Preparation and Characterization of Green Chitosan Nanocomposites and Evaluation of Their Inhibitory Role Against Certain Gram-Negative Bacteria

INTRODUCTION
Recurrent miscarriage remains a serious reproductive health problem worldwide. It is defined as two or more consecutive pregnancy losses before the 20th week of gestation. Recurrent miscarriage is a complex and multifactorial reproductive issue. Known causes of recurrent miscarriage include chromosomal abnormalities, uterine malformations, coagulation disorders, metabolic factors, endocrine disorders, autoimmune disorders, as well as lifestyle habits and general health status. [1-3] It affects a significant number of women during their reproductive years. Although anatomical, genetic, immunological, hormonal, lifestyle, and metabolic factors contribute to miscarriage, microbial infections are a major contributing factor to the risk of recurrent miscarriage. [4] Urinary tract infections (UTIs) are among the most common and important diseases affecting women [5,6］UTIs are often associated with multiple pathogens, including Gram-negative bacteria, Gram-positive bacteria, and some fungi, with an annual prevalence of up to 30% among women and 10% among men [7]. The most common causative agent is Escherichia coli, followed by Klebsiella spp. Staphylococcus [8]. Over the past decade, antibiotic resistance has emerged as a health threat, and the overuse and misuse of antibiotics may lead to the development of resistant bacterial strains [9]. The antibiotic cefotaxime has been widely used to treat urinary tract infections, but its resistance has increased among clinical isolates [10]. Nanotechnology has opened a new path for the development of advanced antimicrobial methods. Chitosan has received considerable attention due to its biodegradability and antimicrobial properties. It is a natural polysaccharide extracted by removing the acetyl group from chitin extracted from shrimp shells [11,12]. Chitosan reacts with negatively charged bacterial cell membranes, leading to membrane rupture and inhibiting bacterial growth [13]. Recent studies have shown that nanotechnology can significantly enhance the biological activity of treatments. Nanotechnology improves antibiotic performance after loading onto nanoparticles [14]. The effectiveness of nanoparticle synthesis is demonstrated in improving biological responses and therapeutic efficacy [15,16]. The importance of nanomaterials derived from natural sources is expected to lead to future advancements in medicine and other industries [17,18]. Based on this, the current study aimed to isolate and identify bacteria causing urinary tract infections (UTI) in women with miscarriages, evaluate antibiotic resistance mechanisms, synthesize chitosan nanoparticles from shrimp shells, and investigate their efficacy before and after loading with the antibiotic cefotaxime (CTX) against resistant bacterial isolates.

MATERIALS AND METHODS
SCTXle Collection
75 clinical urine sampleswere collected from pregnant women and women who had experienced miscarriages while visiting hospitals and health centers. The sampleswere collected in sterile containers and immediately transported to the laboratory under sterile conditions for microbiological examination.

Isolation and Identification of Microorganisms
Urine sampleswere cultured on blood agar and MacConkey agar plates for bacterial isolates and initial identification of each sCTXle. The sampleswere incubated at 37°C for 24 hours under aerobic conditions and identified by morphological characteristics using Gram staining as a baseline. Final identification was performed using the VITEK C-2bioMérieux system according to the manufacturer’s instructions. Susceptibility testing for cefotaxime (CTX) and other antibiotics was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI), and the minimum inhibitory concentration (MIC) values for the tested antibiotics were recorded [19].

Preparation and Identification of Nanoparticles
In this section, chitosan nanoparticles (CSNPs) extracted from shrimp shells were prepared and characterized using atomic force microscopy [20].

Extraction of Chitosan from Shrimp Shells
The shells were collected, washed, dried, and then ground and passed through a sieve to obtain a fine powder. The fine powder was then demineralized with a 1M hydrochloric acid (HCl) solution for 4 hours at room temperature. It was then washed several times with distilled water and left to dry. Protein removal was carried out with a 1M sodium hydroxide (NaOH) solution at 80-90°C with continuous stirring, followed by washing with distilled water. Chitin was then converted to chitosan using a 70% concentrated NaOH solution at 100°C. After this process, it was washed several times with distilled water and then oven-dried at 60°C [21]. Finally, chitosan nanoparticles (CSNPs) were prepared using the direct ion self-assembly method [22]. A 5 mL volume of chitosan solution prepared at a concentration of 0.2 mg/ml (dissolved in 1% acetic acid) was transferred into a glass beaker washed with ionized water. Immediately afterward, a 32 µL volume of cefotaxime (CTX) solution was taken and diluted in 20 mL of deionized water to ensure homogeneous distribution of the molecules [23]. The diluted aqueous cefotaxime (CTX) (20 mL) was then added drop by drop and slowly to the chitosan solution (5 mL) under continuous magnetic stirring for 24 hours at room temperature to ensure homogeneity and binding between the antagonist molecules and the amine groups on the surface of the chitosan [24]. The nanoparticles were isolated by centrifugation at 12,000 rpm for 30 minutes at 4°C. The material was separated to measure loading efficiency, while the nanoparticle preCTXitate was washed three times with deionized water and then freeze-dried to obtain pure nano powder.
The material was then separated to measure loading efficiency. 4. Evaluating the Inhibitory Effect of the Nanoparticle.
In this part, the study assessed how the synthesized nanoparticles inhibit three distinct clinical bacterial isolates: Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, which were grown on Mueller-Hinton agar (MHA) and displayed resistance to uncompleted Cefotaxime (CTX). Additionally, the potential synergistic outcomes following the integration of the green nanoparticle were examined. The experimental samples were categorized into three distinct groups, utilizing four replicates per setup, and evaluating two concentrations across the three tested bacterial pathogens. Treatment 1 (T1): This group involved the administration of uncompleted Cefotaxime (CTX) at a minimum inhibitory concentration (MIC) of 16 μg/ml, which was identified via Vitek sensitivity testing for the investigated Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains [25].Treatment 2 (T2): In this protocol, the specimens were treated with chitosan nanoparticles (CSNPs) at concentrations ranging from 32 to 64 μg/ml for each of the three aforementioned bacterial species. Treatment 3 (T3): This approach utilized chitosan nanoparticles combined with the antibiotic cefotaxime (CTX) at a minimum inhibitory concentration (MIC) of 16 μg/ml and 32 μg/ml against the examined isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

RESULTS AND DISCUSSION
The analytical outcomes of the nanocomposite’s structural profiling demonstrated that the external layer of the three CSNP nanoparticles exhibited a calculated roughness factor (Sa) and root-mean-square (RMS) based on the relevant equation (Table 1). The arithmetic mean height of this pristine nanocomposite surface (Sa) was equivalent to 1.50 nm (Figs. 1 and 2). Following the incorporation of the antibiotic, this parameter shifted to 4.02, indicating a net variance of 2.52\ nm between the pre- and post-loading phases, which serves as a critical indicator for enhanced performance. A higher degree of variance correlates with a more distinctive crystalline architecture post-loading than prior to the process (Table 1) [27]. Table 1 [28] clarify the surface topography and the subsequent proliferation of protrusions, alongside luminous spots that scatter light. This effect causes the post-loading surface matrix to appear dimmer relative to its initial state.Furthermore, the data validate successful loading by tracking the dimensional growth of these protrusions, where the maximal peak expanded by 6.67\ nm. The structural cavities increased in thickness from 6.49\ nm to 23.002\ nm, while their vertical height extended from 6.49\ nm to 20.6\ nm, confirming a well-distributed loading process. This uniform distribution was verified symmetrically, as detailed in Table 1.
The symmetry of the profile height varies based on the Ssk value: a negative Ssk indicates an elevation distribution concentrated above the average reference plane, whereas a positive value reflects a distribution below it. When Ssk equals zero, the peaks and valleys are uniformly balanced around the central plane.
Prior to drug entrapment, the surface features of the synthesized nanocomposite were investigated using SEM (Fig. 3). A homogenous distribution of spherical-shaped, well-dispersed particles with rough surface characteristics was observed. This elevated roughness is crucial for biomedical systems as it expands the surface area-to-volume ratio, thereby enhancing the matrix capacity for Cefotaxime immobilization. The particle sizes were determined to span a range of 115.4–299.0 nm.”
Following the immobilization of cefotaxime, distinct alterations in both surface topography and cluster configuration became evident, as illustrated in Fig. 4. SEM micrographs confirmed the successful integration of the antibiotic onto the nanocomposite matrix, which induced minor particle agglomeration and texture modifications. While the carrier maintained its predominantly spherical cluster morphology, the localized particle dimensions spanned from 73.01 to 304.7 nm. This effective loading efficiency underscores the compatibility and potential of the synthesized matrix as a robust vehicle for targeted drug delivery.”
Recent research indicates that elevated resistance levels have been observed in the most common types of bacteria in pregnant women and those with urinary tract infections(UTI|), specifically Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, for the antibiotic cefotaxime (CTX). Escherichia coli resistance to cefotaxime (CTX) reached 100%, with a minimum inhibitory concentration (MIC) of 32 µg/ml, as shown in Table 2. Escherichia coli resistance to CTX (cefotaxime) is a critical health issue drive by the inappropriate administration of antimicrobials [29]. Cefotaxime resistance is linked to changes in intracellular cefotaxime (CTX) concentration and diminished cellular permeability [30] in Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa exhibits resistance to the antibiotic cefotaxime and possess the capacity to synthesise extended-spectrum beta-lactamase (ESBL) enzymes. These strains also possess resistance mechanisms associated with altered glucose-modified glycolytic pathways, inhibiting glucose breakdown and contributing to increased intracellular glucose accumulation. This leads to genetic mutations and the generation of reactive oxygen species (ROS) [31]. Synergistic treatment combining the antibiotic cefotaxime (CTX) with certain antimicrobial peptides (CTXs) has been shown to reduce the likelihood of resistance development in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Treatment with CTX-loaded nanoparticles, such as chitosan nanoparticles, can inhibit bacterial resistance development, particularly against strains that have already developed resistance due to exposure to low concentrations that suppress bacterial growth without eradication. [32] Antibiotic resistance in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa bacteria isolated from infected women has been studied in many previous research studies. Previous studies [33] showed that Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates from processed cheeses were associated with high levels of drug resistance (MDP). These results mirror those reported by several studies, including one in Nigeria, which showed many antibiotic-resistant bacterial isolates in women with abortions and urinary tract infections and resistance genes [34].
The results of the current study, shown in Table 3 and Fig. 5, indicate significant differences in the inhibition diameters of E. coli bacteria when treated with the antibiotic and chitosan nanoparticles (Chitosan NPs), and utilizing the antibiotic -loaded nanocomposite (Chitosan NPs/CTX). Treatment with the antibiotic (T1) showed the lowest inhibition diameter at (1.25 ± 0.366) mm, indicating high resistance of the bacterial isolates to Cefotaxime. Treatment with chitosan nanoparticles (T2) showed a significant increase in the inhibition diameter at (10.875 ± 1.885) mm, indicating that the nanoparticles possess inhibitory activity against the studied bacteria. Treatment with the nanocomposite with the antibiotic (T3) achieved the highest inhibition diameter at (21.25 ± 0.871) mm, with significant differences (P≤0.05) compared to all other treatments.
Regarding the effect of concentrations on the inhibition diameter, treatment (T3) at the first concentration (C1) recorded an inhibition diameter of (19.75 ± 1.708) mm, while the inhibition diameter increased at the second concentration (C2) to (22.75 ± 2.217) mm. This indicates a direct relationship between increasing concentration and increasing inhibitory activity of the antibody-loaded nanocomposite. These values also significantly exceeded those recorded in treatment (T2), which reached (6.50 ± 1.291) and (15.25 ± 2.630) mm for the first and second concentrations, respectively. The same was true for treatment (T1) at the first and second concentrations, which were (0.50 ± 0.577) and (2.05 ± 0.816) mm, respectively. These results indicate that loading Cefotaxime onto chitosan nanoparticles enhanced its antibacterial efficacy and increased its ability to inhibit the growth of E. coli bacteria.
The results in Table 4 and Fig. 5 showed significant differences in the diameters of inhibition of K. pneumoniae bacteria between the treatment with the antibiotic cefotaxime (CTX), the nano chitosan (Chitosan NPs), and the nanocomposite with the antibiotic (Chitosan NPs/CTX). Treatment with the free antibiotic Cefotaxime (T1) showed the lowest inhibitory activity at concentrations 16 and 32, with an average inhibition diameter of 0.25 ± 0.500 mm for concentration 16 and 1.75 ± 0.957 mm for concentration 2. In contrast, treatment with the chitosan nanoparticle (T2) demonstrated better inhibitory activity than the free antibiotic, with inhibition diameters of 3.50 ± 1.291 mm for concentration 1 and 6.50 ± 1.291 mm for concentration 2. Treatment with the cefotaxime-loaded nanoparticle (T3) showed a clear and significant advantage over the other treatments, recording the highest inhibition diameter values of 7.75 ± 0.957 mm for concentration 1 and 11.50 ± 1.291 mm for concentration 2. As the results show, treatment (T3) at the lower concentration (C1) achieved higher efficacy than all values of free Cefotaxime at both concentrations, as it clearly outperformed treatment (T1). This may be attributed to the ability of nanoparticles to improve the delivery of the antibiotic to bacterial cells and increase its permeability through cell membranes, as well as the synergistic effect between chitosan and Cefotaxime, which contributed to enhancing the sensitivity of K. pneumoniae bacteria and reducing their resistance to the antibiotic.

CONCLUSION
This study demonstrated that urinary tract infection-associated bacteria isolated from pregnant women and women with miscarriage, especially Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, showed high resistance to cefotaxime, indicating a serious therapeutic challenge. Free cefotaxime produced very limited antibacterial activity, confirming the reduced effectiveness of this antibiotic against resistant clinical isolates. Chitosan was successfully extracted from shrimp shells and converted into chitosan nanoparticles, which were then loaded with cefotaxime. AFM and SEM analyses confirmed successful nanoparticle formation and drug loading through clear changes in surface roughness, topography, and particle morphology. These findings support the suitability of chitosan nanoparticles as an effective drug-delivery carrier. Biological testing showed that chitosan nanoparticles alone had measurable antibacterial activity, whereas cefotaxime-loaded chitosan nanoparticles produced significantly greater inhibition against the tested resistant bacteria than either free cefotaxime or unloaded nanoparticles. The strongest effect was observed against E. coli, with inhibitory activity increasing at higher concentrations. Overall, cefotaxime-loaded chitosan nanoparticles appear to be a promising strategy for improving antibacterial efficacy against resistant UTI pathogens. This nanocomposite may offer an alternative approach for controlling antibiotic-resistant infections, although further in vivo, toxicity, and mechanistic studies are needed before clinical application.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.