208S) was used to evaluate the morphology of the nanoparticles and size distribution [18]. AFM was used to determine the beneficial groups and position of each atom on a three-dimensional diagram of the surface topography of a substance at the atomic level (CSPM-AA3000) [19]. EDS tests were done by (Inspect S 50/FEI Company/Netherlands). These assays were achieved at the University of Tehran.


## 2.3. Evaluation of MIC of Amikacin and Bi2O3 NPs

The microdilution assay determined the minimal inhibitory concentration (MIC) of amikacin and bismuth oxide NPs [20, 21].


## 2.4. Antibacterial Activity

The well diffusion technique was utilized to identify the antibacterial effects of amikacin and bismuth oxide NPs againstStaphylococcus aureus and Acinetobacter baumannii isolates at the concentration for sub-MIC (32) μg/ml according to [22, 23].


## 2.5. Fluorescence-Activated Cell Sorting Assay

Bacterial strains were colored by applying the LIVE/DEAD BacLight Bacterial Viability kit corresponding to the production command (Invitrogen AG, Basel, Switzerland). The dilution of SYTO9 was (1 : 60) allowing for prior titrations to get to the best dilution of pigments to be utilized for stain, propidium iodide (PI) (equipment instructions). The samples remained kept for (30) min. at (4°C) and was analyzed by applying a flow cytometer assay (FCA). One unit cell was confined on the up-scatter (FSC-A) versus up-scatter height (FSC-H) plots. The cell remains were rejected. Consequently, 20000 incidents were established in the entry, particularly for single incidents. Unblemished and particular-color controllers were utilized to set a door for PI (+ve cells). FCA was achieved on a BD Verse method, Biosciences, U.S.A., and statistics were examined by applying FACS DIVA computer software (BD), San Jose, CA, U.S.A.


## 2.6. Effect of Sub-MIC of Amikacin and Bi2O3 NPs in Biofilm Formation in Contact Lenses Visualized by AFM

This assay was performed according to previous work, with a few changes to produce biofilm on contact lenses. Briefly, lenses were put in (10) ml of nutrient broth containing bacterial cell growth in a concentration of1.5×108cfu/ml, which exhibited the highest reduction in biofilm formation capacity after treatment with NPs, and amikacin at sub-MIC was chosen for this experiment. For 24 hours, containers were incubated aerobically at (37°C). Decantation was used to remove the media and planktonic cells, followed by two washes with distilled water (DW). The adhering cells were allowed to dry for 30 minutes after being washed twice by DW. The lenses were then stained and prepared for examination under an atomic force microscope. Control positive was created by cultivating lenses with a bacterial culture free of NPs. At the same time, the control negative was prepared by cultivating lenses in nutrient broth without any treatment or bacteria.


## 2.7. Statistical Analysis

At a significance level ofP0.05, an unpaired t-test was used to evaluate the significance level in the observed values across the treatment groups [24].


## 3. Results and Discussion

### 3.1. Characteristics of Bi2O3 NPs

By decreasing bismuth ions in the solution, UV-visible spectroscopy verified the existence of nanoparticles (Figure2). The Bi2O3 NPs were put in a quartz cuvette and monitored for wavelength scanning between (200 and 800) nm with distilled water as a reference. The absorption peak, which is typical of bismuth, was found at 245 nm.Figure 2
UV-visible spectrum of Bi2O3 NPs prepared by laser ablation.SEM was used to examine the morphology of the produced Bi2O3NPs, as shown in Figure 3. The size distribution of the Bi2O3 NPs sample was evaluated using Image J software, and most of the particles were in the 22-24-30 nm range.Figure 3
SEM of Bi2O3NPs prepared by laser ablation.The infrared spectrum of the Bi2O3 NPs is shown in Figure 4. Four peaks appeared, with wavenumbers of 624.87 and 3455.67 cm−1, which are attributed to variation group vibrations of -C=C-, C=O, S-H, and N-H groups. A broad peak at 624.87 cm−1 is associated with an alkyne group (−C=C−), the peak at 1637.06 cm−1 is associated with a carboxylic acid group, the peak at 2084.05 cm−1 is related with the thiol group, and the peaks at 3455.67 cm−1 are associated with amine groups.Figure 4
Infrared spectrum of Bi2O3 nanoparticles.TEM was used to evaluate the size distribution and morphology of nanoparticles. Figure5(a) explains the TEM images of Bi2O3 nanoparticles are spherical-like shape, while Figure 5(b) shows histogram size distribution, most of the particles are in the 5–40 nm, and this was comparable with the SEM result. Figure 5(c) shows energy dispersive spectrum (EDS) explains the chemical composition of the as-synthesized Bi2O3NPs samples. The NPs are mainly composed of Bi and O atoms. The ratio of weight (%) of Bi and O in the as-prepared final NPs sample is 89.8 and 10.2, respectively.Figure 5
TEM image of (a) Bi2O3NPs, (b) size distribution, and (c) EDS spectrum of nanoparticles.
(a)(b)(c)The AFM images for the nanostructures deposited utilizing 150 pulses are shown in Figure6. The topography of the surface of Bi2O3NPs particles was studied, and measurements of surface roughness, particle size nanoparticles rate, and particle size distribution were taken. The average surface roughness is 1.454 nm, with diameters ranging from 5 to 40 nm and a quasispherical shape. These findings are in good agreement with the TEM measurements.Figure 6
AFM of Bi2O3NPs (a) prepared by laser ablation (b) 3D and (c) statistical distribution.
(a)(b)(c)


### 3.2. Evaluation of MIC of Amikacin and Bi2O3 NPs

The MIC is used to identify which antibiotic class is the most effective. The MICs of amikacin and Bi2O3 NPs for S. aureus and A. baumannii isolate examined varied from 64 μg/ml in S. auras isolate, in A. baumannii isolate, and amikacin MICs 16 μg/ml. For S. aureus, the MIC of Bi2O3 NPs is 128 μg/ml (Table 1). Bi2O3NPs had MICs 64 μg/ml in A. baumannii isolate.Table 1
MIC of amikacin and Bi2O3 NPs to inhibit S. aureus and A. baumannii isolate biofilm.
IsolatesAmikacin (μg/ml)Bi2O3 NPs (μg/ml)S. aureus64128A. baumannii1664


### 3.3. Antibiofilm Activity of Amikacin and Bi2O3 NPs

The well diffusion technique occurred used to investigate the synergistic impact of Bi2O3 NPs with antibiotics (Amikacin) at a concentration of 32 μg/ml. The synergistic impact of Bi2O3 NPs against S. aureus is shown in Figure 7. The inhibitory zones were (16) mm, followed by (13) mm for antibiotics alone and (23) mm for Bi2O3 NPs. In addition, Bi2O3 NPs with amikacin have a synergistic impact against A. baumannii. As seen in Figure 7, the zone of inhibition was (12) mm, followed by antibiotics alone (15) mm and Bi2O3NPs (20) mm. The interaction between antibiotics and nanoparticles is most likely to blame for this synergistic impact [25]. For example, silver nanoparticles and amoxicillin were shown to have the most significant synergistic impact in suppressing E. coli growth [26]. This showed that Bi2O3NPs might significantly enhance amikacin invasion and absorption into the cell membrane.Figure 7
Antibacterial activity of Bi2O3 NPs anti-S. aureus and A. baumannii. (a) Control. (b) Bi2O3 NPs at concentration 62.5 μg/ml. (c) Bi2O3 NPs at concentration 125 μg/ml. (d) Amikacin. (e) Bi2O3NPs+amikacin.Furthermore, since NPs need a wide surface area for interactions, they have a higher bactericidal effect than bigger particles, increasing bacterial cytotoxicity. For the identical treatments, the inhibiting area inG+ve bacteria is larger as compared to G−ve bacteria. G+ve bacteria have a different cell wall structure than G−ve bacteria, which could explain why G+ve bacteria are more antibiotic-resistant. Only the exterior peptidoglycan layer of G+ve bacteria is present. G−ve bacteria, on the other hand, have an exterior polysaccharide barrier that keeps all polysaccharide structures intact [27]. Many active compounds, such as hydroxy and amido groups, are found in antibiotic compounds. Chelation is a simple reaction that these groups have with nanoparticles. Through van der Waals contact and other weak interactions, antibiotic molecules can connect. Finally, antimicrobial groups are formed, made up of a nanoparticle core and antibiotic molecules around it. Antimicrobial groups induce more destruction when they act on one location on the surface of bacterial cells [28].


### 3.4. Viability Assay for Bacterial Strains

Flow cytometry of microbial cells was also presented to confirm the bactericidal activity of the prepared nanoparticles. The stains which are applied in this research were SYTO9 and (PI) as DNA-binding pigments, depending on their cell-penetrative property. SYTO9 enters both live and dead cells as various to PI, which can only enter cells whose membrane integrity is conceded, as death/killing cells. The observed FCA is extra receptive compared with conventional plate analysis in the identification of death/killing bacterial cells as the failure in pathogenic cell penetrability is immediately found by PI integration. Unblemished cells as in Figure8 were run to quantitate autofluorescence and set standard energy. At zero time, the experiments have been run to calculate the early proportion of PI (+ve cells). After 6 hr. of treatment with nanoparticles, the possibilities of these bacterial species were examined. Bi2O3NPs determined the maximum cell death ratio as studied by quantification of PI-positive cells as well as the nanoparticles as shown in Figure 8.Figure 8
Effect of bismuth nanoparticles in the viability of bacterial strains. Flow cytometric technique represented determined propidium iodide- (PI-) positive microbial cells in Bi2O3NPs. S. aureus and A. baumannii were treated with Bi2O3 NPs for 6 hrs and dyed with PI and SYTO9 (∗∗∗∗P≤0.0001, Student’s t-test).Flow cytometry of bacterial cells was also used to confirm the bactericidal activity of the nanostructured surfaces. SYTO9 and propidium iodide (PI), the DNA-binding dyes used in this investigation, have different cell-penetrating properties. SYTO9 can infiltrate both live and dead cells, whereas PI can only infiltrate cells whose membrane integrity has been disrupted, i.e., dead cells. It should be noted that the flow cytometric technique is more sensitive than standard plate assay in the detection of dead bacterial cells as the loss in bacterial cell permeability is readily detected by PI incorporation. Unstained cells (Figure8) were run to quantitate autofluorescence and set baseline voltage. Samples were taken during the zeroth hour to determine the initial percentage of PI-positive cells. The viability of these cells was determined after 3 hours of exposure to nanomaterial samples. Quantification of PI-positive cells among nanostructured surfaces revealed that Bi2O3NPs had the highest cell death percentage (Figure 8).


### 3.5. Effect of Sub-MIC Amikacin and Bi2O3 NPs on Biofilm Formation Visualized by AFM

AFM also looked at the contact lenses to see how efficient amikacin and Bi2O3 NPs prevented biofilm development. As demonstrated in Figures 9(a)–9(g), Bi2O3 NPs outperformed amikacin in preventing biofilm development on the lenses. The results of an atomic force microscope (AFM) study of biofilm development in lenses with S. aureus bacteria was (29.6 nm) of high lens surface Figure 9(b). The outcome which did not have any bacteria was (2.14 nm) of lens surface in Figure 9(a). Figure 9(c) shows just A. baumannii bacteria or treatment on the high lens surface (21.4 nm). In Figure 9(d), the highest surface was (9.9 nm) with S. aureus and treated with amikacin antibiotic and with S. aureus and treated with Bi2O3 NPs (3.06 nm) in Figure 9(e). In Figure 9(f), the most significant surface was (12.4 nm) with A. baumannii and treated with Amikacin antibiotic. In comparison, the lowest surface was (8.06 nm) with A. baumannii and treated with Bi2O3 NPs in Figure 9(g). These findings revealed that Bi2O3 NPs were more effective as an antibiofilm agent than amikacin. AFM revealed no S. aureus colonization on any Bi2O3 NP-treated lenses (Figure 9(g)). On the other hand, the untreated contact lenses exhibited colonization of lenses as a multilayer on this lens contact Figure 9(a). AFM was used to measure the adhesion and biofilm stiffness [29]. Surface roughness [30] and topography have all been effective in microbial biofilm research [31]. A few studies have shown that Bi2O3 NPs have strong antibiofilm actions against various pathogens in vitro and have indicated various antibacterial mechanisms for Bi2O3 NPs, including impairing cell division, disturbing the cell wall cytoplasmic membrane, and decomposing DNA [32–34]. By employing AFM image analysis [29], the biofilm formation and surface roughness of P. aeruginosa were investigated by AFM.Figure 9
Antibiofilm formation activity in contact lenses under roughness analysis by AFM image.
(a)(b)(c)(d)(e)(f)(g)The production of harmful ions, shading impact on photosynthetic bacteria, cellular absorption of NPs, physical restraint and cell wall destruction, and oxidative stress are the key processes behind the toxicity of metallic-based nanoparticles, as shown by several biological models. Furthermore, the biological models are used to assess the potential hazards of nanomaterials (cell wall damage, inhibition of cell proliferation, gene expression changes, genotoxicity, and cell death, alteration of function and morphology of organelles, and the presence of oxidative stress bioindicators) [35]. Environmental nanotechnology is defined as the use of biotechnological applications as a low-cost, environmentally friendly, and simple method of producing, manipulating, and utilizing materials with dimensions ranging from 1 to 100 nm [36, 37]. Antimicrobial drug resistance in bacterial infection has driven the development of new treatment options. Among these efforts, nanomaterials have emerged as notable and novel antibacterial agents [38]. In this work, the effectiveness of bismuth nanoparticles in inhibiting the growth of A. baumannii and S. aureus was investigated using the broth microdilution method, which is a standard method that is more accurate, reliable, and easy to interpret than other methods such as the well diffusion method. Because of its poor water solubility, the inhibitory action of elemental bismuth can only be noticed at quite high doses. Nonetheless, our findings revealed that Bi2O3NPs had more efficient antibacterial action against A. baumannii and S. aureus than amikacin, with a MIC of bismuth nanoparticles that was higher than amikacin. Overall, the experimental data suggest that bismuth nanoparticles could be an interesting alternative to combat A. baumannii and S. aureus, which, given the benefits mentioned for bismuth nanoparticles such as inhibiting A. baumannii and S. aureus biofilm formation and higher antibacterial activity compared to amikacin, could be suggested for use in various fields of keratitis, which is commonly associated with contact. Furthermore, additional study is needed into the compatibility of nanoparticles with nature as well as the way of their recovery.


## 3.1. Characteristics of Bi2O3 NPs

By decreasing bismuth ions in the solution, UV-visible spectroscopy verified the existence of nanoparticles (Figure2). The Bi2O3 NPs were put in a quartz cuvette and monitored for wavelength scanning between (200 and 800) nm with distilled water as a reference. The absorption peak, which is typical of bismuth, was found at 245 nm.Figure 2
UV-visible spectrum of Bi2O3 NPs prepared by laser ablation.SEM was used to examine the morphology of the produced Bi2O3NPs, as shown in Figure 3. The size distribution of the Bi2O3 NPs sample was evaluated using Image J software, and most of the particles were in the 22-24-30 nm range.Figure 3
SEM of Bi2O3NPs prepared by laser ablation.The infrared spectrum of the Bi2O3 NPs is shown in Figure 4. Four peaks appeared, with wavenumbers of 624.87 and 3455.67 cm−1, which are attributed to variation group vibrations of -C=C-, C=O, S-H, and N-H groups. A broad peak at 624.87 cm−1 is associated with an alkyne group (−C=C−), the peak at 1637.06 cm−1 is associated with a carboxylic acid group, the peak at 2084.05 cm−1 is related with the thiol group, and the peaks at 3455.67 cm−1 are associated with amine groups.Figure 4
Infrared spectrum of Bi2O3 nanoparticles.TEM was used to evaluate the size distribution and morphology of nanoparticles. Figure5(a) explains the TEM images of Bi2O3 nanoparticles are spherical-like shape, while Figure 5(b) shows histogram size distribution, most of the particles are in the 5–40 nm, and this was comparable with the SEM result. Figure 5(c) shows energy dispersive spectrum (EDS) explains the chemical composition of the as-synthesized Bi2O3NPs samples. The NPs are mainly composed of Bi and O atoms. The ratio of weight (%) of Bi and O in the as-prepared final NPs sample is 89.8 and 10.2, respectively.Figure 5
TEM image of (a) Bi2O3NPs, (b) size distribution, and (c) EDS spectrum of nanoparticles.
(a)(b)(c)The AFM images for the nanostructures deposited utilizing 150 pulses are shown in Figure6. The topography of the surface of Bi2O3NPs particles was studied, and measurements of surface roughness, particle size nanoparticles rate, and particle size distribution were taken. The average surface roughness is 1.454 nm, with diameters ranging from 5 to 40 nm and a quasispherical shape. These findings are in good agreement with the TEM measurements.Figure 6
AFM of Bi2O3NPs (a) prepared by laser ablation (b) 3D and (c) statistical distribution.
(a)(b)(c)


## 3.2. Evaluation of MIC of Amikacin and Bi2O3 NPs

The MIC is used to identify which antibiotic class is the most effective. The MICs of amikacin and Bi2O3 NPs for S. aureus and A. baumannii isolate examined varied from 64 μg/ml in S. auras isolate, in A. baumannii isolate, and amikacin MICs 16 μg/ml. For S. aureus, the MIC of Bi2O3 NPs is 128 μg/ml (Table 1). Bi2O3NPs had MICs 64 μg/ml in A. baumannii isolate.Table 1
MIC of amikacin and Bi2O3 NPs to inhibit S. aureus and A. baumannii isolate biofilm.
IsolatesAmikacin (μg/ml)Bi2O3 NPs (μg/ml)S. aureus64128A. baumannii1664


## 3.3. Antibiofilm Activity of Amikacin and Bi2O3 NPs

The well diffusion technique occurred used to investigate the synergistic impact of Bi2O3 NPs with antibiotics (Amikacin) at a concentration of 32 μg/ml. The synergistic impact of Bi2O3 NPs against S. aureus is shown in Figure 7. The inhibitory zones were (16) mm, followed by (13) mm for antibiotics alone and (23) mm for Bi2O3 NPs. In addition, Bi2O3 NPs with amikacin have a synergistic impact against A. baumannii. As seen in Figure 7, the zone of inhibition was (12) mm, followed by antibiotics alone (15) mm and Bi2O3NPs (20) mm. The interaction between antibiotics and nanoparticles is most likely to blame for this synergistic impact [25]. For example, silver nanoparticles and amoxicillin were shown to have the most significant synergistic impact in suppressing E. coli growth [26]. This showed that Bi2O3NPs might significantly enhance amikacin invasion and absorption into the cell membrane.Figure 7
Antibacterial activity of Bi2O3 NPs anti-S. aureus and A. baumannii. (a) Control. (b) Bi2O3 NPs at concentration 62.5 μg/ml. (c) Bi2O3 NPs at concentration 125 μg/ml. (d) Amikacin. (e) Bi2O3NPs+amikacin.Furthermore, since NPs need a wide surface area for interactions, they have a higher bactericidal effect than bigger particles, increasing bacterial cytotoxicity. For the identical treatments, the inhibiting area inG+ve bacteria is larger as compared to G−ve bacteria. G+ve bacteria have a different cell wall structure than G−ve bacteria, which could explain why G+ve bacteria are more antibiotic-resistant. Only the exterior peptidoglycan layer of G+ve bacteria is present. G−ve bacteria, on the other hand, have an exterior polysaccharide barrier that keeps all polysaccharide structures intact [27]. Many active compounds, such as hydroxy and amido groups, are found in antibiotic compounds. Chelation is a simple reaction that these groups have with nanoparticles. Through van der Waals contact and other weak interactions, antibiotic molecules can connect. Finally, antimicrobial groups are formed, made up of a nanoparticle core and antibiotic molecules around it. Antimicrobial groups induce more destruction when they act on one location on the surface of bacterial cells [28].


## 3.4. Viability Assay for Bacterial Strains

Flow cytometry of microbial cells was also presented to confirm the bactericidal activity of the prepared nanoparticles. The stains which are applied in this research were SYTO9 and (PI) as DNA-binding pigments, depending on their cell-penetrative property. SYTO9 enters both live and dead cells as various to PI, which can only enter cells whose membrane integrity is conceded, as death/killing cells. The observed FCA is extra receptive compared with conventional plate analysis in the identification of death/killing bacterial cells as the failure in pathogenic cell penetrability is immediately found by PI integration. Unblemished cells as in Figure8 were run to quantitate autofluorescence and set standard energy. At zero time, the experiments have been run to calculate the early proportion of PI (+ve cells). After 6 hr. of treatment with nanoparticles, the possibilities of these bacterial species were examined. Bi2O3NPs determined the maximum cell death ratio as studied by quantification of PI-positive cells as well as the nanoparticles as shown in Figure 8.Figure 8
Effect of bismuth nanoparticles in the viability of bacterial strains. Flow cytometric technique represented determined propidium iodide- (PI-) positive microbial cells in Bi2O3NPs. S. aureus and A. baumannii were treated with Bi2O3 NPs for 6 hrs and dyed with PI and SYTO9 (∗∗∗∗P≤0.0001, Student’s t-test).Flow cytometry of bacterial cells was also used to confirm the bactericidal activity of the nanostructured surfaces. SYTO9 and propidium iodide (PI), the DNA-binding dyes used in this investigation, have different cell-penetrating properties. SYTO9 can infiltrate both live and dead cells, whereas PI can only infiltrate cells whose membrane integrity has been disrupted, i.e., dead cells. It should be noted that the flow cytometric technique is more sensitive than standard plate assay in the detection of dead bacterial cells as the loss in bacterial cell permeability is readily detected by PI incorporation. Unstained cells (Figure8) were run to quantitate autofluorescence and set baseline voltage. Samples were taken during the zeroth hour to determine the initial percentage of PI-positive cells. The viability of these cells was determined after 3 hours of exposure to nanomaterial samples. Quantification of PI-positive cells among nanostructured surfaces revealed that Bi2O3NPs had the highest cell death percentage (Figure 8).


## 3.5. Effect of Sub-MIC Amikacin and Bi2O3 NPs on Biofilm Formation Visualized by AFM

AFM also looked at the contact lenses to see how efficient amikacin and Bi2O3 NPs prevented biofilm development. As demonstrated in Figures 9(a)–9(g), Bi2O3 NPs outperformed amikacin in preventing biofilm development on the lenses. The results of an atomic force microscope (AFM) study of biofilm development in lenses with S. aureus bacteria was (29.6 nm) of high lens surface Figure 9(b). The outcome which did not have any bacteria was (2.14 nm) of lens surface in Figure 9(a). Figure 9(c) shows just A. baumannii bacteria or treatment on the high lens surface (21.4 nm). In Figure 9(d), the highest surface was (9.9 nm) with S. aureus and treated with amikacin antibiotic and with S. aureus and treated with Bi2O3 NPs (3.06 nm) in Figure 9(e). In Figure 9(f), the most significant surface was (12.4 nm) with A. baumannii and treated with Amikacin antibiotic. In comparison, the lowest surface was (8.06 nm) with A. baumannii and treated with Bi2O3 NPs in Figure 9(g). These findings revealed that Bi2O3 NPs were more effective as an antibiofilm agent than amikacin. AFM revealed no S. aureus colonization on any Bi2O3 NP-treated lenses (Figure 9(g)). On the other hand, the untreated contact lenses exhibited colonization of lenses as a multilayer on this lens contact Figure 9(a). AFM was used to measure the adhesion and biofilm stiffness [29]. Surface roughness [30] and topography have all been effective in microbial biofilm research [31]. A few studies have shown that Bi2O3 NPs have strong antibiofilm actions against various pathogens in vitro and have indicated various antibacterial mechanisms for Bi2O3 NPs, including impairing cell division, disturbing the cell wall cytoplasmic membrane, and decomposing DNA [32–34]. By employing AFM image analysis [29], the biofilm formation and surface roughness of P. aeruginosa were investigated by AFM.Figure 9
Antibiofilm formation activity in contact lenses under roughness analysis by AFM image.
(a)(b)(c)(d)(e)(f)(g)The production of harmful ions, shading impact on photosynthetic bacteria, cellular absorption of NPs, physical restraint and cell wall destruction, and oxidative stress are the key processes behind the toxicity of metallic-based nanoparticles, as shown by several biological models. Furthermore, the biological models are used to assess the potential hazards of nanomaterials (cell wall damage, inhibition of cell proliferation, gene expression changes, genotoxicity, and cell death, alteration of function and morphology of organelles, and the presence of oxidative stress bioindicators) [35]. Environmental nanotechnology is defined as the use of biotechnological applications as a low-cost, environmentally friendly, and simple method of producing, manipulating, and utilizing materials with dimensions ranging from 1 to 100 nm [36, 37]. Antimicrobial drug resistance in bacterial infection has driven the development of new treatment options. Among these efforts, nanomaterials have emerged as notable and novel antibacterial agents [38]. In this work, the effectiveness of bismuth nanoparticles in inhibiting the growth of A. baumannii and S. aureus was investigated using the broth microdilution method, which is a standard method that is more accurate, reliable, and easy to interpret than other methods such as the well diffusion method. Because of its poor water solubility, the inhibitory action of elemental bismuth can only be noticed at quite high doses. Nonetheless, our findings revealed that Bi2O3NPs had more efficient antibacterial action against A. baumannii and S. aureus than amikacin, with a MIC of bismuth nanoparticles that was higher than amikacin. Overall, the experimental data suggest that bismuth nanoparticles could be an interesting alternative to combat A. baumannii and S. aureus, which, given the benefits mentioned for bismuth nanoparticles such as inhibiting A. baumannii and S. aureus biofilm formation and higher antibacterial activity compared to amikacin, could be suggested for use in various fields of keratitis, which is commonly associated with contact. Furthermore, additional study is needed into the compatibility of nanoparticles with nature as well as the way of their recovery.


## 4. Conclusions

In this study, Bi2O3 NP was prepared by laser ablation technique. The antibacterial activity of the Bi2O3 NPs was determined and compared with amikacin against A. baumannii and S. aureus. S. aureus and A. baumannii, the predominantly bacterium of eye infection, displayed the different capabilities to create biofilms, Bi2O3 NP concentration of 32 μg/ml had antibacterial activity against S. aureus and A. baumannii. This result was confirmed by studying the effect of Bi2O3 NPs in bacterial biofilm formation in eyes contact lenses using the atomic force microscopy (AFM) technique. Taken together, Bi2O3 NPs could be used as a preservative for contact lenses as eye drops prevent the formation of microbial biofilms in contact lenses. Furthermore, Bi2O3 NPs were more effective as antibiofilm agents than amikacin in vitro by AFM, and the use of Bi2O3 NPs as contact lens preserving instead of chemicals preservatives. Taken together, Bi2O3 NPs could be used as promising therapeutic or preservative material for contact eye lenses to prevent the formation of microbial biofilms.



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*Source: 8511601-2022-07-01.xml*