Abstract

General Background: Antibiotic resistance is a critical global health issue, and innovative approaches are needed to combat multidrug-resistant (MDR) bacteria. Specific Background: Nanotechnology has emerged as a promising strategy to enhance antibiotic efficacy and reduce resistance. Knowledge Gap: However, there is limited understanding of how metal oxide nanoparticles (NPs) like Fe2O3 and CuO can be utilized to improve the performance of antibiotics such as sulfadiazine. Aims: This study aimed to synthesize Fe2O3 and CuO nanoparticles, conjugate them with sulfadiazine, and evaluate their antibacterial efficacy against MDR Pseudomonas aeruginosa. Results: The nanoparticles were synthesized via chemical precipitation, with Fe2O3 and CuO having mean crystal sizes of 41.40 nm and 44.83 nm, respectively. When bound to sulfadiazine, the crystal sizes were 42.62 nm (Fe2O3) and 38.77 nm (CuO). The minimum inhibitory concentration (MIC) values for sulfadiazine-bound CuO and Fe2O3 NPs ranged from 16-32 μg/ml, significantly lower than the 64-128 μg/ml observed for standard sulfadiazine. Hemolysis assays confirmed the biocompatibility of these nanocomposites at tested concentrations. Novelty: The study reveals that Fe2O3 and CuO nanoparticles significantly enhance sulfadiazine's antibacterial activity against MDR P. aeruginosa, suggesting a potential method to bypass traditional resistance mechanisms. Implications: The study suggests that nanoparticle-conjugated antibiotics could be a promising solution for combating antibiotic resistance, potentially reducing its negative impact on public health.

Highlights:

1. Nanoparticles reduce sulfadiazine's MIC against MDR Pseudomonas aeruginosa.
2. Fe2O3 and CuO nanoparticles enhance antibiotic efficacy.
3. Hemolysis assays confirm nanocomposites' safety and biocompatibility.

Keywords: Nanotechnology, Antibiotic Resistance, Fe2O3 Nanoparticles, CuO Nanoparticles, MDR Pseudomonas aeruginosa

Introduction

According to statistics on infections brought on by MDR bacteria, bacterial resistance to antibiotics has multiplied significantly in recent years. Numerous studies have demonstrated the grave threat that antibiotic resistance poses to human health [1]. Microorganisms can develop insensitivity to antibiotics at deadly dosages, known as multidrug resistance (MDR). MDR has become a significant concern about the efficiency of antibiotics against pathogenic diseases [2]. MDR bacteria's situation has worsened due to the lack of new antibacterial medicines being developed [3]. Further supporting the development of bacterial tolerance is the treatment of MDR bacteria using ineffective antibiotics [4]. Antibiotic resistance in bacteria reduces the drug's therapeutic effectiveness for treating life-threatening infectious diseases and raises the overall cost of therapeutic approaches [5].

A variety of ailments, including bloodstream infections, urinary tract infections, infections of burn wounds, and lung infections, are linked to Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium that can cause a wide range of life-threatening infections. P. aeruginosa is essential for healthcare-associated infections in hospitals [6]. High morbidity and mortality are linked to the multidrug resistant (MDR) strain infections that are becoming more prevalent and have few therapeutic choices [7,8]. Low outer membrane permeability, the development of efflux pumps, and the generation of enzymes that inactivate antibiotics all contribute to the intrinsic resistance. The development of acquired resistance may result through mutational alterations or via the horizontal transmission of resistance genes by mobile genetic elements (MGEs), such as integrins, transposons, or plasmids [8]

The available information indicates that interactions with DNA significantly impact Ag+ effectiveness as an antibacterial agent. Silver compounds, such as silver sulfadiazine, provide Ag+ when frequently given externally as a treatment. The quantities of sulfadiazine applied topically effectively against most bacteria that cause burns and chronic wound infections. Fortunately, despite the wide range of reactivity, exposure to therapeutic doses of Ag+ usually does not endanger human health [9-11]. Most Ag+-targeted bacterial sites are proteinaceous, where changes in amino acid residues have a variety of impacts, including structural damage, interference with metabolic and replicative processes, and others [12-14]. The U.S. Food and Drug Administration (FDA) has authorized the antibacterial medication sulfadiazine to treat and prevent a number of bacterial illnesses, including ulcers, toxoplasmosis encephalitis, urinary tract infections, and other ailments [15,16].More precisely and steadily than conventional antibiotic molecules, pure antibiotics created at the nanoscale can cross bacterial cell membrane barriers [17].

The science of nanotechnology, which deals with materials at the molecular or nanoscale, is still in its infancy. In fields including biology, medicine, chemistry, and physics, it is currently working extraordinary wonders [18]. Materials' physical and chemical properties are radically changed when they are shrunk to the nanoscale. The increased surface area changes these attributes since it enhances the reactivity of materials at the nanoscale [19]. These bactericidal pathways are influenced by the size, shape, composition, and surface chemistry of the NPs. The ability of NPs to interact with the cell wall or membrane of bacteria is crucial for increasing the treatment's efficacy [20].Numerous bactericidal mechanisms can be used by NPs to carry out their function, making it difficult for bacteria to build resistance to them [21]. Due to their unique physical, chemical, optical, and mechanical characteristics, inorganic nanoparticles have generated a lot of interest [22].When combined with nanoparticles (NPs) or used alone, pure antibiotics made at the nanoscale can pass through bacterial cell membrane barriers more precisely and steadily than conventional antibiotic compounds [23].

Methods

Bacterial diagnosis and Antibiotic Susceptibility

Clinical samples were taken from patients who were resting wounds at the Baquba Teaching Hospital in Iraq. To identify bacteria isolates, biochemical testing, morphological diagnosis, microscopy, and culture were employed. The VITEK® 2 compact device, however, served as the foundation for the confirmatory identification test, and it was also used to test the samples for antibiotic susceptibility.

Synthesis of Fe2O3 or CuO Nanoparticles

In 50 mL of ethanol, salts of iron or copper were dissolved in a 0.25 M solution. In 50 mL of ethanol, NaOH dissolves in a 0.5 M solution. For 30 minutes at room temperature, the second solution was continuously distilled into the first solution while being continuously stirred with a magnetic stirrer. accurate and set pH = 7 using drops of diluted HCI acid, followed by filtration, water washing, and room-temperature drying. For Fe2O3 NPs, a gel is created, allowed to set for five days, then dried at 100°C for five hours before being burned. Regarding the CuO NPs, they were afterward filtered, cleaned with water, and allowed to air dry. then torched for three hours at 700°C to produce a black precipitate.

Loading of sulfadiazine with nanoparticles

0.5 gm of the prepared Nano oxides CuO and Fe2O3 were taken and mixed with 100 mL of deionized water for each oxide using an ultrasonic device for 10 minutes; after that, each was added to 2 gm of sulfadiazine in 100 mL of deionized water and placed on the engine magnetically for two hours, at room temperature, then placed in an ultrasound machine for three hours, and dried at 45°C. (Sulfadiazine 99.9% obtained from Al-Kindi Pharmaceutical Company in Nahrawan/Iraq).

Preparation of bacterial inoculum

The overnight BHI broth medium, which is similar to the McFarland 0.5 standard and yields turbidity comparable to that of a bacterial suspension containing 1.5 × 108 colony forming units (CFU)/ml, was used to prepare bacterial suspensions for inoculation. In reality, an OD 600 between 0.08 and 0.1 matches a McFarland 0.5 standard match.

Minimum Inhibitory Concentration

Depending on references [24,25], the stock solution of Sulfadiazine and NPs Sulfadiazine were prepared. Serial concentrations with twice the progressive value rate (8-16-32-64-128-256-512-1024µg/ml) were then prepared and loaded into defined rows of 96 well microplates from the antibiotic stock solution. Three replicates’ wells were assigned to each treatment with a negative control and a positive control. The three wells of microplates for tests with equal conditions were used for each bacterial isolates testing. The test wells were composed of serial dilutions of antibiotics, Mueller-Hinton broth and bacterial suspension. The negative control wells for each case consisted of the serial dilutions of antibiotics, Mueller-Hinton broth and no bacterial suspension. The positive control wells consisted of Mueller-Hinton broth and the bacterial suspension without antibiotic. After inoculation and incubation at 37°C for 24 hours, the plates were subjected to scanning at 630 nm of wavelength ELISA reader.

Hemolysis assay

The hemolysis assay was used to screen for nano-antibiotics at different concentrations (16, 32, 64, 128, 256, and 512µg/ml) in order to detect harmful or dangerous compounds. Human blood type O cells were obtained from the blood bank at Baquba teaching hospital and prepared by washing several times with PBS. Following the last wash, red blood cells (RBC) were diluted to 1/10 of their volume with PBS to create the blood cell suspension.

The assay was performed by mixing 0.3 mL of the RBC solution with 1.2 mL of antibiotics were prepare in the current study with the above-mentioned concentrations, 1.2 mL of distilled water was set as a positive control and 1.2 mL of PBS as a negative control. The mixtures were vortexed, left for 2 h at room temperature, and then centrifuged at 4,000 x g for 10 min at 4°C. Absorbance of the supernatants was measured at 540 nm in a UV-Vis spectrophotometer [26]. The percentage of hemolysis of each fraction was calculated using the expression below:

Figure 1.

Result and Discussion

Characterization by X-ray diffraction

The copper oxide X-ray spectrum in Figure (1) 44.83 nm was the average crystal size. Figure (2) displays the Fe2O3 XRD. The crystals had an average size of 41.40 nm. Figure (3) shows that the XRD and Sulfadiazine were associated with CuO NPs and the mean crystal size was 38.77 nm. Figure (4) shows that the XRD and Sulfadiazine were associated with Fe2O3 NPs and the mean crystal size was 42.62 nm.

Figure 2.

Characterization by energy-dispersive X-rays

Figure (5) illustrates how energy-dispersive X-rays were used to determine the fraction of elements contained in CuO NPs. The high-purity copper oxide was found in the findings. Figure (6) illustrates the constituents that were identified in Fe2O3 NPs. High-purity iron oxide was visible in the results. Energy-dispersive X-ray (EDX) NPs revealed the proportion of elements in sulfadiazine bound to CuO NPs; the results are shown in Figure (7) and highlight the great purity of the sulfadiazine linked to CuO NPs. The outcomes that demonstrated the sulfadiazine's great purity when bound to Fe2O3 NPs are shown in Figure (8).

Figure 3.

Diagnosis of bacterial isolates and antimicrobial resistance

Twenty-five isolates of Pseudomonas aeruginosa were identified from clinical samples of burn and wound patients. It was diagnosed and its resistance to antibiotics was detected by the Vitek 2 device, where five most resistant isolates of each type were selected. The five isolates of P. aeruginosa (P1-P5) showed resistance pattern against Cefotaxime, Amikacin, Gentamicin, Ticarcillin-Clavulanate, Piperacillin, Cefepime, Ciprofloxacin, Tobramycin, Ceftazidime, Levofloxacin, Polymyxin, and Meropenem. Ten most resistant isolates were selected, five isolates of them (P1-P5) showed resistance pattern against Cefotaxime, Amikacin, Gentamicin, Ticarcillin-Clavulanate, Piperacillin, Cefepime, Ciprofloxacin, Tobramycin, Ceftazidime, Levofloxacin, Polymyxin, and Meropenem. Also, three of them (P6-P8) showed resistance pattern against Cefotaxime, Amikacin, Ticarcillin-Clavulanate, Ciprofloxacin, Ceftazidime, Levofloxacin and Polymyxin. While two isolates (P9, P10) were resistance to Amikacin, Ticarcillin-Clavulanate, Piperacillin, Ciprofloxacin, Tobramycin and Polymyxin. Based on the above all selected isolates considered multi drug resistant.

Determination of Minimal Inhibitory Concentration

The results of standard Sulfadiazine gradient for P. aeruginosa isolates under study shown three isolates (P2,P5,P6) (30%) with MIC 128μg/ml and seven isolates (P1,P3,P4,P7,P8,P9,P10) (70%) with MIC 64μg/ml. Whereas the results of MIC with regard to Sulfadiazine bound to CuO NPs were determined for these isolates were detected that four isolates (P5,P7,p9,p10) (40%) with MIC 16μg/ml and six isolates (P1,P2,P3,P4,P6,P8) (60%) with MIC 32μg/ml (Table 1). While all ten isolates (100%) shown same MIC 32μg/ml for sulfadiazine bound to Fe2O3 NPs (Table 1).

Sulfadiazine bound to CuO NPs and sulfadiazine bound to Fe2O3 NPs showed increase in the inhibitory against P. aeruginosa activity compared to the standard antibiotics, this thing reduces the effectiveness of the resistance mechanisms, whether innate or acquired.

The presence of oxides gives the antibiotic a positive charge, which makes it more affinity for binding to the outer membrane of the bacteria with a negative charge. It is also possible that the reason is that these modifications do not enable the bacteria to benefit from antibiotic resistance enzymes that are responsible for destroying these molecules by binding to them, because they are enzymes specialized in molecules with certain chemical structures, and any modification in the base substance disrupts the enzyme’s attachment to these molecules.

Determination Hemolysis assay

The antibiotics prepared under this study (sulfadiazine bound CuO NPs and sulfadiazine bound Fe2O3 NPs) were the subject of a cytotoxicity test for the purpose of ensuring their safety. The results showed that the percentage of the hemolytic activity of the sulfadiazine-bound CuO NPs with concentrations (16, 32, 64, 128, 256, and 512µg/ml) were (2%, 3%, 7%, 9%, 13%, and 15%) respectively. In contrast, the percentage of the hemolytic activity of the sulfadiazine bound Fe2O3 NPs with the same concentrations were (7%, 7%, 10%, 12%, 17%, and 35%) respectively. These results suggest that the compound was safe and non-toxic at all concentrations except 500μg/ mL, which showed a toxic effect in the form of fractured platelets Figure 13&14. the attention that this concentration of 500μg/ml is much higher than MIC for isolates under study 32μg/ml.

Figure 4.Hemolysis test sulfadiazine bound CuO NPs

Figure 5.Hemolysis test sulfadiazine bound Fe2O3 NPs

Conclusion

Based on our findings, we can conclude that increase in the effectiveness of Sulfadiazine bound to CuO NPs and sulfadiazine bound to Fe2O3 NPs showed increase in the inhibitory against P. aeruginosa activity compared to the standard antibiotics, this thing reduces the effectiveness of the resistance mechanisms, whether innate or acquired. the small size of the antibiotic increased its surface area, which plays an important role in increasing its effectiveness of it. This may be due to its ease of permeability from the membrane of bacteria cells, as well as accumulating inside the cell in greater concentrations, which makes it difficult for bacterial cells to get rid of them through the resistance system using flow pumps responsible for the disposal of harmful substances outside the bacterial cell. Furthermore, these nanoparticles have no toxicity.

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